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Strukturna i funkcionalna analiza gena za alfa-1-antitripsin u bolestima pluća čoveka | Structural and functional analysis of alpha-1-antitrypsin gene in human lung diseases | Alfa-1-antitripsin je inhibitor serin proteaza čija je osnovna biološka funkcija inhibicija
elastaze neutrofila – enzima koji učestvuje u degradaciji elastina i koji dovodi do oštećenja
tkiva pluća. Deficijencija i disfunkcija alfa-1-antitripsina leže u osnovi više oboljenja od
kojih su najčešći emfizem pluća i oboljenja jetre, a mogu predstavljati i faktor rizika za
nastanak karcinoma pluća. Ovaj rad je imao za cilj da ispita postojanje strukturnih promena
u kodirajućim egzonima gena za alfa-1-antitripsin kod ispitanika sa emfizemom pluća i kod
ispitanika sa karcinomom pluća, kao i da istraži funkcionalne posledice otkrivenih promena
i njihov eventualni značaj u nastanku i razvoju bolesti.
Strukturnom analizom gena za alfa-1-antitripsin DGGE metodom i DNK sekvenciranjem
detektovano je prisustvo deficijentnih varijanti Z, S i Mmalton, kao i dve nove varijante
označene kao G320R i V321F.
Elektroforezom u denaturišućem poliakrilamidnom gelu uočeno je da varijante G320R i
V321F migriraju brže u odnosu na WT varijantu, a analizom u denaturišućem
poliakrilamidnom gelu sa 5M ureom pokazano je da ove varijante verovatno imaju
smanjenu hidrofobnost u odnosu na WT varijantu. Analizom formiranih kompleksa između
alfa-1-antitripsina i elastaze pankreasa u SDS gelovima, utvrđeno je da se inhibitorna
aktivnost ispitivanih varijanti ne razlikuje od inhibitorne aktivnosti WT varijante proteina.
Nativna elektroforeza preparata alfa-1-antitripsina inkubiranih na različitim temperaturama
pokazala je da mutirane varijante proteina imaju blago smanjenu termostabilnost u odnosu
na WT protein, a elektroforeza proteina eksprimiranih u COS7 ćelijskoj liniji pokazala je da
varijante G320R i V321F pod fiziološkim uslovima ne formiraju polimere. Metodom
imunofluorescencije na tranzijentno transfekovanim COS7 ćelijama pokazano je da su
varijante G320R i V321F, kao i WT protein, lokalizovane u Goldžijevom aparatu i da se ne
zadržavaju u endoplazmatičnom retikulumu.
Rezultati ovog rada potvrđuju pretpostavku da su retke varijante alfa-1-antitripsina
verovatno zastupljenije nego što se pretpostavlja i ukazuju na to da su metode
genotipizacije neophodne u pravilnoj detekciji ali i u proceni učestalosti varijanti alfa-1-
antitripsina. Rezultati funkcionalne analize varijanti G320R i V321F ukazuju na to da ove
dve novootkrivene varijante nemaju uticaja na funkciju proteina, ali je prisutan efekat na
strukturu proteina. Funkcionalna analiza varijanti G320R i V321F alfa-1-antitripsina
predstavlja prve podatke o funkcionalnoj analizi ovog regiona u proteinu. | Alpha-1-antitrypsin is a serine protease inhibitor whose main biological function is
inhibition of neutrophil elastase – enzyme capable of digestion of elastin and involved in
lung tissue destruction. Deficiency and disfunction of alpha-1-antitrypsin is associated with
emphysema and liver disease, and can represent a risk factor for development of lung
cancer. The objective of this work was to perform structural analysis of alpha-1-antitrypsin
gene in subjects with emphysema and lung cancer, as well as to perform functional analysis
of discovered variants in order to estimate their role in disease development.
Structural analysis of alpha-1-antitrypsin gene, using DGGE and DNA sequencing,
revealed presence of deficient variants Z, S and Mmalton, as well as two novel variants –
G320R and V321F.
Denaturing PAGE analysis showed that variants G320R and V321F exibited increased gel
mobility compared to WT variant, and electrophoresis in the presence of 5M urea showed
that these variants effected the protein structure, probably by changing its hydrophobicity.
Nondenaturing electrophoresis of heat treated proteins showed that both variants had
slightly decreased thermostability when compared to WT variant and non-denaturing
PAGE of COS7 expressed proteins showed that these variants did not form polymers under
physiological conditions.
Inibitory activity of variants G320R and V321F, determined by analysis of SDS-PAGE
resistant complexes between alpha-1-antitrypsin and porcine pancreas elastase, was same as
in WT variant.
Immunofluorescence of transiently transfected COS7 cell line showed that variants G320R
and V321F, as well as WT protein, were predominantly localized at Golgi apparatus
indicating that they are not retained in endoplasmic reticulum.
Results of this study indicate that rare alpha-1-antitrypsin variants are probably more
frequent than it is assumed and highlight the need for use of genotyping methods in
detection of alpha-1-antitrypsin variants. Results of functional analysis of novel variants
G320R and V321F showed that although the effect on the protein structure was present,
their functional activity was not impaired. Functional analysis of G320R and V321F
variants is the first functional study of this region of alpha-1-antitrypsin. | true |
Uticaj delimičnog isušivanja korenova na ekspresiju NCED, TAO1 i EIL1 gena i rastenje divljeg tipa i flacca mutanta paradajza (Lycopersicon esculentum Mill.) | The effect of partial root-zone drying on expression of NCED, TAO1 and EIL1 genes and growth in tomato wild-type and flacca mutant (Lycopersicon esculentum Mill.) | Cilj ovog istraţivanja je bio karakterizacija fizioloških procesa koji su u osnovi
regulacije rastenja i razvića biljaka paradajza pod dejstvom delimiĉnog isušivanja
korenova (Partial root-zone drying -PRD), ali i regulisanog deficita navodnjavanja
(Regulated deficit irrigation - RDI). Cilj je bio i da se u ova ispitivanja ukljuĉe i
molekularna istraţivanja, koja bi, uz analizu procesa rastenja i drugih fizioloških
procesa (vodnog reţima, razmene gasova, akumulacije i distribucije stres hormona
ABA, kao i usvajanja i distribucije jona), znaĉajno doprinela razumevanju PRD i RDI
efekata na paradajz. Zato je praćena ekspresija gena ukljuĉenih u biosintetski put ABA
(TAO1 i NCED1) i transdukciju signala etilena (EIL1). Eksperimentalni model u ovim
istraţivanjima je bio paradajz (Lycopersicon esculentum Mill.), kao kultura koja ima
znaĉajne potrebe za vodom i kod koje suša moţe da izazove znaĉajne efekte na rastenje
i razviće. Odabrani su divlji tip (WT), sorta Ailsa Craig, i mutant flacca koji se odlikuje
smanjenom sintezom hormona ABA. Biljke paradajza divljeg tipa i mutanta flacca su
nakon proklijavanja presaĊene u posebno dizajnirane saksije, koje su podeljene na dve
polovine plastiĉnom ploĉom, ĉime je omogućeno razdvajanje korenovog sistema u dva
hidrauliĉno izolovana kompartmenta. Biljke su gajene u komercijalnom supstratu
(Klasmann Potground H) u uslovima fitotronske komore (temperatura dan/noć 26/17
oC, relativna vlaţnost vazduha 60%, osvetljenost (PAR) 250 µmolm-2s-1 i duţina
fotoperioda od 14 h). Koliĉina vode u supstratu je merena pomoću TDR proba (Time
domain reflectometer, TRASE, Soil Moisture Equipment Corp., USA). Primenjivana su
tri razliĉita tretmana zalivanja. Optimalno navodnjavanje (Full irrigation - FI) - supstrat
u celoj rizosferi je svaki dan zalivan do optimalnog vodnog kapaciteta, odnosno,
sadrţaja vode u supstratu od 36%. Delimiĉno isušivanje korenovog sistema (PRD) -
supstrat u polovini korenovog sistema je zalivan do optimalnog vodnog kapaciteta, dok
u drugoj polovini nije zalivan sve dok se sadrţaj vode u supstratu nije spustio na 18-
20%, kada je vršena inverzija strana zalivanja. Regulisani deficit navodnjavanja (RDI) -
biljke su zalivane do vrednosti vode u supstratu od 18%. Vodni potencijal listova i
otvorenost stoma su mereni u danu PRD okreta (promene strane zalivanja korena).
Ukupni potencijal vode u listu (Ψl) meren je u komori pod pritiskom (Soil Moisture
Equipment Corp., Santa Barbara, CA, USA). Provodljivost stoma je merena aparatom
porometrom (AP4, Delta-T, Cambridge, U.K.). Na kraju ekperimenta izmerena je sveţa
i suva masa korena, stabla i listova i odreĊen odnos suve mase izdanka i korena. Sadrţaj
abscisinske kiseline u listovima je analiziran ELISA testom uz korišćenje visoko
specifiĉnog monoklonalnog antitela (MAC 252). Apsorpcija je oĉitavana na ELISA
ĉitaĉu (Sunrise, Tecan) na 405nm. Sadrţaj Ca, Mg, Fe, Mn, Zn i Cu odreĊivan je
atomsko-apsorpcionom spektrofotometrijom, K plamenofotometrijski, dok je sadrţaj N
odreĊivan metodom po Kjeldahl-u. Ekspresija gena je praćena u danu PRD okreta.
Izolacija RNK je uraĊena primenom RNA-ase plant mini kit-a (Quiagen, Germany). Za
sintezu cDNK je korišćen First strand cDNA synthesis kit (Fermentas, Lithuania).
Ekspresija gena je praćena primenom Real Time-PCR (SYBR-Green tehnika; RT-PCR
7500, Applied Biosystems, UK). Rezultati su pokazali da je PRD tretman, kao i RDI,
doveo do redukcije rastenja biljaka. Kada su u pitanju sveţa i suva masa organa, kod
biljaka divljeg tipa je došlo do redukcije u oba tretmana, dok je kod flacca mutanta,
PRD tretman doveo do povećanja biomase, za razliku od RDI, koji je doveo do
redukcije. Analiza rasta vegetativnih organa je pokazala da su biljke flacca mutanta bile
znaĉajno niţe (za ca 40%) i sa manjom obrazovanom sveţom i suvom masom u odnosu
na divlji tip. Analiza vodnog reţima biljaka, na osnovu rezultata potencijala vode () u
listovima i koliĉine vode u supstratu, je pokazala da su RDI biljke bile izloţene većem
stepenu stresa u odnosu na PRD biljke i to kod oba ispitivana genotipa. Rezultati
dnevne dinamike provodljivosti stominih ćelija (gs) su ukazali da se reakcije na PRD
tretman mogu okarakterisati kao reakcije brţeg odgovora, a reakcije na RDI kao
reakcije sporijeg odgovora stominih ćelija na sušu. Analiza reakcija stominih ćelija u
odnosu na promene potencijala vode u listovima je pokazala da su reakcije zatvaranja
stoma, u uslovima RDI, pratile opadanje potencijala vode u listovima, što ukazuje da su
ove reakcije stominih ćelija kod oba genotipa rezultat dejstva hidrauliĉnih signala.
Rezultati analize efekata PRD tretmana na vodni reţim biljaka su pokazali da su
hemijski signali bili odgovorni za reakcije stominih ćelija. Kod divljeg tipa, veći efekat
na povećanje sadrţaja ABA u listovima imao je PRD tretman, dok je kod flacca
mutanta to bio RDI tretman. Sliĉno povećanje u sadrţaju ABA u listovima kod oba
genotipa prilikom PRD tretmana ukazuje da flacca mutacija ne pogaĊa transport ABA
iz korena u nadzemni deo. Merenja koncentracije jona, uglavnom, nisu pokazala
znaĉajan efekat primenjenih sistema deficita navodnjavanja na usvajanje, akumulaciju
ili distribuciju jona. Najznaĉajniji je bio uticaj PRD koji je kod oba genotipa ubrzao
translokaciju Mn iz korena u listove, što moţe biti od znaĉaja za adaptivne metaboliĉke
reakcije biljaka na PRD, jer je Mn znaĉajan antioksidacioni agens. TakoĊe je PRD
doveo i do veće akumulacije N kod flacca mutanta. Molekularna ispitivanja ekspresije
gena u listovima su pokazala razlike izmeĊu tretmana, tokom njihovog trajanja i izmeĊu
ispitivanih genotipova. Analiza ekspresije sva tri gena u PRD tretmanu je pokazala
genotipske razlike. Sliĉan obrazac ekspresije TAO1, NCED i EIL1 kod biljaka divljeg
tipa ukazuje na moguće sinergistiĉko dejstvo signalnih puteva za ABA i etilen. Sliĉan
trend promena nije pronaĊen kod flacca mutanta. Redukovana NCED i znaĉajna EIL1
ekspresija verovatno reflektuje porast u produkciji etilena, koji moţe da utiĉe na signale
i biosintezu ABA. Kod RDI tretmana, obrazac ekspresije gena u listovima divljeg tipa i
flacca mutanta je bio sliĉan, što ukazuje na mogućnost sinergistiĉkog efekta signalnih
puteva za ABA i etilen. Pošto je kod flacca mutanta bila izraţena ekspresija TAO1 gena
i utvrĊena akumulacija ABA u listovima, naši rezultati su pokazali da neki drugi gen iz
TAO familije, a ne TAO1 kodira enzim abscisinsku aldehidnu oksidazu kod flacca
mutanta. Generalno, svi ovi rezultati su doprineli razumevanju efekata suše indukovane
PRD i RDI tretmanima na paradajz. U budućim istraţivanjima bi trebalo analizirati više
gena, posebno onih koji su ukljuĉeni u put biosinteze i/ili u signalnu mreţu ABA i
etilena. | The aim of presented PhD thesis was to investigate the physiological mechanisms that
are in the base of tomato growth and development under partial root-zone drying (PRD)
and regulated deficit irrigation (RDI). Investigations included plant growth, water
regime, gas exchange, abscisic acid (ABA) content in the leaves of tomato and ions
content in different tomato plant organs. The aim of presented PhD thesis was, also, to
assess the expression of TAO1, NCED and EIL1 genes in the leaves of tomato under
drought treatments of PRD and RDI. The investigated genes are involved in the
biosynthesis and signaling pathways of abscisic acid (ABA) and ethylene as key
hormones in the reactions of plants to drought. Experiments were done with tomato
plants (Lycopersicon esculentum Mill.), wild type (Ailsa Craig) and flacca mutant.
Tomato was chosen because of its significant needs for water and because drought may
have significant effects on its growth and development. Tomato mutant flacca, which is
chosen for investigation, is defective in the last step of ABA biosynthesis. As a
consequence, the endogenous level of ABA in flacca tissues is significantly lower than
in wild type. Tomato plants wild type (WT) and flacca mutant were raised from seed
and transplanted into 10 L pots (one plant per pot) filled with commercial compost
(Klasmann Potground H) in a growth chamber (photoperiod was 14 h; light intensity at
plant level 250 µmolm-2s-1, temperature 26/17 oC and relative humidity 60%). For PRD
experiments the pots were specially designed in such a way that they were separated
with plastic sheets into two equally sized compartments. Washed roots of the seedlings
were divided into approximate halves and repotted into these two hydraulically
separated compartments. The volumetric soil water content (θ) of both compartments of
each pot was measured daily using TDR probes (time domain reflectometer, TRASE,
Soil Moisture Equipment Corp., USA
After transplantation, plants were subjected to
following irrigation treatments:
full irrigation (FI), in which the whole root system
was irrigated to full substrate holding capacity (θ of 36%); ) partial root-zone drying
(PRD), where one half of the root was irrigated to reach θ of 36% while the other half
was allowed to dry, and the irrigation was shifted when θ of the dry side had decreased
to 18-20%.
deficit irrigation (RDI), in which water was evenly applied to the whole
root system to rich θ of 18%. Leaf water potential and stomatal conductance were
measured in day when PRD shift occurred. Leaf water potential was measured by
pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, CA, USA); while
stomatal conductance was measured by porometer (AP4, Delta-T, Cambridge, U.K.). At
the end of experimental period, fresh and dry weight of roots, stems and leaves were
measured and the shoot/root ratio determinated. Measurement of ABA content in the
leaves of investigated plants was done by ELISA method using a MAC 252 monoclonal
antibody for ABA. Plate contents were read at 405 nm by ELISA reader (Sunrise,
Tecan). Ca, Mg, Fe, Mn, Cu and Zn content in leaves and roots was measured by atomic
absorption spectrofotometry, while N content determination was done by Kjeldahl
method. Samples of leaves, for gene expression analysis, were taken after every PRD
shifting period. Isolation of RNA was done by using RNAeasy Plant Mini Kit (Quiagen,
Germany) according to the manufacturer’s instructions. For synthesis of cDNA, the
First Strand cDNA Synthesis Kit (Fermentas, Lithuania) was used according to the
manufacturer’s instructions. Expression of these genes was monitored by using RT-PCR
(Real Time PCR SYBR-Green technique, RT-PCR 7500, Applied Biosystems, UK).
Results showed that PRD treatment, like RDI, reduced plant growth. In WT plants, both
treatments reduced plant organs biomass, while in flacca RDI reduced plant organs
biomass and PRD increased it. Analysis of plant vegetative organs growth showed that
flacca plants were smaller and with smaller fresh and dry biomass, compared to WT.
Analysis of plant water regime, done on the base of leaf water potential and soil water
content measurements, showed that RDI plants were exposed to higher degree of stress,
compare to PRD plants, in both genotypes. Results in daily dynamics of stomatal
conductance suggested that reactions to PRD treatment may be characterized as faster
response reactions, while to RDI as slower response reactions. Measurements of leaf
water potential and stomatal conductance showed that the effects of regulated water
deficit on stomatal closure were a consequence of plant water regime changes
(hydraulic effect). The effects of partial root-zone drying on stomatal closure were a
consequence of drought induced chemical effects. Drought also induced increase in leaf
ABA content. In WT plants, bigger effect on increase in leaf ABA content had PRD,
while in flacca it was RDI. The similar increase of leaf ABA content in both genotypes
under PRD conditions indicated that the flacca mutation did not affect ABA transport
from the root to the shoot. Deficit irrigation treatments did not showed significant effect
on ions content and distribution. The most significant effect was effect of PRD, which
increased Mn translocation from roots to leaves. This may be important for adaptive
metabolic reactions of plants to PRD, because Mn is a powerful natural antioxidant.
PRD also increased N content and translocation in flacca plants. Results of gene
expression confirmed differences in expression of investigated genes under applied
treatments, during the treatments` duration as well as between investigated WT and
flacca plants. The most significant differences between WT and flacca were found
under PRD treatment. The similar expression pattern of all genes in WT plants could
indicate synergistic signaling pathways for ABA and ethylene. In flacca reduced NCED
and significant EIL1 expression might reflect the increase in ethylene production, which
could influence ABA signaling and production occurred under PRD. Under RDI
treatment the pattern of all genes in WT and flacca leaves was similar and once again it
raises the possibility of synergistic effect of ABA and ethylene signaling pathways.
Since flacca mutant showed both expression of TAO1 gene and accumulation of ABA
in the leaves, our results indicated that other gene from TAO family and not TAO1 are
responsible for encoding enzyme abscisic aldehyde oxidase in flacca. In general, all
these results provide novel insights into the understanding of tomato response to
drought induced by PRD and RDI. Future investigations should analyze more genes that
underlie the ABA and ethylene biosynthetic and/or signaling pathways. | true |
Studija asocijacije genetičkih varijanti u regionima 7q36, 8q24 i 17q12 sa rizikom za razvoj i progresiju karcinoma prostate | Association study of common genetic variants in regions 7q36, 8q24 and 17q12 with prostate cancer | Karcinom prostate je drugi najčešće dijagnostikovan maligni tumor muškaraca u svetu, i
šesti po broju smrtnih slučajeva. Standardni dijagnostički i prognostički parametri KP
su serumska vrednost prostata specifičnog antigena (PSA), Glison gradus sistem (GGS),
i klinički stadijum KP. Studije asocijacije na čitavom genomu identifikovale su preko
30 SNP asociranih sa rizikom za razvoj i progresiju KP. Genetičke varijante
predstavljaju potencijalne genetičke markere koji bi se pored standardnih prognostičkih
parametara koristili u dijagnostici i praćenju bolesnika sa KP. Rezultati replikativnih
studija slučajeva i kontrola potvrdili su da pojedinačne genetičke varijante mogu imati
različit stepen asocijacije sa rizikom za razvoj i/ili progresiju KP u različitim
populacijama.
Ova studija imala je za cilj da proceni moguću asocijaciju genotipova i alela
genetičkih varijanti u regionu 7q36 (rs1799983, rs2070744, NOS3 -764A>G, NOS3 -
714G>T, rs3918226, NOS3 -649G>A), regionu 8q24 (rs1447295, rs4242382,
rs6983267, rs7017300, rs7837688) i regionu 17q12 (rs3760511, rs7501939) sa rizikom
za razvoj i progresiju KP.
Studija je obuhvatala 150 bolesnika sa KP, 150 bolesnika sa benignom
hiperplazijom prostate (BPH) i 100 muškaraca bez kliničkih znakova bolesti prostate
(kontrolna grupa). Genotipizacija genetičkih varijanti vršena je metodom RFLP i
metodom automatskog sekvenciranja.
Za svih pet SNP u regionu 8q24 (rs1447295, rs4242382, rs6983267, rs7017300 i
rs7837688), kao i za rs7501939 iz regiona 17q12 pokazana je asocijacija sa rizikom za
razvoj KP. U našoj studiji, od 13 analiziranih genetičkih varijanti, za samo dve,
rs3760511 (region 17q12) i rs1799983 (region 7q36), pokazana je asocijacija sa
standarnim prognostičkim parametrima KP. Rezultati naše studije pokazali su
asocijaciju genetičkih varijanti rs3760511 (region 17q12) i rs3198266 (region 7q36) sa
rizikom za progresiju KP.
Rezultati ove studije pokazali su naučnu opravdanost studije slučajeva i kontrola
u populaciji Srbije. Doprinos nastavka istraživanja asocijacije genetičkih varijanti sa
rizikom za razvoj i progresiju KP je u definisanju panela minimalnog broja SNV koji bi
se koristili kao nestandardni prognostički parametri u dijagnostici i kliničkom praćenju
bolesnika sa KP. | Prostate cancer (PCa) is the second most frequently diagnosed malignant tumor, and the
sixth leading cause of cancer-related deaths among men worldwide. Standard diagnostic
and prognostic parameters are serum PSA level, Gleason gradus score, and the clinical
stage of PCa. Genome-wide association studies (GWAS) have identified over 30 single
nucleotide polymorphisms (SNPs) associated with human PCa. Genetic variants have
emerged as potential genetic markers that could be used along with standard prognostic
parameters in PCa diagnostics and outcome prediction.
This study aimed to evaluate possible association between genotypes and alleles
of genetic variants at 7q36 (rs1799983, rs2070744, NOS3 -764A>G, NOS3 -714G>T,
rs3918226, and NOS3 -649G>A), 8q24 (rs1447295, rs4242382, rs6983267, rs7017300,
rs7837688) and 17q12 (rs3760511, rs7501939) with PCa risk and progression.
150 patients with PCa, 150 patients with benign prostatic hyperplasia (BPH),
and 100 men without clinical signs of prostate diseases (control group) were recruited as
study participants. The genotyping was performed by using PCR-RFLP analysis and bi-
directional DNA sequencing.
Our study has shown association for all five analized variants in the region 8q24
(rs1447295, rs4242382, rs6983267, rs7017300 and rs7837688) and rs7501939 in the
17q12 region with the risk of PCa. Among 13 variants analyzed in our study, two
(rs3760511 at 17q12 and rs1799983 at 7q36) have shown evidence of association with
standard prognostic parameters of PCa. Our study yielded evidence of association of
rs3760511 (17q12) and rs3198266 (7q36) with the risk of PCa progression.
The results of our study have shown the scientific racionale for conducting case-
control studies in Serbian population. The primal contribution of continuing reasearch
regarding association between genetic variants and PCa risk and progression is in
defining the minimal panel of single nuclotide variants which would be used as non-
standard prognostic parameters in PCa diagnostics and outcome prediction. | true |
Promena boje cveta perunike (Iris germanica L.) genetičkom modifikacijom biosinteze karotenoida | Alteration of flower color of Iris germanica L. through genetic modification of carotenoid biosynthetic pathway | Boja cveta perunike (Iris germanica L.) je određena na osnovu dva odvojena
biohemijska puta sinteze biljnih pigmenata. Na osnovu biosinteze karotenoida dobijaju
se cvetovi žute, narandžaste i ružičaste boje, dok biosinteza antocijanina dovodi do
plave, ljubičaste i bordo boje cvetova. Crvene perunike ne postoje u prirodi, i do danas
nisu dobijeni kultivari sa ovom bojom primenom konvencionalnih načina
oplemenjivanja. U ovoj disertaciji ispitana je mogućnost primene metoda genetičkog
inženjeringa sa ciljem dobijanja perunika sa crvenim cvetovima. Za ostvarenje ovog
osnovnog cilja razvijeni su protokoli za regeneraciju perunike (I. germanica L. ‘Skating
Party’) iz ćelijskih suspenzija, protokoli za efikasnu genetičku transformaciju kao i
genetičku transformaciju sa ciljem modifikacije biosintetskog puta karotenoida kod
perunika.
Za genetičku transformaciju perunika su korišćene dve nezavisne metode, jedna
zasnovana na biolističkoj transformaciji (bombardovanje mikroprojektilima) pomoću
PDS-1000/He (Particle Delivery System) i druga zasnovana na Agrobacterium-
posredovanoj transformaciji. Ovo su prve poznate metode za genetičku transformaciju
vrsta roda Iris, koji će omogućiti dalje oplemenjivanje ovih ukrasnih vrsta primenom
savremenih molekularnih metoda. Protokol zasnovan na Agrobacterium-posredovanoj
transformaciji se pokazao kao bolji od biolističke transformacije u smislu efikasnosti i
jednostavnosti primene. Ispitana je osetljivost ćelija perunika na dejstvo nekoliko
antibiotika i pokazano je da su higromicin i gentamicin (G418) najpogodniji za selekciju
transformisanih ćelija. Soj A. tumefaciens LBA 4404 koji nosi superbinarni vektor
pTOK233 je bio najefikasniji za transformaciju perunika. Više od 300 morfološki
normalnih transgenih perunika je dobijeno posle 6 meseci. Oko 80% regeneranata je
pokazalo pozitivnu reakciju za marker gen ß-glukuronidazu (GUS) i gen za neomicin
fosfotransferazu II (NPTII) (paromomicin-rezistencija) koji se nalaze na plazmidu za
transformaciju. Integracija transgena u genom jedra transformisanih perunika je
potvrđena Southern blot-analizom.
Sa ciljem razvoja metoda za modifikaciju karotenoidnog biosintetskog puta kod
perunika, prvo je transformisan kultivar ‘Fire Bride,’ koji ima ružičaste cvetove sa
bakterijskim genom za fitoen sintazu (crtB) iz Pantoea agglomerans pod kontrolom
promotorskog regiona gena za kapsantin-kapsorubin sintazu iz Lilium lancifolium
(PLlccs). Ovaj pristup imao je za cilj da poveća protok prekursora karotenoida i dovede
do povećanja koncentracije likopena i na taj način izazove pojavu tamnijih nijansi
ružičaste ili crvenih nijansi cvetova perunika. U kalusnim kulturama ovog kultivara
došlo je do povećane ekspresije crtB gena i promene boje kalusa od žute u ružičasto-
narandžastu i nijanse crvene usled povećane akumulacije likopena. Cvetovi transgenih
biljka dobijenih iz transgenih kalusa ovog kultivara su pokazali značajniju promenu boje
plodnika i cvetnih drški (od zelene ka narandžastoj) kao i antera (od bele ka ružičastoj).
Nisu uočene značajnije promene u boji perigona cvetova (standardima i folovima) u
odnosu na kontrolne biljke. Rezultati ukazuju da su ovi geni u zelenim delovima
cvetova perunike pod drugačijom regulacijom od delova cveta koji nisu zeleni. Kao
generalni zaključak dobijamo da se ektopijska ekspresija gena crtB može uspešno
koristiti za delimično menjanje boje delova cvetova kultivara perunike ‘Fire Bride’ ali
nije najpogodaniji metod za značajnije menjanje boje u standardima i folovima.
Kao drugi pristup genetičkom inženjeringu promene boje cvetova sa ciljem
modifikacije sadržaja karotenoida u cvetovima ispitana je pogodnost gena za kapsantin-
kapsorubin sintazu (Llccs) iz perijanta tigrastog ljiljana (Lilium lancifolium
‘Splendens’). Narandžasta boja cvetova tigrastog ljiljana nastaje, pre svega usled
akumulacije dva κ-ksantofila, kapsantina i kapsorubina. Kapsantin-kapsorubin sintaza
(CCS) je ključni enzim koji katalizuje konverziju anteraksantina i violaksantina u
kapsantin i kapsorubin. Klonirana je sekvenca Llccs gena pomoću brzog umnožavanja
cDNK krajeva (RACE) i heterolognog ne-degenerativnog prajmera dizajniranog na
osnovu sekvence gena za likopen β-ciklazu (LllcyB) koji ima veliku homologiju sa
Llccs. Dobijena je cDNK od Llccs gena dužine koja je iznosila 1785 bp sa otvorenim
okvirom čitanja od 1425 bp koji kodira peptid od 474 aminokiseline sa predviđenom
NH2-terminalnom sekvencom tranzitnog peptida (TP). Primenom metode reverzne
transkripcije (RT-PCR) pokazana je prostorno i vremenski regulisana ekspresija Llccs
gena koja je prisutna u cvetovima, ali ne i u vegetativnim delovima L. lancifolium.
Stabilna, pojačana ekspresija Llccs gena u kalusnim kulturama I. germanica, koji inače
prirodno akumuliraju nekoliko ksantofila uključujući violaksantin, prekursor
kapsorubina koji daju kalusima žutu boju, dovela je do promene boje kalusa ka
narandžasto-crvenoj. Dobijena izmenjena boja kalusa je nastala usled ektopijske
akumulacije κ-ksantofila, kapsantina i kapsorubina, što je potvrđeno analizom pomoću
HPLC i UPLC-MS/MS sa odgovarajućim standardima.
Da bi testirali sposobnost novo-kloniranog Llccs gena da promeni boju cvetova
transformisan je paradajz (Solanum licopersicum L.) kao model sistem. Cvetovi
paradajza prirodno akumuliraju anteraksantin i violaksantin, prekursore kapsantina i
kapsorubina, ali njih same ne akumuliraju. Boja cvetova transgenog paradajza je
promenjena od prirodno žute u različite nijanse narandžaste. Dobijeni rezultati
nedvosmisleno pokazuju da ektopijska ekspresija Llccs gena može efikasno da
preusmeri biosintezu karotenoida ka kapsantinu i kapsantin sličnom karotenoidu u
cvetovima. Ovo predstavlja prvi uspešan pokušaj promene boje cvetova kod neke biljne
vrste preko metaboličkog inženjeringa biosinteze kapsantina i kapsantin-sličnog
karotenoida.
Naučna oblast:
Biologija
Uža naučna oblast: Fiziologija biljaka
UDK broj:
577.21: 582.579.2(043.3)
581.1: 602.6(043.3) | Flower color in irises (Iris germanica L.) is determined by two distinct
biochemical pathways. The carotenoid biosynthetic pathway generates yellow, orange
and pink flowers, while the anthocyanin biosynthetic pathway produces blue, violet and
purple flowers. Red iris flowers do not exist naturally, and conventional breeding
methods have thus far failed to produce them.
This study examines genetic engineering approaches as a potential avenue for
the development of a red iris that has not yet been explored. In the course of the work,
protocols were developed for genetic transformation of I. germanica, and genetic
modifications to the carotenoid biosynthetic pathway in irises.
Two independent methods for the genetic transformation of irises were
developed; one based on biolistic (microprojectile bombardment) using PDS-1000/He
(Particle
Delivery
System),
and
the
other
using
Agrobacterium-mediated
transformation. These are the first known protocols for the genetic transformation of I.
germanica, which will allow this ornamental crop to benefit from modern molecular
applications.
The
Agrobacterium-mediated
protocol
demonstrated
significant
advantages in both efficiency and ease of use over the biolistic transformation protocol.
In the course of development of the Agrobacterium-mediated protocol, a series
of selection agents were tested. Hygromycin and geneticin (G418) were identified as the
most suitable antibiotics for selecting transformed iris cells. A. tumefaciens strain
LBA4404, carrying the superbinary vector pTOK233, was identified as the most
efficient strain/vector combination for transformation of iris, of those tested.
More than 300 morphologically normal transgenic iris plants were obtained in
about 6 months. About 80% of the regenerants tested positive for the β-glucuronidase
(GUS) marker gene and the neomycin phosphotransferase II (NPTII) (paromomycin-
resistance) gene carried by the transformation plasmid. The integration of the
transgenes into the nuclear genome of the transformed iris plants was confirmed by
Southern blot analysis.
With the goal of developing methods for modifying the carotenoid biosynthetic
pathway in irises using genetic engineering a pink iris cultivar, ‘Fire Bride,’ was
transformed with the bacterial phytoene synthase gene (crtB) from Pantoea
agglomerans under control of the promoter region of a gene for capsanthin-capsorubin
synthase from Lilium lancifolium (PLlccs). This approach aimed to increase the flux of
carotenoid precursors into the carotenoid biosynthetic pathway, and ultimately lead to
elevated levels of lycopene, thus generating shades of darker pink or red in the flowers.
Iris callus tissue overexpressing the crtB gene showed a color change from yellow to
pink-orange and red, which was shown to be due to an increased accumulation of
lycopene. Transgenic plants regenerated from the calli showed prominent color changes
in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to
pink). However, the color of the standards and falls showed no significant differences
when compared to the control plants. This suggests that carotenogenesis in non-green
parts of iris flowers is regulated differently compared to the green tissues. Altogether,
the results demonstrated that ectopic expression of a bacterial phytoene synthase gene
can be used to successfully alter the color of some flower parts in I. germanica ‘Fire
Bride’ and produce new flower traits. However, it is not a suitable method to produce
significant color change in the standards and falls.
A second approach for modifying iris flower color tested the feasibility of using
a gene for capsanthin-capsorubin synthase (Llccs) from the flower tepals of tiger lily
(Lilium lancifolium ‘Splendens’). The orange color of L. lancifolium ‘Splendens’
flowers is primarily due to the accumulation of two κ-xanthophylls, capsanthin and
capsorubin. The capsanthin-capsorubin synthase (CCS) catalyzes the conversion of
antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. The Llccs
gene sequence was cloned using rapid amplification of cDNA ends (RACE) and a
heterologous non-degenerate primer based on the sequence of a gene for lycopene β-
cyclase (LllcyB) that has high homology with Llccs. The full-length cDNA of Llccs was
1785 bp long and contained an open reading frame of 1425 bp that encoded a
polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence.
Analysis by reverse transcription-PCR (RT-PCR) revealed that Llccs expression is
spatially and temporally regulated; it was observed in the flower buds and flowers of L.
lancifolium, but not in the vegetative tissues.
Stable overexpression of the Llccs gene in yellow callus tissue of I. germanica,
which normally accumulates several xanthophylls including violaxanthin, resulted in
transgenic calli whose color changed to red-orange. This color change was due to the
ectopic accumulation of the κ-xanthophylls capsanthin and capsorubin, as confirmed by
HPLC and UPLC-MS/MS analyses with authentic standards.
To test the ability of the newly cloned Llccs gene to change flower color, the
Llccs-transgenic plants of tomato (Solanum lycopersicum L.) were generated. Tomato
flowers accumulate both, antheraxanthin and violaxanthin, the precursors of capsanthin
and capsorubin, respectively, but do not naturally accumulate capsanthin or capsorubin.
The Llccs-transgenic tomato flowers changed from their natural yellow color to
different shades of orange. The results demonstrate that the ectopic expression of the
Llccs gene can effectively reroute the biosynthesis of carotenoids towards capsanthin
and capsanthin-like carotenoid in flower tissues. This represents the first successful
alteration of flower color of any species through metabolic engineering of capsanthin
and capsanthin-like carotenoid biosynthesis.
Scientific field:
Biology
Specific scientific field:
Plant Physiology
UDC number:
577.21: 582.579.2(043.3)
581.1: 602.6(043.3) | true |
Ekološka istraživanja juvenilnog inćuna, Engraulis encrasicolus (Linnaeus, 1758) u Kotorskom zalivu | Ecological study of the juvenile anchovy, Engraulis encrasicolus (Linnaeus, 1758) in the Bay of Kotor | U ovoj disertaciji prikazani su rezultati dvogodišnjeg istraživanja ekoloških
karakteristika juvenilnog inćuna, Engraulis encrasicolus (L.), na području Kotorskog
zaliva. S obzirom da se ribolov ove vrste na području Kotorskog zaliva obavlja
tradicionalnom metodom, korišćenjem mreža potegača sa vrlo malim okom na mreži
(6 mm), koja ima viševjekovnu tradiciju, jedan od glavnih ciljeva bio je da se odredi
da li se može i u kojoj mjeri dozvoliti ovaj tip ribolova a da se ne ugrozi populacija
inćuna na području Kotorskog zaliva.
Uzorkovanje je vršeno iz komercijalnih ulova mrežama potegačama u periodu
jun 2004. – jul 2005. godine i jul 2006. – jun 2007. godine. Analizirano je ukupno
4203 jedinki inćuna. U analiziranim ulovima inćun je u obije istraživane godine bio
zastupljen sa 37%, dok je ulov po jedinici napora (CPUE) iznosio 150 kg po potezu.
Struktura istraživane populacije inćuna u odnosu na dužinske klase kretala se u
rasponu od od 5,2 do 13,2 cm, sa srednjom dužinom od 8,6 cm. Faktor alometrije
dužinsko-težinskog odnosa za cjelokupan analiziran uzorak iznosi b = 3,122, dok se
vrijednost faktora kondicije kretala u rasponu od 0,981 do 1,017, sa srednjom
vrijednošću od 1,003 ± 0,009.
Po prvi put su na ovom području, i uopšte na južnom Jadranu, analizirani
otoliti juvenilnog inćuna i određena je starost u danima. Na osnovu očitavanja otolita
izračunati su parametri rasta (L∞ = 10,41, K = 3,36 i t0 = -0,074 ), i određena je dužina
pri kojoj dolazi do metamorfoze iz larvalnog u juvenilni stadijum (32,41 mm), a
takođe je određena i dnevna stopa rasta juvenilnog inćuna (0,41 mm/danu). Izračunati
su i brzina rasta otolita i njihova asimptotska dužina (L∞ = 1918 µm), dok je
maksimum rasta otolita 62 dana nakon izlijeganja jedinke.
Procenjeni parametri rasta za juvenilni dio populacije modalnom progresijom
iznose L∞ = 9,21, K = 4,51 i t0 = -0,04 za period 2004-2005. godine i L∞ = 10,94, K =
2,25 i t0 = -0,175 za period 2006-2007. godine. Istom metodom procijenjeni su i
parametri rasta za cijelu populaciju inćuna, i to za period istraživanja 2004-2005.
V
godine (L∞ = 15,41, K = 0,59 i t0 = -0,761 ), odnosno 2006-2007. godine (L∞ = 15,36,
K = 0,98 i t0 = -0,399 ). Izračunat je indeks uspješnosti rasta (φ’), i srednja vrijednost
ovog indeksa na osnovu svih izračunatih parametara rasta iznosi 2,416.
Izračunate trenutne stope smrtnosti za populaciju inćuna u obije godine bile su
slične (u prvoj istraživanoj godini Z = 6,65, u drugoj Z = 5,99), i njihove vrijednosti
pokazuju visok stepen ukupne smrtnosti populacije inćuna na području Kotorskog
zaliva. Izračunata je i stopa prirodnog mortaliteta (M = 5,95), kao i ribolovni
mortalitet (F = 0,7).
Povećanje veličine oka sa 6 mm u 2004-2005. godini na 8 mm u 2006 - 2007.
godini dovelo je i do povećanja L50% sa 7,359 cm na 8,47cm, kao i t50% sa 0,285 na
0,421 godina, čime je omogućeno većem broju jedinki da dostignu polnu zrelost.
Analizom reproduktivnih karakteristika inćuna putem ispitivanja GSI i
histologije gonada, utvrđeno je da reproduktivni ciklus ove vrste na području
Kotorskog zaliva traje od aprila do oktobra mjeseca. Ženke su brojnije u odnosu na
mužjake i odnos polova je 0,92. Mikroskoskom analizom jajnika inćuna utvrđena je
najmanja dužina dostizanja polne zrelosti za ženke koja iznosi 7,5 cm i to je za sada
najmanja mikroskopski potvrđena dužina na Jadranu.
Procijenjena je i biomasa inćuna (B = 433.2 t), koeficijent ranjivosti q =
0,006371, kao i stopa eksploatacije E = 0,106. Na osnovu ovih podataka izračunat je
optimalni ribolovni napor (fopt) za ovo područje i on iznosi 78 potezanja mreže u toku
jednog mjeseca, što znači da bi broj izdatih dozvola za ovaj tip ribolova trebalo da
bude 8 do 9 dozvola godišnje. | This thesis presents the results of a two-year research of ecological parameters
of juvenile European anchovy, Engraulis encrasicolus (L.), in the Bay of Kotor.
Considering that the anchovy fisheries in this region have been for centuries
performed using beach seines with very small mesh size (6 mm), one of the main
goals of the thesis was to determine whether this type of fisheries can be allowed to
continue, and to what extent, in order to preserve the anchovy population in the Bay
of Kotor.
Samples were collected from commercial beach seine catches in periods from
June 2004 to July 2005, and from July 2006 to June 2007. A total of 4203 specimens
were studied. Anchovy made up 37% of the total catch in both sampling periods,
while the catch per unit effort (CPUE) was estimated to be 150 kg per haul. The
length frequency of the studied sample ranged from 5.2 to 13.2 cm total length (TL),
with an average length of 8.6 cm TL. The power coefficient of length–weight
relationship was estimated to be b = 3.122, while the relative condition factor ranged
from 0.981 to 1.017, with an average value of 1.003±0.009.
This is the first time that the otoliths of juvenile anchovy were analysed, not
only in the area, but in the southern Adriatic Sea in general. Age in days was
determined, and, based on the results obtained from otolith readings, growth
parameters (L∞ = 10.41 cm, K= 3.36, t0 = −0.074) were estimated, as well as the
length at which larvae metamorphose to the juvenile stage (32.41 mm), and the daily
growth rate of the juvenile anchovy (0.41 mm/day). Otolith growth rate and their
asymptotic length (L∞ = 1918 μm) were also estimated, and the maximum otolith
growth rate was 62 days after hatching.
Estimated growth parameters for juveniles using modal progression were L∞ =
9.21 cm, K= 4.51, t0 = −0.04 for the period 2004-2005, and L∞ = 10.94 cm, K= 2.25,
t0 = −0.175 for the period 2006-2007. The same method was used to estimate growth
parameters for the entire anchovy population for 2004-2005 (L∞ = 15.41 cm, K= 0.59,
t0 = −0.761) and 2006-2007 (L∞ = 15.36 cm, K= 0.98, t0 = −0.399). Growth
VII
performance index (φ’) was also estimated, and its mean value based on all growth
parameters was φ’ = 2.416.
Instantaneous mortality rate were similar in both study periods (Z = 6.65 in
2004-2005, and Z = 5.99 in 2006-2007), and they indicate a high level of the total
mortality within the Bay of Kotor. Natural mortality was estimated to be M = 5.95,
and fishing mortality was F = 0.7.
The increase in mesh size from 6 mm in 2004-2005 to 8 mm in 2006-2007
lead to an increase of L50% from 7.359 cm to 8.47 cm, and of t50% from 0.285 to
0.421 years, which allowed a greater number of individuals to reach sexual maturity.
The analysis of reproductive characteristics using the gonadosomatic index
(GSI) and gonad histology showed that the reproductive cycle of this species in the
Bay of Kotor lasts from April to October. Females were more numerous compared to
males, and the sex ratio was estimated to be 0.92. Microscopic analysis of female
ovaries was used to estimate the minimum length at first maturity, which occurs at 7.5
cm. This is the smallest microscopically confirmed length at first maturity for the
anchovy in the Adriatic Sea.
Biomass of anchovy was estimated at B = 433.2 t, the coefficient of
vulnerability at q = 0.006371), and the exploitation rate at E = 0.106. Based on this
data, the optimum fishing rate (fopt) for the area was estimated at 78 hauls per month.
This implies that a number of licences for this type of fisheries should be limited to 8
or 9 licences pr year. | true |
Procena angiogenih i anti-inflamatornih efekata ekstrakorporealne terapije zvučnim udarnim talasima na animalnom modelu ishemije kožnih režnjeva | Evaluation of angiogenic and anti-inflammatory effects of extracorporeal shockwave therapy in animal model of ischemic skin flaps | Skoro 30 godina terapija zvučnim udarnim talasima (engl. Extracorporeal Shock
Wave Therapy, ESWT) je u upotrebi u kliničkoj praksi kao efikasan tretman za razbijanje
kamena u urinarnom traktu. Ova tehnologija je kasnije razvijena i kao efikasan
neinvazivan tretman za različite indikacije u ortopediji i traumatologiji uključujući i
komplikovane rane koje ne zarastaju. Udarni zvučni talasi su tranzijentni, kratkotrajni
akustički pulsevi sa visokim pikom pritiska, koji se dostiže u veoma kratkom vremenu,
mereno nanosekunadama (jedan bilioniti deo sekunde). Udarni zvučni talasi koji se
koriste u medicini se generišu putem tri tipa generatora i fizičkih rešenja:
elektrohidraulički, piezoelektrični i elektromagnetni čiji je krajnji cilj da prenese fizičku
energiju do ciljnog tkiva.
Odloženo zarastanje, ili rane koje ne zarastaju predstavljaju veliki teret za svakog
pacijenta, značajno umanjujući njegov kvalitet života. Velika briga o pacijentu i rani je
neophodna u ovim slučajevima i ovo je veliki problem za društvo u smislu smanjene
produktivnosti ljudi kao i smislu velikih finansijskih izdataka za zdravstvo. Sa druge
strane, teške povrede kao one koje se viđaju u oružanim sukobima i saobraćajnim
nesrećama predstavljaju glavni uzrok smrti u mlađoj adultnoj populaciji. Tehnološki
napredak, i napredak u dostupnim terapeuticima, koji dovodi do inicijalnog povećanja
preživljavanja pacijenata nije praćen razvojem lekova koji su usmereni protiv moguće
prateće sistemske inflamacije, koja često progresira do sindroma sistemskog
inflamatornog odgovora i sindroma propadanja organskih sistema koji mogu dovesti i do
smrtnog ishoda. Zbog svega prethodno pomenutog, finansijski pristupačni, neinvazivni i
efikasni tretmani su neophodni da bi se postiglo ubrzavanje i potpuno zarastanje
problematičnih rana kao i redukovala cena lečenja. Zbog toga je neophodno utvrditi
potencijalnu primenljivost ESWT u relevantnim animalnim modelima i kod pacijenata, i
ispitati i razjasniti biološke mehanizme ove terapije.
Nekoliko eksperimentalnih i kliničkih studija je pokazalo efikasnost ESWT za
ubrzavanje opopravka ostećenog tkiva i regeneraciju različitih rana. Međutim, biološki
2
mehanizmi kojima se ovi procesi ubrzavaju su ostali neobjašnjeni. U ovom radu, su
ispitani efekti ESWT na molekularnom, ćelijskom i sistemskom nivou kako bi proširili i
produbili znanja o mehanizmu delovanja ove terapije što je glavni cilj ove disertacije.
Jedan od ciljeva je bio i da se utvrdi značaj vremena aplikacije tretmana i da se ispita
efekat ESWT na neoangiogenezu i sprečavanje procesa nekroze tkiva kao posledice
ishemije. Zbog opšteprihvaćenog mišljenja o opasnosti primene ESWT na abdomen i
izbegavanja njene primene za različite indikacije u oblasti abdomena, pokušali smo da
utvrdimo da li je primena ESWT bezbedna i upotrebljiva u ovoj regiji kako bi se proširila
lista indikacija primene ove terapije. Procenjivan je imunomodulatorni efekat ESWT u
modelu inflamacije i gastrointestinalnog dismotiliteta izazvanog teškom traumom (engl.
Tissue Bone Matrix model, TBX) kao i potencijalnu kliničku vrednost ESWT u supresiji
inflamacije i postoperativnog ileusa izazvanog hirurškom manipulacijom crevima.
U ovom radu pokazani su pozitivni efekti ESWT u sva tri animalna modela,
modelu ishemičnih režnjeva kože, TBX modelu i modelu postoperativnog ileusa.
Rezultati su potvrdili povećanje perfuzije, supresiju formiranja edema, kao i angiogeni
potencijal ESWT u modelu ishemije kožnih režnjeva. Dodatno je pokazano da ovi efekti
nisu ograničeni samo na tkivo pod direktnim uticajem udarnog zvučnog talasa već da se
efekti mogu uočiti i na udaljenim tkivima i ćelijama kao i na sistemskom nivou. Mi smo
uspešno razvili i opisali TBX model i koristeći ga pokazali da ESWT utiče na inflamaciju
i nakon velike traume. Pretretman sa ESWT ima značajne protektivne efekte protiv
veoma nepovoljnih efekata teške traume modulacijom inflamacije i u oštećenom tkivu
kao i na sistemskom nivou. Zbog nepredvidljivosti vremena traume, mi smo se okrenuli
modelu koji je klinički mnogo praktičniji zbog precizno definisanog vremena hirurške
intervencije, modelu postoperativnog ileusa. U ovom modelu, pokazani su protektivni
efekti primene ESWT kao pretretmana, kao i pozitivni efekti na očuvanje
gastrointestinalnog dismotiliteta izazvanog hirurškim manipulisanjem gastrointestinalnim
traktom i supresiju inflamatornog odgovora i to na molekularnom (promena u ekspresiji
nekoliko gena i proteina), ćelijskom (smanjena infiltracija aktivirnih leukocita) i
sistemskom nivou (kroz modulisanje serumskim nivoima pro i antiinflamatornih citokina
i hemokina).
3
ESWT je obećavajuća, relativno nova i neinvazivna terapija čija klinička upotreba
konstanto raste poslednjih nekoliko decenija. Ona pokreće sopstvene mehanizme za
borbu protiv povreda i sprečava negativne efekte posledične inflamacije putem
modulacije imunskog odgovora, pospešivanja procesa neoangiogeneze kao i brzine
sazrevanja novoformiranih krvnih sudova. Dalja izučavanja su neophodna da bi se u
potpunosti razumeo mehanizam delovanja i njena primenljivost sa posebnim osvrtom na
primenljivost na gastrointestinalni trakt. | For almost 30 years, extracorporeal shock wave therapy (ESWT) has been
implemented clinically and has proven to be an effective treatment for urinary stones.
This technology has also emerged as an efficacious non-invasive treatment modality for
several orthopedic and traumatic indications including problematic soft tissue wounds.
Shock waves are produced as transient short-term acoustic pulses with high peak pressure
and a very rapid rise to peak pressure on the order of magnitude of nanoseconds (one
billionth of a second). Shock waves for use in medicine can be generated using different
physical principles - electro-hydraulic, piezoelectric and electromagnetic – with the goal
of delivering physical energy to target tissue of interest in order to create a desired
biological effect.
Delayed/non-healing or chronic wounds constitute a burden for each patient
affected, significantly impairing quality of life (QOL). Intensive multi-modality wound
care is required, and this places an enormous burden on society in terms of lost
productivity and healthcare costs. Severe traumatic wounds seen in civilian and combat
related injuries are a major cause of death amongst the younger population.
Technological and therapeutic advances that lead to improved initial survival of patients
with life-threatening injuries were unparalleled in therapeutics development to fight
concomitant systemic inflammation that accompanies these injuries. This systemic
inflammatory response to wounding often leads to development of a well-known
syndrome - systemic inflammatory response syndrome (SIRS) - and can progress to
multiple organ dysfunction syndrome (MODS) and death. Because of all of the
aforementioned, cost-effective, non-invasive, and efficacious treatments are imperative to
achieve both (accelerated and complete) healing of problematic wounds and reduce
treatment-related costs to the human and the healthcare system. Therefore, the potential
of therapeutic shock wave application in relevant animal models and patients has to be
determined in addition to the elucidation of the biological mechanisms involved.
5
Several experimental and clinical studies have demonstrated efficacy of
extracorporeal shock wave therapy in terms of accelerating tissue repair and regenerating
various types of wounds. However, the biological mechanism(s) by which this treatment
modality exerts its therapeutic effects remains unclear. We herein present the evaluation
of molecular, cellular and systemic effects of ESWT in order to broaden our
understanding of these mechanisms, the main goal of this thesis. We also aimed to
determine the importance of the timing of application of shock waves, and to evaluate the
neo-angiogenic potential of ESWT and its efficacy in preventing tissue necrosis after
ischemic injury. Due to the mainstream reluctance to apply ESWT to the abdomen, for
fear of barotrauma, we aimed to investigate the safety and feasibility of ESWT
application to the gastrointestinal tract and to extend the list of potential indications for
this promising therapeutic modality. Specifically, we investigated the immunomodulatory
effects of ESWT in a model of severe peripheral tissue trauma (TBX model) and the
potential clinical value of ESWT in suppressing inflammation and limiting post-operative
ileus caused by surgical manipulation of the abdominal contents.
We have demonstrated the positive biological effects of ESWT in models of
ischemic skin flaps, peripheral tissue trauma-induced inflammation and post-operative
ileus. Our results confirm shock wave-associated increases in ischemic tissue perfusion,
suppression of soft tissue edema formation, and the angiogenic potential of ESWT. In
addition, we have shown that these effects are not limited to the tissue directly treated
with shockwaves, but remote tissue and systemic effects were also documented. We have
successfully developed a novel TBX model and used it to test if shock waves can
modulate the inflammatory response. Shock wave pre-conditioning has significant
protective effects against the debilitating effects of severe trauma, through modulation of
inflammation in damaged tissue as well as through reduction of systemic inflammatory
response. Due to the unpredictable nature of severe wounding we turned our attention to
a well-described model of post-operative ileus due to the ability to precisely control the
nature and the timing of injury. We have clearly shown the protective and positive effects
of ESWT pre-conditioning in terms of the preservation of gastrointestinal motility despite
surgical manipulation and suppression of the inflammatory response to surgery on a
molecular, cellular and systemic level.
6
ESWT is a promising non-invasive therapeutic modality whose clinical
application has steadily increased over the years. Shock waves trigger the body’s own
mechanisms to promote tissue repair and regeneration whilst preventing the negative
effects of both local and systemic inflammation through its immunomodulatory and
neoangiovenic capacity and its capacity to affect maturation of newly formed blood
vessels. More mechanistic and clinical trials are needed to fully understand its
mechanisms and its applicability to gastrointestinal disorders. | true |
Ekološka i toksikološka istraživanja crnomorske haringe (Alosa immaculata Bennett, 1835) u Dunavu u Srbiji | Ecological and toxicological study of the Pontic shad (Alosa immaculata Bennett, 1835) in the Danube River in Serbia | Crnomorska haringa (Alosa immaculata, Bennett 1835) je anadromna vrsta koja
veći deo života provodi u jako zagađenom severoistočnom delu Crnog mora, a radi
mresta migrira u Dunav. Populacije crnomorske haringe imaju opadajući trend
brojnosti, tako da se prema IUCN Crvenoj listi ugroženih vrsta tretira kao ranjiva vrsta
(VU). Njen migratorni put radi mresta skraćen je izgardnjom brane Đerdap II do 863
rkm Dunava. Iako je komercijalno značajna, istraživanja na crnomorskoj haringi
sprovode se povremeno.
Za izradu ovog rada, migranti crnomorske haringe prikupljeni su tokom aprila i
maja 2004., 2006., 2007. i 2008. godine na 863 rkm Dunava, neposredno ispod brane
Đerdap II.
Struktura populacije u odnosu na dužinske klase i dužinsko-težinski odnosi
analizirani su na osnovu merenja totalne dužine (TL) i ukupne težine tela (W) jedinki
koje su sakupljene u 2004. i 2006. godini. Opseg variranja totalnih dužina tela
ulovljenih jedinki iznosio je od 24,2 do 38,7 cm, sa dominacijom klase od 32-32,5 cm.
Dobijeni faktori alometrije dužinsko-težinskog odnosa manji su od tri (b<3) za oba pola
i za obe sezone uzoraka.
Ova studija je imala za cilj da obezbedi i prve podatke o geometrijsko-
morfometrijskim karakterima crnomorske haringe, vrednostima gonadosomatskog
(GSI) i hepatosomatskog indeksa (HSI) kao i Fultonovog faktora kondicije (K) u
Dunavu u Srbiji. Zabeležena je statistički značajna razlika između GSI, HSI i K između
oba pola i u obe sezone uzorkovanja. Razlike nisu pronađene samo za vrednosti K
između mužjaka i ženki u 2004. godini i za vrednosti GSI i K između ženki
uzorkovanih u 2004. i 2006. godini. Vrednosti Fultonovog faktora kondicije u ovom
istraživanju bile su najniže u poređenju sa vrednostima koje su do sada zabeležene za
crnomorsku haringu.
Ekološka i toksikološka istraživanja crnomorske haringe u Dunavu u Srbiji
V
Uzrasna struktura populacije određena je na osnovu očitavanja 28 krljušti i 30
kičmenih pršljenova nasumično izabranih jedinki. Starost analiziranih primeraka kretala
se od 2+ do 6+, sa dominacijom jedinki starosti 3+ i 4+. Očitavanje starosti sprovela su
dva interpretatora sa iskustvom u određivanju starosti kod različitih vrsta riba i tri
interpretatora sa iskustvom za određivanje starosti korišćenjem krljušti i pršljenova kod
crnomorske haringe. Interpretatori sa iskustvom čitanja starosti kod haringe imali su
manje vrednosti indeksa prosečne procentualne greške (IAPE) i indeksa koeficijenta
varijacije (ICV). Nisu nađene statistički značajne razlike za ponovljivost očitavanja kod
svakog pojedinačnog interpretatora u određivanju starosti na osnovu krljušti i
pršljenova. Prema tome, iskustvo interpretatora ima veći uticaj na preciznost nego što to
ima izbor koštane strukture pomoću koje bi se determinisala starost. Determinacija
starosti kod riba zahteva usvajanje standardnih protokola koji moraju uključiti i
interakcije između interpretatora.
Geometrijsko-morfometrijska analiza pokazala je da postoje statistički značajne
razlike u obliku tela jedinki na oba nivoa: sezone tj. godine uzorkovanja (nested
ANOVA: F=13,84; P<0,001) i pola (nested ANOVA: pol u okviru jedne sezone tj.
godine uzorkovanja, F=1,71; P<0,01). Najznačajniji parametri za razdvajanje uzoraka iz
2004. i 2006. godine bile su razlike u obliku glave i u poziciji peraja. Ženke su u odnosu
na mužjake imale veći intenzitet dorzo-ventralne izduženosti srednjeg dela tela, i
istureniji gornji dorzalni deo tela. UPGMA fenogram izveden na osnovu matrice
Prokrustovih distanci za oblik tela pokazuje da je grupa koja obuhvata mužjake iz 2004.
godine razdvojena u odnosu na ostale tri grupe. Polni dimorfizam crnomorske haringe,
utvrđen u ovom istraživanju, u skladu je sa literaturnim nalazima kod drugih vrsta iz
istog roda.
Određivanja nivoa kontaminacije teškim metalima i mikroelemenata sprovedeno
je na tkivima crnomorske haringe uzorkovanim tokom aprila i maja 2007. i 2008. Mišić,
jetra i škrga su pripremljeni korišćenjem mikrotalasne digestije, dok je analiza Al, As,
B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pb, Sr i Zn urađena pomoću
indukovano spregnute plazme – optičko emisione spektrometrije (ICP-OES). Statistički
značajne razlike u koncentracijama teških metala i mikroelemenata utvrđene su među
različitim tkivima, kao i među polovima. Koncentracije Al, Sr, Ba, Mg i Li su bile
najveće u škrgama, dok su koncentracije Cd, Cu, Zn, Fe i B bile najveće u jetri.
Ekološka i toksikološka istraživanja crnomorske haringe u Dunavu u Srbiji
VI
Koncentracija većine analiziranih elemenata bila je najniža u mišićnom tkivu, osim za
As čija je koncentracija najveća upravo u ovom tkivu. Ova studija je pokazala da su
koncentracije Cd i As u mišićnom tkivu crnomorske haringe bile iznad maksimalne
dozvoljene koncentracije propisane zakonskom regulativom koja je prihvatljiva za
ljudsku ishranu. | The Pontic shad (Alosa immaculata Bennett 1835) is an anadromous species that
spends most of its life cycle in the heavily polluted northwestern part of the Black Sea
and migrates into the Danube River to spawn. Populations of the Pontic shad have a
declining trend, and according to the IUCN Red List of Threatened Species the species
is classified as vulnerable (VU). Although its migratory way upstream the Danube is
shortened to 863 rkm after the construction of the Iron Gate II dam and even though it
represents a commercially important fish species in the lower section of the Danube, the
research on the Pontic shad was performed only sporadically in this area.
For the purpose of this study, Pontic shad migrants were collected during April
and May 2004, 2006, 2007, and 2008, immediately downstream from the Iron Gate II
dam.
The length frequency distribution and length-weight relationship were
determined according to the measured total body length (TL) and body weight (W) of
individuals sampled in 2004 and 2006. The length-weight allometric factor (b) was
lower than 3 (b<3) for both sexes and both sampling seasons. The length frequency
ranged from 24.2 to 38.7 cm, with the domination of the length class of 32-32.5 cm.
This study was also aimed at providing the very first data on geometric
morphometrics, gonadosomatic index (GSI), hepatosomatic index (HSI), and Fulton's
condition factor (K) of Pontic shad migrants from the Danube in Serbia. There were
statistical differences in GSI, HSI, and K between males and females in both sampling
seasons, except for K between males and females in 2004, and for GSI and K between
females in 2004 and 2006. The Fulton's condition factor estimated in this study was the
lowest recorded for the Pontic shad ever.
The age frequency distribution was estimated from 28 scales and 30 vertebrae of
randomly chosen individuals. Age of analysed fish ranged from 2+ to 6+, with the
domination of age classes 3+ and 4+. Reading of scales and vertebrae was done by two
Ekološka i toksikološka istraživanja crnomorske haringe u Dunavu u Srbiji
VIII
interpreters experienced in fish aging and by three more interpreters experienced in shad
aging. Interpreters experienced in shad age determination had lower values of the index
of average percent error (IAPE) and index of coefficient of variation (ICV). In regard to
within-interpreter repeatability, no significant differences were found between scale and
vertebrae as structures for age determination. The results of this study indicate that the
experience in age determination has more impact on precision of age determination than
the choice of specific structure, scale or vertebra. More work is needed on adopting
standard protocols that must include some sort of common interaction between age
interpreters.
Geometric morphometry revealed differences in shape that were statistically
significant for both levels: seasons/years (nested ANOVA: F = 13.84; P < 0.001) and
sex (nested ANOVA: sex nested into season/year, F = 1.71; P < 0.01). The major
differences between seasons/years were in the shape of the head and the position of the
fins. The most significant difference of females compared to males was the dorso-lateral
expansion of the mid-body section, with emphasis on the higher dorsal part of the body.
The UPGMA phenogram derived from the matrix of Procrustes distances for body
shape showed that group "males 2004" were separated from the other three groups. The
sexual dimorphism determined in this study was in accordance with findings on other
shad species.
To assess the heavy metal and trace elements contamination level, individuals of
the Pontic shad were sampled during April and May 2007 and 2008. Muscle, liver and
gill tissue samples were prepared using the microwave digestion, and the analysis of Al,
As, B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pb, Sr, and Zn was carried out
using the inductively coupled plasma – optical emission spectrometry (ICP-OES).
Significant differences in concentrations of analysed elements were observed between
different tissues, as well as between sexes. The concentration of Al, Sr, Ba, Mg, and Li
was highest in gills, and of Cd, Cu, Zn, Fe and B in the liver. Although muscle had the
lowest concentration of most of the analysed elements, it had the highest concentration
of As. This study has shown that the concentrations of Cd and As in the muscle tissue of
the Pontic shad exceeded the maximum acceptable concentration for human
consumption.
Ekološka i toksikološka istraživanja crnomorske haringe u Dunavu u Srbiji
IX | true |
Fitohemijska i genetička varijabilnost vrsta iz sekcije Serpyllum (Mill.) Benth. roda Thymus L. (Lamiaceae) u Srbiji | Phytochemical and genetical variability of species from section Serpyllum (Mill.) Benth. genus Thymus L. (Lamiaceae) in Serbia | Rod Thymus je po broju vrsta jedan od najznaĉajnijih u okviru familije
Lamiaceae. U zavisnosti od pristupa autora broj vrsta unutar roda varira, meĊutim
ukoliko se izabere pristup minimalne varijabilnosti, prema raspoloţivim podacima
opisano je 215 vrsta ovog roda. Vrste ovog roda podeljene su u osam sekcija. U flori
Srbije opisana je, do sada, 31 vrsta i preko 100 infraspecijskih taksona, koje sve
pripadaju podsekciji Serpyllum, sa izuzetkom vrsta Thymus comptus i Th. striatus (Sect.
Hyphodromi) i Th. vulgaris (Sect. Thymus). Reproduktivna izolacija izmeĊu vrsta ovog
roda je izuzetno slaba što ĉini taksonomska istraţivanja još teţim.
Vrste roda Thymus koriste se kao lekovite i aromatiĉne biljke u tradicionalnoj i
zvaniĉnoj medicini u svim krajevima u kojima se javljaju. Cilj ovog rada je bio da se
opišu razliĉiti hemotipovi unutar vrsta i populaciju ovog roda kao i da se analizom
površinskih flavonoida i AFLP markera poboljša uvid u genetiĉke i evolutivne odnose
izmeĊu populacija vrsta ovog roda sa podruĉja Srbije. U ovo istrţivajne ukljuĉene su
populacije vrsta Th. pulegioides (Subsect. Alternantes), Th. glabrescens, Th.
maschallianus i Th. pannonicus (Subsect. Isolepides), Th. balcanus i Th. praecox
(Subsect. Peudomarginati)
Analiza sastava etarskih ulja vršena je pomoću TD-GC-MS sistema, a jedinjenja
su odreĊen uporeĊivanjem RI i/ili masenog spektra sa literaturnim podacima.
Identifikovana su ukupno 103 jedinjenja. Identifikovane su, po brojnosti, tri glavne
grupe
jedinjenja:
seskviterpenski
ugljovodonici,
oksigenovani
monoterpenski
ugljovodonici i monoterpenski ugljovodonici. Statistiĉka obrada sastava etarskih ulja
ukljuĉila je korelacije, analizu glavnih komponenti i klaster analizu. Statistiĉka obrada
je pokazala da se, po relativnoj zastupljenosti jedinjenja, mogu razlikovati geraniolni,
fenolni, germakren-D, citralni, linaloolni, (E)-kariofilen, α-terpinil acetat i linalil
acetatni hemotip.
Analiza površinskih flavonoida vršena je pomoću LC-DAD i LC-APCI-MS.
UtvrĊeno je prisustvo 15 površinskih flavonoida. Ispitivane populacije se mogu podeliti
vi
u dve grupe. U prvoj grupi su vrste koje imaju niske i srednje koliĉine površinskih
flavonoida, kao i one kod kojih su se površinski flavonoidi nalazili samo u tragovima.
Kod ove grupe su najzastupljeniji flavoni sa 5,6-diOH-7-OMe i 5,6-diOH-7,8-diOMe
supstitucionom šemom u A prstenu. U drugu grupu bi se mogle svrstati populacije koje,
uglavnom, imaju malu koliĉinu površinskih flavonoida i kod kojih je su najzastupljeniji
flavoni sa 5-OH-6,7-diOMe i 5-OH-6,7,8-triOMe supstitucionom šemom u A prstenu.
Prvoj grupi pripadaju populacije vrsta Th. pulegioides, Th. glabrescens i Th. balcanus,
kao i neke populacije Th. pannonicus, dok drugoj grupi pripadaju populacije Th.
marschallianus, neke populacije Th. pannonicus i Th. praecox.
AFLP markeri su korišćeni da bi se utvrdili genetiĉki odnosi izmeĊu odabranih
populacija vrsta roda Thymus, sekcija Serpyllum. Analiza molekularne varijanse
(AMOVA) je pokazala da najviše genetiĉkog diverziteta potiĉe od razlika meĊu
individuama unutar populacija, dok je ostatak diverziteta gotovo jednako raspodeljen
izmeĊu vrsta i izmeĊu populacija unutar vrsta. Dvosmerna AMOVA je dalje pokazala
da je diferencijacija meĊu vrstama bila znaĉajna u svim sluĉajevima osim izmeĊu Th.
marschallianus i Th. pannonicus. Sve jedinke koje su pripadale Th. pulegioides
(Podsekcija Alternantes) formirale su dobro podrţanu kladu, koja je najviše divergirala
od ostalih vrsta. Prema dobijenim rezultatima podsekcija Isolepides je polifiletska. Th.
glabrescens se jasno odvaja od druge dve vrste ove podsekcije (Th. marschallianus, Th.
pannonicus). Th. marschallianus i Th. pannonicus, iako se jasno razlikuju morfološki
po indumentumu, na molekularnim nivo se ne mogu razdvojiti. Vrste koje pripadaju
podsekciji Pseudomarginati su formirale monofiletsku kladu, mada ova nije bila
podrţana visokom bootstrap vrednošću. Ova klada se dalje delila u tri dobro podrţane
klade koje su predstavljale pojedinaĉne vrste (Th. balcanus, Th. praecox i Th.
moesiacus).
vii
Primena AFLP markera, u sluĉaju izabranih vrsta roda Thymus, se pokazala
prikladnom za razrešavanje kompleksnih genetiĉkih odnosa koji ukljuĉuju i ĉeste
hibridizacije meĊu vrstama, mada striktna retikulatna evolucija ne moţe biti odreĊena.
Svi pokazatelji populacione genetike u sekcij Serpyllum ukazuju da je reproduktivna
izolacija meĊu vrstama slaba i ukljuĉuje izraţen retikulatni sistem, naroĉito u
sluĉajevima kada se dve ili više vrsta javljaju simpatriĉki. | In terms of the number of species, the genus Thymus is one of the most
important genera within the Lamiaceae family. The number of species may vary
depending on the approach of the author, but if the concept of minimal variability is
chosen 215 species have been described. This genus is divided into 8 sections. In Flora
of Serbia, 31 species and more than 100 ifraspecific taxa have been described, all of
which belong to section Serpyllum, except Thymus comptus and Th. striatus (Sect.
Hyphodromi) and Th. vulgaris (Sect. Thymus). Reproductive isolation between species
seems to be weak, which makes taxonomical studies in this genus even more difficult.
Species of genus Thymus are used as aromatic and medicinal plants in both
traditional and modern medicine in all regions where they grow. The aim of this work
was to describe different chemotypes within species and population of this genus and,
by analysis of surface flavonoids and AFLP markers, provide better insight into genetic
and evolutional relationships among the populations and species of the genus Thymus
from Serbia. This work included populations of Th. pulegioides (Subsect. Alternantes),
Th. glabrescens, Th. maschallianus and Th. pannonicus (Subsect. Isolepides), Th.
balcanus and Th. praecox (Subsect. Peudomarginati).
Analysis of essential oils was done by TD-GC-MS system, and compounds were
identified by comparing RI and/or mass spectra with published data. 103 compounds
were identified. Three major group of essential oil compounds were identified:
monoterpene hydrocarbons, oxygenated monoterpene hydrocarbons and sesquiterpene
hydrocarbons. Based on relative abundance of compounds, Corelation, PCA and Cluster
analysis were performed, and it revealed the existence of several chemotypes, such as
geraniol, phenol, germacrene-D, citral, linalool, (E)-caryophyllene, α-terpinyl acetate
and linalyl acetate chemotype.
Analysis of surface flavonoids was done by LC-DAD i LC-APCI-MS. Fifteen
different flavones were identified. The analysed populations can be divided into two
main profile groups; the first group comprises populations containing generally low and
medium concentrations, as well as thoese with trace concentrations of surface
ix
flavonoids. In this group the dominant flavones were with a 5,6-diOH-7-OMe and 5,6-
diOH-7,8-diOMe A-ring substitution pattern. The second group comprises populations
containing generally low quantities of surface flavonoids, and with dominant 5-OH-6,7-
diOMe and 5-OH-6,7,8-triOMe A-ring substitution pattern. Th. pulegioides, Th.
glabrescens i Th. balcanus, and some populations of Th. pannonicus belong to first
group, while Th. marschallianus, some populations of Th. pannonicus and Th. praecox
belong to the second group.
Amplified fragment length polymorphism (AFLP) markers were used to assess
genetic relations of 32 Thymus populations belonging to seven species of genus
Thymus, section Serpyllum from Serbia. Analysis of molecular variance (AMOVA)
revealed that most of the genetic diversity was attributable to differences among
individuals within populations, while the remaining variability was almost equally
distributed among species and among populations within species. The pairwise
AMOVA analyses further showed that species differentiation was significant in all
cases, except between Th. marschallianus and Th. pannonicus. All the individuals
belonging to Th. pulegioides (subsection Alternantes) formed a well-supported clade,
the most divergent from all other species in this study. According to these results, the
subsection Isolepides is polyphyletic. Th. glabrescens is clearly separated from the other
two species of this subsection (Th. marschallianus, Th. pannonicus). Although
morphologically distinguishable by leaf indumentum, individuals belonging to Th.
marschallianus and Th. pannonicus could not be differentiated at the molecular level.
The species belonging to subsection Pseudomarginati formed a monophyletic clade,
although not supported by high bootstrap value. The clade is further divided into three
well-supported clades representing distinct species (Th. balcanus, Th. praecox and Th.
moesiacus).
x
AFLP markers demonstrated, in the case of selected species of genus Thymus,
that they could be suitable for studying complex genetic relationships, including
frequent interspecies hybridization events, although strict reticulate evolutionary history
could not be determined. All the population genetic parameters in the section Serpyllium
suggests that the reproductive incompatibility between species is very weak indicating
strong reticulating system, especially in the case when two or more species occur
sympatrically. | true |
Uticaj promenljivog magnetnog polja na motorno ponašanje pacova u laboratorijskim uslovima | Effect of an alternating magnetic field on motor behaviour of rats in laboratory conditions | Brojne studije su pokazale da promenljiva magnetna polja ekstremno niskih frekvencija (ENF-MP) utiču na motorno ponašanje eksperimentalnih životinja i ljudi, ali još uvek nisu u potpunosti razjašnjene veze između promena u neurotransmiterskim sistemima odgovornim za kontrolu motorne aktivnosti i promena u ponašanju. Cilj ovih istraživanja je ispitivanje efekata akutnog (1, 3 i 7 dana) i hroničnog (od začeća do starosti od oko 3 meseca) izlaganja ENF-MP (50 Hz, 0,5 mT) na različite parametre motornog ponašanja (lokomocija, stereotipija, uspravljanje i rotacije) odraslih pacova Wistar soja. Analizirana je i aktivnost dopaminskih D1 i D2 receptora u strijatumu i serotoninskih 5-HT2A receptora u prefrontalnoj kori velikog mozga, kao i koncentracije nukleotida (IMP, cAMP, ATP i GTP) u kori velikog mozga i strijatumu ovih životinja. Rezultati istraživanja akutnih efekata ENF-MP ukazuju na: (1) povećanu lokomociju i stereotipiju (1 dan) i smanjeno uspravljanje životinja (1, 3 i 7 dana), (2) smanjenu lokomociju i stereotipiju (3 i 7 dana), kao i rotacije životinja (7 dana) izazvane primenom dopaminergičkog agoniste amfetamina (AMPH, 1,5 mg/kg, i.p.), (3) smanjen afinitet i povećanu gustinu 5-HT2A receptora (1, 3 i 7 dana) i (4) smanjen sadržaj cAMP (3 i 7 dana) i IMP (7 dana), kao i stimulisano stvaranje ATP (1 i 3 dana) u kori velikog mozga. Hronično izlaganje ENF-MP: (1) povećava motornu aktivnost životinja, (2) smanjuje uspravljanje životinja izazvano primenom AMPH, (3) povećava afinitet D1 receptora i smanjuje afinitet 5-HT2A receptora i (4) povećava koncentraciju cAMP u kori velikog mozga i strijatumu. Na osnovu rezultata možemo zaključiti da spoljašnja ENF-MP modulišu motorno ponašanje eksperimentalnih životinja direktnim i/ili indirektnim uticajem na sinaptičku neurotransmisiju u kori velikog mozga i strijatumu, pri čemu karakter ovih promena zavisi od dužine izlaganja ENF-MP. | Numerous studies have shown that alternating magnetic fields of extremely low frequency (ELF-MF) affect motor behaviour of experimental animals and humans, but the connections between changes in neurotransmitter systems involved in motor control and changes in behaviour are not yet completely explained. The purpose of this study is to examine the effects of acute (1, 3, and 7 days) and chronic (from conception to about 3 months old) exposure to ELF-MF (50 Hz, 0.5 mT) on different parameters of motor behaviour (locomotion, stereotypy, rearing, and rotations) of adult Wistar rats. Dopamine D1 and D2 receptors in the striatum and serotonin 5-HT2A receptors in the prefrontal cortex, as well as concentrations of nucleotides (IMP, cAMP, ATP, and GTP) in the cerebral cortex and striatum of these animals were also analysed. Results concerning the acute ELF-MF effects reveal: (1) increased locomotion and stereotypy (1 day), and decreased rearing of animals (1, 3 and 7 days), (2) decreased locomotion and stereotypy (3 and 7 days), as well as rotations of animals (7 days) induced by application of dopaminergic agonist amphetamine (AMPH, 1.5 mg/kg, i.p.), (3) decreased affinity and increased density of 5-HT2A receptors (1, 3 and 7 days), and (4) reduced cAMP (3 and 7 days) and IMP content (7 days), as well as stimulated ATP production (1 and 3 days) in the cerebral cortex. Chronic ELF-MF exposure: (1) enhances motor activity of animals, (2) decreases AMPH-induced rearing of animals, (3) increases D1 receptors affinity and decreases 5-HT2A receptors affinity, and (4) increases cAMP concentrations in the cerebral cortex and striatum. It can be concluded that an external ELF-MF modulates motor activity of experimental animals by direct and/or indirect influence on synaptic neurotransmission in the cerebral cortex and striatum, whereby the character of these changes depends on the duration of ELF-MF exposure. | false |
Fiziološki i biohemijski aspekti regeneracije košutice (Fritillaria meleagris L.) in vitro | Physiological and biochemical aspects of regeneration snake's head fritillary (Fritillaria meleargis L.) in vitro | Proučavana je in vitro regeneracijia košutice (Fritillaria meleagris), višegodišnje
lukovičaste geofite. Indukcija morfogeneze in vitro košutice postignuta je u kulturi zrelih
zigotskih embriona, segmenata lukovica kao i bazalnih delova listova in vitro formiranih
lukovica. U kulturi zrelih zigotskih embriona regeneracija biljaka je postignuta procesom
somatske embriogeneze i organogeneze u isto vreme i na istom eksplalntatu na hranljivoj
podlozi bez regulatora rastenja ili hranljivoj podlozi obogaćenoj sa TDZ. U kulturi
segmenata in vitro formiranih lukovica indukcija morfogeneze postignuta je na hranljivoj
podlozi sa 2,4-D ili TDZ na svetlosti ili u mraku. Regeneracija biljaka je bila postignuta na
obe hranljive podloge putem somatske embriogeneze i organogeneze, s tim da je
regeneracija putem organogeneze bila uspešnija na hranljivoj podlozi obogaćenoj sa TDZ.
U kulturi bazalnih delova listova indukovana je somatska embriogeneza na hranljivim
podlogama sa 2,4-D, KIN ili 2,4-D i KIN. Anatomska istraživanja su pokazala da je
somatska embriogeneza direktna, a somatski embrioni imaju višećelijsko poreklo i nastaju
od epidermalnih i subepidermalnih slojeva ćelija bazalnih delova lista. Ispitan je uticaj
niskih temperatura (15 i 4 °C), povećane koncentracije saharoze u hranljivoj podlozi kao i
predtretman sa GA3 na rastenje, razviće i prevazilaženje dormancije in vitro formiranih
lukovica košutice. Pokazano je da predhodno gajenje lukovica na sniženim temperaturama i
hranljivoj podlozi sa 4,5 % saharoze pozitivno utiče na rastenje i umnožavanje lukovica.
Pored toga, predtretman rastvorom GA3 pre izlaganja lukovica niskoj temperaturi (4 °C)
dovodi do stimulacije umnožavanja i klijanja lukovica. Izlaganjem in vitro formiranih
lukovica košutice niskoj temperaturi dolazi do promena u sadržaju šećera (saharoze,
glukoze i fruktoze), fotosintetičkih pigmenata i poliola. Ispitana je aktivnost antioksidativnh
enzima (SOD, CAT, GR i POX) tokom prevazilaženja dormancije lukovica gajenjem na
niskoj temperaturi. Analize su pokazale da enzimi antioksidativnog stresa aktivno učestvuju
u procesima prekidanja dormancije in vitro formiranih lukovica košutice, kao i
aklimatizacije lukovica na ex vitro uslove. Enzimi antioksidativne zaštite su aktivni i tokom
indukcije morfogeneze košutice u kulturi segmenata lukovica, a njihova aktivnost zavisi od
sastava hranljive podloge i predtretmana kome su lukovice prethodno izložene.
Zimogramskom detekcijom esteraza tokom indukcije morfogeneze in vitro u kulturi
segmenata lukovica utvrđeno je prisustvo 6 izoformi i njihova aktivnost kao i zastupljenost
pojedinih izoformi zavise takođe od predtretmana na kojima su lukovice predhodno gajene
kao i od regulatora rastenja u hranljivoj podlozi. Praćena je promena sadržaja
arabinogalaktanskih proteina (AGP) u eksplantatima tokom indukcije morfogeneze in vitro
u kulturi bazalnih delova listova i segmenata lukovica na dve hranljive podloge obogaćene
sa 2,4-D i KIN ili TDZ. Koncentracija AGP u eksplantatima se povećava već posle 7 dana
gajenja bazalnih delova listova odnosno posle 21 dana gajenja segmenata lukovica na
hranljivim podlogama sa regulatorima rastenja. Koncentracija AGP je veća u bazalnim
segmentima lista gajenim na hranljivoj podlozi sa 2,4-D i KIN nego na hranljivoj podlozi
sa TDZ. Analizom profila AGP dobijenog ukrštenom elektroforezom pokazano je prisustvo
samo jednog tipa AGP tokom indukcije morfogeneze in vitro.
Naučna oblast: Biologija
Uža naučna oblast: Fiziologija biljaka
UDK broj: 581.1(043.3) | We have investigated in vitro regeneration of snake’head fritillary (Fritillaria
meleagris L.), a perennial bulbous geophyte. The induction of morphogenesis in vitro of
snake’head fritillary was achieved in mature zygotic embryo culture, scale segment and leaf
base culture of in vitro formed bulbs. Plant regeneration via somatic embryogenesis and
organogenesis was obtained in mature zygotic embryo culture on a growth regulator-free
medium or on medium supplemented with TDZ. The induction of morphogenesis in vitro
was achieved in scale segment culture of the in vitro formed bulbs on media supplemented
with 2,4-D or TDZ, grown either on light or in darkness, with more efficient regeneration
on media supplementned with TDZ. Somatic embryogenesis was induced in leaf base
culture of the in vitro formed plants on media with 2,4-D, KIN or 2,4-D and KIN.
Anatomical studies revealed that the somatic embryogenesis was direct, with somatic
embryos of multicellular origin formed from epidermal and subepidermal leaf base cells .
The effect of low temperature (4 and 15 °C), higher concentration of sucrose in the
nutritional media and GA3 pretreatment on growth, differentiation and dormancy breaking
of the in vitro formed bulbs was investigated. It was shown that pre-cultivation of the in
vitro regenerated bulbs at lower temperatures and higher concentration of sucrose in the
nutrition media (4,5 %) have stimulatory effect on growth and multiplication of the bulbs.
Also, GA3 pretretment followed by cultivation at low temperature (4 °C) had a stimulatory
effect on multiplication and germination of the bulbs. Cultivation at low temperature breaks
dormancy of the bulbs and causees changes in the sugar content (sucrose, glucose and
fructose), photosinthetic pigments and poliols. Activity of antioxidative enzymes (SOD,
CAT, GR i POX) during dormancy breaking was investigated. It was shown that these
enyzmes are actively involved in dormancy breaking of the in vitro formed bulbs, and in
the process of acclimatization of the bulbs to ex vitro conditions. Antioxidative enzymes
were active during the induction of morphogenesis in vitro in bulb segment culture and
their activity depended on the nutritional media and the pretreatment to which the bulbs
were exposed. During morphogenesis in vitro in the scale segment culture of snake’s head
fritillary, up to 6 esterase isoforms have been detected, depending on the pretreatment and
media composition. The content of arabinogalactan proteins (AGPs) in explants during the
induction of morphogenesis in vitro in leaf base and scale segment cultures at media
supplemented either with 2,4-D and KIN or with TDZ was determined. Concentration of
AGPs increased after seven days of cultivation of explants on media with growth regulators
in the leaf base culture and 21 days in the scale segment culture. In the leaf base culture,
concentration of AGPs in explants was higher on a medium with 2,4-D and KIN than on a
medium with TDZ. The AGP profile obtained by crossed electroforesis reveiled the
presence of one AGP type during induction of morphogenesis in vitro of F. meleagris.
Scientific field: Biology
Specific scientific field: Plant Physiology
UDC number: 581.1(043.3) | true |
Floristička, taksonomska i ekološka istraživanja dezmidnih algi Srbije | Floristical, taxonomical and ecological study of desmid algae in Serbia | Doktorska disertacija obuhvata istraživanja dezmidnih algi na teritoriji Srbije. Algološki
uzorci (299) za ovo istraživanje sakupljeni su u periodu od 2007. do 2011. godine sa 37
lokaliteta.
Istraživanje je obuhvatilo različite tipove vodenih staništa: tresave, veštačke
akumulacije, slane bare i ribnjake. Sa najvećeg broja lokaliteta sakupljano je više
uzoraka iz različitih zajednica algi: planktona, bentosa, epifita.
U radu je data sinonimika i opis svih zabeleženih taksona dezmidnih algi na teritoriji
Srbije od 1883. godine do danas, tip staništa koja naseljavaju i rasprostranjenje u Srbiji.
Pregledom literaturnih podataka i uzoraka sakupljenih ovim istraživanjem ustanovljeno
je prisustvo 611 taksona dezmidnih algi na teritoriji Srbije, od čega je u ovom
istraživanju utvrđeno 244 taksona iz 23 roda. Svi do sada zabeleženi taksoni dezmidnih
algi svrstani su u 5 familija sa 25 rodova. Najveći broj taksona je utvrđen u rodovima
Cosmarium (250), Staurastrum (102) i Closterium (86).
Prvi put je za floru algi Srbije zabeleženo prisustvo dva nova roda (Cosmocladium i
Heimansia) sa ukupno 3 nove vrste.
Prvi put je za floru algi Srbije zabeleženo 69 taksona dezmidnih algi. Rodovi sa
najvećim brojem novih taksona za floru algi Srbije su Cosmarium (25), Staurastrum
(12) i Closterium (9).
Pripadnici dezmidnih algi su nađeni u svim ispitivanim uzorcima.
Statističkom obradom podataka utvrđeno je da postoji značajna korelisanost između
trofičnosti i kiselosti staništa sa strukturom zajednica dezmidnih algi na istraživanim
lokalitetima. | The work compements and develops investigations on the desmid algae of Serbia.
Algological samples (299) for this study were collected from 37 localities during the
years 2007-2011.
The samples of phytoplankton, benthos and epiphyte were collected from pit bogs,
reservoirs, salt marshes and fish ponds.
Detailed description of all desmid taxa which were found in Serbia since 1883 as well
as their distribution in Serbia, habitats and saprobities are given.
Total of 611 desmid taxa were rocorded for the territory of Serbia, 244 of which are
dealt with in this work. All desmid taxa were classified in 5 families with 25 genera.
The most numerous genera are Cosmarium (250), Staurastrum (102) and Closterium
(86).
For the first time two new genera (Cosmocladium and Heimansia) with 3 new species
have been recorded in Serbia.
The presence of 69 desmid taxa have been recorded for the fisrt time for territory of
Serbia. The most numerous genera were Cosmarium (25), Staurastrum (12) and
Closterium (9).
In this investigation desmids have been found in all collected samples.
Statistical research were shown that there are significant correlation between trophic
status and acidity of water with structure of desmids community. | true |
Uticaj elektromagnetnog polja (2 mT, 50 Hz) na spontanu bioelektričnu aktivnost neurona antenalnog lobusa adulta Morimus funereus (Insecta, Coleoptera) | The influence of electromagnetic field (2 mT, 50 Hz) on spontaneous bioelectrical activity of antennal lobe neurons of adult Morimus funereus (Insecta, Coleoptera) | Spoljašnja magnetna polja (MP) narušavaju ravnotežu bioloških sistema, čak i
tokom kraćeg vremena izlaganja, što za posledicu ima poremećaje na različitim nivoima
organizacije. Na ovaj način nastali poremaćaji akumuliraju se u organizmima, što je jedan
od razloga za traženje objašnjenja bioloških efekata MP. S druge strane, veoma važna
činjenica koja nas usmerava na što bolje razumevanje ove problematike je potreba za
zaštitom ljudskog zdravlja zbog uvođenja novih tehnologija u svakodnevni život,
industriju i medicinu, kao i zbog terapijske upotrebe MP.
Pretpostavka je da se interakcija spoljašnjih MP i organizama dešava na svim
nivoima organizacije, uključujući i nervni sistem. Stoga je cilj ovih istraživanja bio
ispitivanje uticaja elektromagnetnog polja (2 mT, 50 Hz) različitih karakteristika (trajanje,
talasni oblik) na spontanu bioelektričnu aktivnost neurona antenalnog lobusa adulta
Morimus funereus. Aktivnost neurona (pozadinskih/neuronske populacije i najbližih
registrujućoj elektrodi) je registrovana kroz nekoliko faza u kojima smo adulte izlagali
sinusoidalnom ili kvadratnom MP različitog trajanja: 5, 10 i 15 min (MP-5, MP-10, MP-15,
respektivno). Uticaj MP smo procenjivali na osnovu promena u neuronskoj aktivnosti, a
procenjivali smo i (i)reverzibilnost promena između tripleta formiranih od određenih
eksperimentalnih faza. U tripletima koji slede vremenski tok eksperimenta, a
podrazumevaju delovanje MP određenog trajanja (MP-5, MP-10 i MP-15, respektivno),
testirali smo i njihove međusobne razlike.
Rezultati istraživanja su pokazali da primenjeno MP, bez obzira na dužinu
izlaganja i talasni oblik, menja aktivnost neurona antenalnog lobusa. U sinusoidalnom
MP, te promene su najizraženije nakon delovanja MP-15, a u kvadratnom MP, nakon
delovanja MP-5. Pokazano je da je kontrolna aktivnosti pozadinskih neurona značajno
promenjene nakon delovanja sinusoidalnog MP-5, kao i nakon delovanja MP-10, a
značajno je manja nakon delovanja MP-15. S druge strane, sinusoidalno MP, bez obzira na
dužinu izlaganja izaziva značajnu inhibiciju kontrolne aktivnosti najbližih neurona.
Promene kontrolne neuronske aktivnosti su značajno manje nakon delovanja kvadratnog
MP različitog trajanja (i MP-5 i MP-10 i MP-15). U odnosu na aktivnost neposredno nakon
izlaganja sinusoidalnom MP, povećanje neuronske aktivnosti 35 min nakon izlaganja je za
pozadinske neurone značajno u MP-5, a za najbliže neurone u MP-10. Promene neuronske
aktivnosti 35 min po prestanku izlaganja kvadratnom MP-5 u odnosu na aktivnost
neposredno nakon izlaganja su značajne i kod pozadinskih i kod najbližih neurona. S
druge strane, neuronska aktivnost 35 min po prestanku izlaganja kvadratnom MP-10 je
kod pozadinskih neurona bila značajno veća, a kod najbližih neurona značajno manja u
odnosu na aktivnost neposredno nakon izlaganja. Promene neuronske aktivnosti koje
nastaju usled izlaganja sinusoidalnom/kvadratnom MP različitog trajanja, u većini
slučajeva su imale ireverzibilan karakter. Sinusoidalno MP različitog trajanja ne izaziva
značajne promene faktora reverzibilnosti između tripleta eksperimentalnih faza koji
podrazumevaju delovanje MP određenog trajanja. Nasuprot tome, kvadratno MP značajne
razlike izaziva kod najbližih neurona i to između tripleta koji uključuju delovanje MP-10 i
MP-15.
Na osnovu svega navedenog, možemo zaključiti da su jedinke M. funereus osetljive
na delovanje i sinusoidalnog i kvadratnog elektromagnetnog polja (2 mT, 50 Hz) različitog
trajanja i da njihova reakcija zavisi od karakteristika primenjenog polja, kao i od
individualnih specifičnosti jedinki ove vrste. Takođe, naši rezultati ukazuju na činjenicu
da M. funereus ima sposobnost da detektuje i odgovara na spoljašnja magnetna polja. | External magnetic fields (MF) disturb the equilibrium of biological systems, even
during short-term exposure, resulting in disturbances at different levels of organization.
These changes accumulate in organisms, which is one of the reasons for testing biological
effects of MF. On the other hand, the fact that modern man is exposed to increasing
number of MF sources due to the use of new technologies in everyday life, industry,
medicine, as well as therapeutic application of MF, makes this topic even more important,
in terms of human health protection.
It is assumed that the interaction between external MF and organisms takes place
at all levels of organization, including the nervous system. Thus, the aim of this study was
to investigate the influence of electromagnetic field (EMF) of different characteristics
(exposure duration, waveform) on spontaneous bioelectrical activity of antennal lobe
neurons of adult Morimus funereus. Neurons activity (background/neuronal population
and those nearest to the recording electrode) was registered through several phases of
exposure of adult longhorn beetles to sine wave or square wave MF of different exposure
duration: 5, 10 and 15 minutes (MP-5, MP-10 and MP-15, respectively). Estimation of MF
influence was based on registered changes of neuronal activity. (I)reversibility of such
changes was tested through comparison the triplets formed by particular experimental
phases. Changes in triplets that follow the time course of the experiment, with reference
to MF influence of different exposure duration (MP-5, MP-10 and MP-15, respectively),
were also tested.
Results of this study showed that the applied MF, regardless of exposure duration
and waveform, modifies the activity of antennal lobe neurons. In sine wave MF, the most
prominent changes are recorded after MP-15, while in square wave MF such changes
were recorded after MP-5. Control activity of the neuronal population was significantly
changed both after MP-5 and MP-10, as well as significantly decreased after MP-15. Sine
wave MF, regardless of exposure duration, significantly inhibits control activity of
nearest neurons. Changes in control neuronal activity are significantly lower in square
wave MF of different exposure duration (MP-5, MP-10, MP-15). Compared to the activity
detected immediately after exposure to sine wave MF, the activity of neuronal population
and nearest neurons is significantly increased 35 minutes after cessation of MF-5 and MP-
10 exposure, respectively. In case of square wave MF, changes in the activity of neuronal
population and nearest neurons are significant 35 minutes after cessation of MF-5
exposure compared to the activity detected immediately after exposure to this MF. On
the other hand, the neuronal activity detected 35 minutes after cessation of MF-10
exposure was significantly higher in the neuronal population and significantly lower in
the nearest neurons compared to the activity detected immediately after exposure to this
square wave MF. Changes in the activity of neurons exposed to sine wave or square wave
MF of different duration were mostly irreversible. Sine wave MF of different duration
does not change reversibility factors between the triplets formed by particular
experimental phases. In contrast, in square wave MF, reversibility factors of the nearest
neurons are significantly changed between the triplets formed by MP-10 and MP-15
experimental phase.
Results of our experiment imply that M. funereus individuals are sensitive to both
sine wave and square wave EMF (2 mT, 50 Hz) of different duration, whereby their
reactions depend on the characteristics of the applied MF and specificity of each
individual. In addition, it can be presumed that adult M. funereus has the ability to detect
and respond to external MF. | true |
Adaptivan ili stabilan odgovor Amy-genotipova Drosophila subobscura u uslovima diskretnih i naglih promena sredine | Adaptive or stable response of Amy - genotypes of Drosophila subobscura in the conditions of discrete and abrupt changes of environment | U savremenoj biologiji opšte je prihvaćen stav da se organizmi i populacije ne mogu posmatrati izvan svojih sredina. U dinamici genofonda populacije značajan je stepen uspešnosti odgovora različitih genotipova na promene uslova sredine. Stoga je sagledavanje mogućih vidova interakcije različitih genotipova i sredina značajno za razumevanje mikroevolucionih procesa koji se odigravaju u okviru prirodnih populacija. Jedinke iste vrste, čije je razviće u različitim sredinama, mogu se značajno razlikovati po fenotipskim karakterima. Ovakva fenotipska diferencijacija između grupa jedinki čije se razviće odvijalo u različitim staništima, može biti uzrokovana genetičkim razlikama između grupa, ili isključivo efektima sredine na fenotip jedinki. Postojanje jasne biohemijsko – fiziološke uloge α–amilaze i jasno definisanog supstrata njenog delovanja pruža mogućnosti da se sagledaju mogući vidovi interakcije različitih genotipova i sredine, što može biti od značaja za razumevanje mikroevolucionih procesa koji se odigravaju u okviru prirodnih populacija. Polimorfizam Amy–lokusa kod Drosophila subobscura uzet je kao model sistem za eksperimentalno populaciono – genetičko razmatranje stepena uspešnosti odgovora različitih genotipova na vrstu i brzinu promene sredine, kako bi se razmatrali oblici, pravci i efekti interakcije genotip/sredina. U našim istraživanjima analiziran je uticaj različitih koncentracija skroba kao i uticaj postepenih i naglih promena koncentracije skroba na komponente adaptivne vrednosti. Dužina razvića i preživljavanje, pored specifične aktivnosti α–amilaze kod jedinki Drosophila subobscura homozigotnih ili heterozigotnih za “spori” ili “brzi” amilazni alel, mogu biti takođe validni pokazatelji načina odgovora na različite koncentracije skroba u medijumu na kome jedinke kompletiraju životni ciklus. Norme reakcija za prosečne vrednosti analiziranih karakteristika ukazuju na postojanje značajnog sredinskog uticaja kao i da je u njihovoj osnovi genetička varijabilnost jedinki Drosophila subobscura. Na osnovu rezultata analize odgovora Amy-genotipova Drosophila subobscura može se zaključiti da su genotipovi, prosečno, fenotipski plastični za analizirane komponente adaptivne vrednosti u svim posmatranim sredinskim uslovima. | In modern biology, it is the generally accepted that organisms and populations can not be observed separately from their environments. Effectiveness of responses of different genotypes to environmental changes is essential for the dynamics of population gene pool. Therefore, consideration of the possible forms of interaction among different genotypes and environments is important for understanding microevolutionary processes occurring within natural populations. Individuals of the same species that develop in different environments, can significantly differ phenotypically. This phenotypic differentiation can be caused by genetic differences between the groups or environmental effects. The existence of clear biochemical - physiological role of α–amylase and clearly defined substrate of its action allows the analysis of possible interactions between different genotypes and environment, which can be significant for understanding of microevolutionary processes occurring within natural populations. Polymorphism of Amy–locus in Drosophila subobscura is taken as a model system for the experimental population – genetic study the effectiveness of responses of different genotypes to type and dynamic of environmental changes, to consider forms, directions and effects of genotype/environment interactions. Our research deals with the effects of different starch concentrations, as well as gradual and abrupt changes of starch concentrations on fitness components. Development time, viability and specific activity of α–amylase in Drosophila subobscura, which are homozygous or heterozygous for “slow” or “fast” amylase allele, can also be good indicators of response to different concentrations of starch in substrate on which life cycle of individuals is completed. Obtained reaction norms, for the average values of analyzed traits, reveal significant environmental effect and the presence of genetic variability of Drosophila subobscura individuals. Based on the analysis of responses of Amy-genotypes of Drosophila subobscura, it can be concluded that the genotypes are phenotypically plastic for the analyzed fitness traits in all observed environmental conditions. | false |
Spektralna analiza elektrokortikalne aktivnosti mozga pacova u modelu intoksikacije aluminijumom | Spectral analysis of electrocortical brain activity in rat model of aluminium intoxication | Cilj ove teze je prikaz metoda za kvalitativnu i kvantitativnu analizu moždane aktivnosti. Ujedno je matematički urađena biomedicinska aproksimacija rezultata. Animalni model intoksikacije aluminijumom je korišćen zbog rasprostranjenosti aluminijuma u prirodi i okruženju. Aluminijum ima selektivno neurotoksično dejstvo na pojedine delove mozga. Ujedno neurotoksičnost aluminijuma ima za posledicu izmenjenu aktivnost celog mozga, ali i specifično dejstvo na pojedine moždane funkcije. Animalni model nudi mogućnost više parametara i može se koristiti za izučavanje neurotoksičnosti aluminijuma pod različitim okolnostima. Procesi koji prate neurotoksičnost mogu se porediti sa promenama koje se javljaju u akutnim i hroničnim stanjima bolesti. Spektralna analiza zasnovana na Furijerovoj transformaciji daje opis promena elektrokortikalne aktivnosti po frekventnim oblastima. Poređenje spektara snage i njihova statistička analiza omogućavaju kvantifikaciju promena. Praćena je elektrokortikalna aktivnost parijetalnog korteksa i malog mozga kako bi se obuhvatila sva tri parametra intoksikacije: akumulacija u tkivu, lokalno izmenjena aktivnost neurona i promena regulatorne funkcije - plastičnost i adaptabilnost mozga. Eksperimentalni dizajn podrazumeva variranje parametara intoksikacije (aplikacija aluminijuma, moždana struktura, doza, starost, soj, fiziološko stanje). Na osnovu dobijenih podataka definisani su efekti neurotoksičnosti aluminijuma. Varijabilnost parametara fizioloških promena i efekata neurotoksičnosti su matematički analizirani. Rezultati ukazuju da promena moždane aktivnosti može biti graduisana tako da može poslužiti kao dijagnostičko-prognostički parametar patofiziološkog stanja. Ova studija ima multidisciplinarni značaj jer obrađuje različite aspekte - biološki, fiziološki i medicinski. | The aim of the study was quantitative and qualitative analysis of electrocortical brain activity in rat model of aluminium intoxication. We used animal model of aluminium intoxication becouse aluminium toxicity is widespread in nature. Aluminium has selective effects on different brain regiones. Animal model included different variables, so it can be used to study neurotoxicity in different conditiones. Also, processes due to neurotoxicity can be compared with state of some acute or chronic diseases. Spectral analysis was based on Fast Fourie Transform and changes of electrocortical activity was obsreved in relation to frequency ranges. Comparation of power spectra and statistic analyses was used for quantification of changes in brain activity. We recorded electrocortical activity of parietal cortex and cerebellum to include all parameters of intoxication: acumulation in tissue, changes of neuronal local group activity and changes in regulatory function - plasticity and adaptability of brain. Experimental procedure included different parametars of intoxication - aplication of aluminium, brain structure, doses, age, straint and physiological state of animals. Based on obtained data we define effects of neurotoxicity. Biological parameters, such as neurotoxical effects, physiological change and intoxication are matematicaly analyzed. Results suggest that the change of brain activity can be used in diagnoses and prognoses of pathophisiological state. This study presents synthesis of biological, physiological and medical aspect. | false |
Zaštitni efekat probiotika na modelu toksičnosti kadmiuma kod pacova | Protective effects of probiotics in the model of cadmium toxicity in rats | Kadmijum je široko prisutni toksikant životne sredine, koji izaziva različite poremećaje
u biološkim sistemima, uključujući tu i bubrežnu disfunkciju i degeneraciju tkiva jetre. Sa
druge strane, pretpostavlja se da su pozitivne osobine probiotskih bakterija u vezi sa njihovom
sposobnošću da vezuju različite ligande, što dovodi do poboljšanja crevne mikrobijalne
ravnoteže i drugih koristi za domaćina. Imajući u vidu pomenuta, ova studija je sprovedena da
istraži zaštitni efekat probiotika protiv kadmijum-indukovanog toksičnosti kod pacova.
Muški pacovi Rattus norvegicus Wistar soja, težine 130±10 g, gajeni su u standardnim
laboratorijskim uslovima (22±2°C, režim 12h svetlo/12h mrak) u metaboličkim kavezima (3
pacova/kavezu). Životinje su podeljene u četiri grupe: 1) kontrolne jedinke; 2) životinje
tretirane probioticima; 3) životinje koje su primale kadmijum u formi CdCl2; i 4) životinje
tretirane probioticima i kadmijumom. U krvi je određivana koncentracija kadmijuma, aktivnost
alanin aminotransferaze (ALT) i aspartat aminotransferaze (AST), kao i nivoi adrenalina i
noradrenalina. U homogenatima tkiva jetre i bubrega određivane su koncentracije kadmijuma,
cinka i bakra, kao i aktivnosti ukupne superoksid dismutaze (SOD) i katalaze (CAT). Takođe,
ispitivan je i histomorfološki profil tkiva jetre i bubrega u svim grupama životinja. U fecesu
eksperimentalnih životinja određivana je koncentracija kadmijuma i mikrobijalni profil
odabranih vrsta bakterija crevne mikroflore. Na kraju, Comet testom je testirana genotoksičnost
kadmijuma in vitro and in vivo.
Na osnovu naših rezultata mogu se izvesti sledeći zaključci:
1. Davanje kadmijuma eksperimentalnim životinjama u formi kadmijum hlorida i
koncentraciji 70ppm u pijaćoj vodi izaziva značajno smanjenje u porastu telesne
mase tokom 5 nedelja eksperimenta. Istovremeno, između eksperimentalnih grupa
nema značajnih promena u količini popijene vode i pojedene hrane.
2. Istovremeno davanje probiotika zajedno sa kadmijumom dovodi do povećanja
koncentracije kadmijuma u fecesu i pratećeg smanjenja njegove koncentracije u krvi,
jetri i bubregu. Istovremeno, administracija kadmijuma dovodi do značajnog
povećanja koncentracije cinka u jetri i bubregu.
3. Kadmijum izaziva značajne histopatološke promene tkiva jetre. Parenhim tkiva jetre
je izmenjen, sinusoidi prošireni, naročito u proksimalnom delu centralne vene, a
hepatocite često sadrđe nukleuse sa kondenzovanim hromatinom, bez prisustva
nukleolusa. Kupferove ćelije pokazuju morfološke znake aktivacije. Davanje
probiotika zajedno sa kadmijumom smanjuje intezitet nabrojanih promena, iako one
ostaju evidentne.
4. Kadmijum povećava aktivnost ALT i AST u krvi eksperimentalnih životinja, što
dodatno ukazuje na oštećenja jetre. Međutim, davanje probiotika zajedno sa
kadmijumom vraća aktivnost oba enzima na kontrolni nivo.
5. Kadmijum izaziva značajne histopatološke promene i tkiva bubrega. U epitelu
proksimalnih tubula uočavaju se nekrotične i nabubrele ćelije. Davanje probiotika
zajedno sa kadmijumom smanjuje intezitet nabrojanih promena.
6. Pod delovanjem kadmijuma aktivnost katalaze je smanjena u oba tkiva, i jetri i
bubregu Sa druge strane, obrazac promena aktivnosti SOD pokazuje tkivnu
specifičnost: dok je pod delovanjem kadmijuma aktivnost ovog enzima u jetri
povećana, u bubregu je ona smanjena. Davanje probiotika zajedno sa kadmijumom
dovodi do restitucije aktivnost ovih enzima blizu kontrolnog nivoa
7. Kadmijum značajno povećava koncentraciju adrenalina i noradrenalina u krvi
ispitivanih životinja. Istovremeno, sami probiotici ne menjaju koncentraciju ni
jednog niti drugog kateholamina. Davanje probiotika zajedno sa kadmijumom
smanjuje kadmijumom izazvano povećanje oba kateholamina.
8. U kulturi hepatocita kontrolnih životinja (in vitro) i hepatocitima životinja tretiranih
kadmijumom (in vivo) ovaj metal izaziva značajan porast oštećenja DNK. Davanje
probiotika zajedno sa kadmijumom značajno smanjuje ovaj efekat kadmijuma.
9. Kadmijum smanjuje koncentraciju svih ispitivanih bakterija crevne mikroflore.
Davanje probiotika zajedno sa kadmijumom značajno smanjuje ovaj efekat samo u
slučaju laktobacila, povećavajući njihovu, kadmijumom smanjenu koncentraciju u
fecesu.
10. Na osnovu svega može se zaključiti da probiotici aktivno učestvuju u uklanjanju
kadmijuma iz organizma, verovatno ga vezujući proteinima ćelijskog zida.
11. Neophodna su dalja istraživanja u cilju određivanja specifične kombinacije
probiotika koja bi bila najefikasnija u uklanjanju kadmijuma, ali i drugih teških
metala i karcinogena. | Cadmium is a ubiquitous environmental toxicant that causes a variety of disturbances
in biological systems, including renal dysfunction and liver tissue degeneration. On the other
hand, it is supposed that beneficial properties of probiotic bacteria are related to their capacity
to adhere or bind different targets, thus leading to improved intestinal microbial balance and
other benefits to host. Bearing aforementioned in mind, the present study was undertaken to
investigate the protective effect of probiotic supplementation against cadmium-induced
toxicity in rat.
Male Wistar rats Rattus norvegicus, weighing 130±10 g, were acclimated to 22±2°C in
metabolic cages (3 rats/cage) and maintained under a 12h light/12h dark cycle. Animals were
divided into four groups: 1) controls; 2) probiotics treated; 3) CdCl2 treated; and 4) probiotics
+ CdCl2 treated.
The cadmium concentrations were obtained in the blood, liver, kidney, and feces, as
well as the blood alanine aminotransferase (ALT), aspartate aminotransferase (AST),
adrenaline and noradrenaline activities. Furthermore, histomorphological changes of liver and
kidney were determined, as well as the level of total supeoxide dismutase (SOD) and catalase
(CAT) activities, and zinc and cooper concentration. Finally, cadmium genotoxicity were
determined both in vitro and in vivo by Comet test, as well as the alterations in the fecal
microflora content.
Based on our results we can conclude that:
1. Exposure of rats to cadmium chloride (70ppm) alone resulted in significant decrease
in body weight gain compared with the control group. The body weight in co-treated
animals was increased compared with the cadmium treated group. Overall, there were
not significant changes between all of the experimental groups in both food and water
intake.
2. The present results revealed that probiotics in a mixture with cadmium acted
beneficially to an organism, increasing the cadmium concentration in feces, and
consequently decreasing its concentration in blood and both liver and kidney. On the
other hand, administration of cadmium alone caused a significant increase of zinc
concentrations in both organs, kidney and liver.
3. Administration of cadmium showed alterations in histopathological changes in liver.
Parenchyma of liver was evidently changed after Cd-treatment. Sinusoids were
dilated, especially in the proximity of the central vein, and hepatocytes often contain
nuclei with compacted chromatin, while nucleoli were not visible. Kupffer cells
showed morphological characteristics of activation. At the same time, in co-treated
rats the degenerative changes of liver parenchyma are extenuated but still evident.
4. It was showed in this study that administration of cadmium increased the levels of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of the
cadmium treated animals. On the other hand, adding probiotics simultaneously with
cadmium chloride restored the altered values to the normal level.
5. The histopathological changes in kidneys of rats were also evaluated. In the cadmium
treated group, proximal tubules epithelium consists of swollen or necrotic cells,
necrosis of the whole tubules was reported, there is no visible pas-positive brush
border, nor inducing glomerular shrinkage. At the same time, in co-treated rats the
degenerative changes of kidney are extenuated. There were no differences observed
between the probiotics and the control group.
6. The activities of antioxidant enzymes such as total superoxide dismutase (SOD) and
catalase (CAT) were measured in. The activity of CAT decreased in both liver and
kidney in the cadmium treated animals, with no changes in the co-treated group
compared to the control group. On the other hand, pattern of SOD activity changes
differ between liver and kidney: in liver, cadmium causes SOD activity increase,
while in kidney this enzyme activity decreases. However, in the co-treated animals
SOD activities back to control level.
7. Adrenaline and noradrenaline were significantly increased in animals treated with
CdCl2 compared to the controls, while probiotics alone did not affect the amount of
these catecholamines. At the same time, levels of both adrenaline and noradrenaline
decreased in the co-treated animals.
8. The treatment by cadmium chloride resulted in significant increase of DNA damage
in both exposures in vitro and in vivo. On the contrary, cadmium given
simultaneously with probiotics produced a significant decrease in genotoxicity which
was induced by cadmium.
9. Administration of cadmium resulted in significantly reduced numbers of all different
types of microflora which were accounted for in this study. At the same time, adding
probiotic to Cd resulted in increased only of lactobacilli comparing to Cd treated
animals.
10. Overall, present results indicate that probiotics actively contribute in cadmium
removal from an organism, probably by binding to their bacterial cell wall, as
proposed earlier.
11. Further researches are needed to define specific probiotic combination that would be
most effective in binding Cd2+ and other heavy metals, as well as different toxins and
carcinogens. | true |
Fiziološki i morfološki efekti ekspresije AtCKX gena u transformisanim biljkama krompira (Solanum tuberosum L.) gajenim in vitro | Physiological and morphological effects of expression of AtcKX genes in transformed potato (Solanum tuberosum l.) plants grown in vitro | Krompir (Solanum tuberosum L.) je po značaju četvrta poljoprivredna kultura
na svetu gajena za potrebe ljudske ishrane. Ekonomski značaj krompira potiče od
krtola, jestivih podzemnih modifikacija bočnih izdanaka koji omogućavaju
nakupljanje rezervi skroba. Brojna istraživanja potvrdila su da biljni hormoni
citokinini
igraju
značajnu
ulogu
u
kontroli
tuberizacije.
Citokinin
oksidaza/dehidrogenaza (CKX) je ključni enzim katabolizma citokinina kod
biljaka, a transgene biljke koje eksprimiraju gene za ovaj enzim predstavljaju
dragocen model sistem za izučavanje citokininske homeostaze. Krompir (sorta
"Désirée") transformisan je genima AtCKX1 i AtCKX2 poreklom iz Arabidopsis
thaliana u cilju utvrđivanja efekata promenjene citokininske homeostaze na
morfološke i fiziološke parametre transformisanih biljaka krompira gajenih in vitro,
a naročito na proces tuberizacije. Dobijene su dve AtCKX1 i tri AtCKX2 linije sa
visokim nivoom ekspresije transgena, odnosno aktivosti CKX u proteinskim
ekstraktima izdanaka i korenova. Ekspresija gena AtCKX1 i AtCKX2 dovela je do
značajnih promena u hormonskoj homeostazi transformisanih biljaka krompira
gajenih in vitro, ali ne i do snižavanja nivoa ukupnih endogenih citokinina. Umesto
toga, kod većine transformisanih linija dolazi do značajnog sniženja nivoa
bioaktivnih citokinina – slobodnih baza i ribozida, kao i citokininskih nukleotida.
Pored toga, značajno je promenjen i nivo endogene indol-sirćetne kiseline (IAA).
Izmenjena hormonska homeostaza odražava se na morfologiju biljaka, kao i na
procese vezane za tuberizaciju, pa kod pojedinih transformisanih linija dolazi do
izmena u regulaciji tuberizacije in vitro, kako u uslovima dugog dana, tako i u
kontinuiranom mraku. Kod tri od pet transformisanih linija došlo je do
prevazilaženja inhibitornog uticaja svetlosti na indukciju krtola, pa su se krtole
pojavile već u toku prvih 30 dana pri uslovima dugog dana, što se nikada ne
događa kod kontrolnih biljaka ove sorte krompira. Transformisane AtCKX biljke
formirale su u proseku manji broj krtola po izdanku nego kontrolne biljke, što
ukazuje na smanjenu inicijaciju krtola kod biljaka sa sniženim nivoima bioaktivnih
citokinina, a može biti posledica povećanog odnosa između endogene IAA i
bioaktivnih citokinina. Efekat na inicijaciju krtola je mnogo izraženiji u uslovima
kontinuiranog mraka, nego dugog dana. Prečnik i masa krtola kod većine
transformisanih linija se nisu značajno razlikovali od vrednosti kod kontrolnih
biljaka. Samo jedna od pet transformisanih linija, AtCKX1-36a imala je u uslovima
dugog dana krtole manjeg prečnika i mase. Citokinini različito utiču na pojedine
faze u procesu tuberizacije kod krompira. Sniženje nivoa bioaktivnih citokinina
dovodi do ranije indukcije krtola kod AtCKX biljaka, ali ovaj efekat nije ujedno
praćen stimulatornim efektima na inicijaciju i uvećavanje krtola. Potrebna su dalja
istraživanja transgenih biljaka koje bi eksprimirale kako kataboličke (AtCKX) tako i
gene biosinteze citokinina (ipt), kako bismo identifikovali regulatorne mehanizme
pomoću kojih citokinini utiču na različite faze procesa tuberizacije kako u
indukujućim, tako i u neindukujućim uslovima. | Potato (Solanum tuberosum L.) is the world's fourth most important food
crop. Its economical importance arises from its potential to develop tubers, edible
storage organs derived from modified underground lateral shoots, which
accumulate starch. Extensive research has provided evidence that plant hormones
cytokinins have an important role in control of tuberization. Cytokinin
oxidase/dehydrogenase (CKX) is the key enzyme of cytokinin catabolism in
plants, thus transgenic plants expressing CKX genes have provided a valuable
model system for the research of cytokinin homeostasis. Potato (cv. "Désirée")
plants have been transformed with AtCKX1 and AtCKX2 genes from Arabidopsis
thaliana, in order to determine the effects of altered cytokinin homeostasis on the
morphological and physiological features of the transformed potato plants grown
in vitro, with particular emphasis on the tuberization process. A high level of
transgene expression and/or CKX activity in shoots and roots have been
confirmed in two AtCKX1 and three AtCKX2 lines. The expression of AtCKX genes
has reflected on significant changes in the hormonal homeostasis of the
transformed potato plants grown in vitro, but did not lead to the decrease of total
endogenous cytokinin levels. However in the majority of the AtCKX lines, the
levels of bioactive cytokinins (nucleobases and ribosides), as well as cytokinin
nucleotides, were significantly decreased. In addition, the level of endogenous
indole-3-acetic acid (IAA) was also significantly changed. The altered hormonal
homeostasis reflected on the morphological level, including the tuberization
process. Tuberization was altered in AtCKX potato lines both in long-day
conditions, and in continuous darkness. In three of the five AtCKX lines, the
overcoming of the inhibitory effect of light on tuber induction has been observed.
In these lines, tubers appeared already during the first 30 days in long-day
conditions, which has never been observed in control plants of this potato cultivar.
The AtCKX plants developed less tubers per shoot in comparison to control plants,
indicating decreased tuber initiation in plants with lower endogenous levels of
bioactive cytokinins. It is possible that this was a consequence of the increased
ratio of IAA vs. bioactive cytokinins. The effect on tuber initiation was more
pronounced in continuous darkness, than in long-day conditions. Tuber diameter
and mass did not significantly differ from control values in the majority of
transformed AtCKX lines. Only one of the five transformed lines, AtCKX1-36a
developed tubers of smaller diameter and mass. Cytokinins have different effects
on particular steps in the tuberization process in potato. Lower bioactive cytokinin
levels lead to early tuber induction in AtCKX plants, but this effect is not followed
by enhanced tuber initiation and growth. Further research on potato plants that
would express either cytokinin catabolism (AtCKX) or biosynthesis (ipt)
transgenes, is required to identify the regulation mechanisms by which cytokinins
affect particular steps of the tuberization process in both tuber-inducing and non-
inducing conditions. | true |
Uticaj B vitaminskih kompleksa i ribavirina na odgovor nervnih i glijskih ćelija i oporavak motornih funkcija nakon povrede mozga pacova | The effect of vitamin B complexes and ribavirin on response of neuronal and glial cells and recovery of motor function after injury to the rat brain | Traumatska povreda mozga (TPM) predstavlja veliki zdravstveni problem
savremenog društva jer je vodeći uzrok smrti i invaliditeta kod mlađih osoba. Uprkos
znatnim istraživačkim i kliničkim naporima, još uvek ne postoji ni jedan dovoljno
uspešan terapijski pristup koji utiče na sve patofiziološke procese koji prate moždanu
traumu, odnosno, koji može ukloniti sve njene posledice. Stoga se uspešan tretman u
borbi protiv TPM danas pokušava pronaći kombinacijom različitih terapijskih
tretmana. Poznato je da je oporavak izgubljene funkcije nakon povrede mozga odraslih
veoma spor i nepotpun usled nepovratnog uništenja nervnih ćelija i ograničene
mogućnosti remodelovanja postojećih i formiranja novih neuronskih veza. Glavna
prepreka za uspešniji oporavak posle oštećenja je nepermisivna sredina glijskog ožiljka
koji se formira nakon povrede, a koji sprečava izrastanje novih nervnih vlakana i
uspostavljanje novih neuronskih veza. Glijski ožiljak čine gusta mreža glijskih ćelija
(astrocita i oligodendrocitnih prekursorskih ćelija) i nepropustan vanćelijski matriks
(ECM). Cilj ove studije bio je da se ispita dejstvo kompleksa vitamina B (B1, B2, B3, B6 i
B12) i ribavirina (RBV, purinskog nukleozidnog analoga) na oporavak motornih
funkcija, posle jednostrane ablacije senzomotorne kore (ASK) prednjeg mozga pacova.
Mužjacima Wistar soja, starim 2.5 meseca, urađena je ASK prema sledećim
koordinatama: 2mm ispred, 4 mm iza bregme i 4 mm lateralno od središnje linije u
dubinu do pojave bele mase mozga. Operisane životinje podeljene su, prema tretmanu
nakon ASK, u 6 grupa: L grupa – tretirana fiziološkim rastvorom; LK1 grupa –
tretirana kombinacijom vitamina B1 (33 mg/kg/dan), B6 (33 mg/kg/dan) i B12 (0,5
μg/kg/dan); LK2 grupa – tretirana kombinacijom vitamina B2 (7,5 mg/kg/dan) i B3
(500 mg/kg/dan); LK grupa – tretirana kombinacijom svih pet vitamina: B1, B2, B3, B6
i B12 u prethodno navedenim dozama; LR grupa – primala je RBV (30 mg/kg/dan);
LKR grupa – tretirana kombinacijom svih 5 B vitamina i RBV u prethodno navedenim
dozama. Prva injekcija B vitamina data je i.p. 15 minuta nakon ASK, a potom na svaka
24 sata tokom sledećih 14 dana, dok je RBV aplikovan od 3 dana nakon ASK, jednom
dnevno do kraja eksperimenta. Uticaj primenjenih tretmana na oporavak nakon ASK
praćen je testiranjem ponašanja (test prelaska preko grede - BW test), RT-PCR i
Western blot analizom, kao i različitim imunohistohemijskim metodama.
Ispitivano je dejstvo različitih kombinacija B vitamina i RBV na oporavak
koordinacije lokomocije nakon ASK. Pokazano je da primena B vitamina ubrzava
oporavak lokomotornih funkcija, ali sa različitom efikasnošću: LK ≥ LK2 > LK1, dok su
kombinacije B vitamina i RBV imale sledeću efikasnost: LK ≥ LKR > LR.
Jedan od ciljeva ovog istraživanja bio je da se utvrdi efekat različitih
kombinacija B vitamina i RBV na odgovor nervnih ćelija i na sinaptičku plastičnost.
Procesi plastičnosti praćeni su primenom specifičnih markera izrastanja aksona,
dendrita i formiranja sinapsi (GAP43, MAP2, sinaptofizin). Kompleks svih 5 B
vitamina (LK), a naročito njegova kombinacija sa RBV (LKR) imaju najizraženiji efekat.
Njihova primena podstakla je izrastanja aksona i dendrita, grananje dendrita kao i
formiranje novih sinapsi. To je verovatno doprinelo stvaranju novih neuronskih veza
čime se može objasniti zapaženi brži oporavak motorike zadnjeg kontralateralnog
ekstremiteta. S druge strane, B vitamini (pre svega kombinacija B2+B3), njihove
kombinacija sa RBV (LKR), kao i sam RBV (LR), pokazali su izrazito povoljan efekat u
očuvanju neurona, što se ogledalo kako u njihovoj morfologiji (imunohistohemijsko
bojenje sa antitelom na NF-H i SMI31), tako i u smanjenju broja degenerisanih neurona
(Fluoro-Jade B obeležavanje, koje boji samo degenerisane neurone).
Posebno je ispitivan uticaj kompleksa B vitamina i RBV na proces reaktivne
glioze i formiranje glijskog ožiljka u regionu oko mesta povrede. Pokazano je da
tretmani ovim supstancama smanjuju broj reaktivnih astrocita i oligodendrocitnih
prekursorskih ćelija i odlažu formiranja ožiljnog tkiva koje se intenzivno formira
između 7. i 14. dana posle ASK. Kao specifični markeri reaktivnih astrocita korišćeni su
GFAP, vimentin i S100B. U 14. danu posle operacije, pored povećanja broja reaktivnih
astrocita, uočeno je značajno povećanje ekspresije GFAP i vimentina u svim operisanim
grupama, kako na genskom, tako i na proteinskom nivou. Tretman različitim
kombinacijama B vitamina (a najviše LK), kao i RBV smanjio je broj reaktivnih
astrocita. Ekspresija S100B proteina takođe je značajno smanjena pod dejstvom
kompleksa B vitamina i RBV i to sa sledećom efikasnošću LKR >> LR > LK. Sva tri
tretmana dovode do značajnog smanjenja broja reaktivnih astrocita i menjaju njihovu
morfologiju ka mirujućoj formi astrocita.
Takođe, praćen je i odgovor komponenti ECM nakon povrede mozga.
Primenom antitela na hondroitin sulfatne proteoglikane – CSPG (4S i 6S CSPG,
fosfakan, neurokan i brevikan), koji su elementi ECM, uočeno je značajno povećanje
njihove ekspresije u širokom pojasu oko mesta lezije. Dvojnim imunofluorescentnim
bojenjem sa GFAP-om povećana ekspresija ovih CSPG uočava se oko reaktivnih
astrocita, koji ih i izlučuju. Tretman kombinacijom svih pet B vitamina nakon ASK
redukuje ekspresiju fosfakana, neurokana i brevikana na usku zonu oko mesta
povrede, što doprinosi povećanju permisivnosti moždanog parenhima i omogućava
izrastanje aksona i formiranje novih sinaptičkih veza, kao i uspostavljanju izgubljenih
funkcija.
Rezultati dobijeni u okviru ove doktorske teze pokazuju da primena ispitivanih
supstanci, iako one deluju posredstvom različitih mehanizama, poboljšava oporavak
nakon ASK. To ukazuje da bi kombinacija vitamina B (B1, B2, B3, B6i B12) i RBV mogla
da bude efikasna dopuna postojećih pristupa terapiji povrede mozga, što zahteva dalju
kliničku proveru. | Traumatic brain injury (TBI) is a major health problem in modern society, being
a leading cause of death and disability among young people. Despite huge basic
research and clinical efforts an effective treatment for TBI has not been established yet,
that will counteract all of pathophysiological processes accompanying brain trauma.
Therefore, a best possible present treatment that would act on several secondary
processes in TBI is based on a combination of different therapeutic approaches.
A recovery of lost functions after brain injury is slow and incomplete in adults,
due to the irreversible destruction of nerve cells and the limited ability of brain tissue
for remodeling of existing and formation of new synaptic connections. The main
obstacle for a successful recovery is existence of non-permissive glial scar, which
emerges after a brain trauma preventing axonal sprouting and the establishment of
new neural circuits. Glial scar consists of a dense network of glial cells (astrocytes and
oligodendrocyte precursor cells) and dense extracellular matrix (ECM). The aim of this
study was to explore the effects of vitamin B complex (B1, B2, B3, B6 and B12) and
ribavirin (RBV, a purine nucleoside analogue) on the recovery of motor function after
unilateral sensorimotor cortex ablation (SCA) of rat forebrain.
Two and a half-month old male Wistar rats underwent SCA at the following
coordinates: 2 mm before, 4 mm behind the bregma and 4 mm lateral to the midline,
and to the depth of the white matter. After the surgery, rats were randomly divided
according to treatment protocol into 6 groups: group L – treated with saline solution;
LC1 group - treated with a combination of vitamin B1 (33 mg/kg/day), B6 (33
mg/kg/day) and B12 (0,5 mg/kg/day); LC2 group - treated with a combination of
vitamin B2 (7.5 mg/kg/day) and B3 (500 mg/kg/day), LC group - treated with a
combination of all five vitamins: B1, B2, B3, B6 and B12 in the above mentioned doses,
LR group - received RBV (30 mg/kg/day); LCR group - treated with a combination of
5 B vitamins and RBV in the above doses. The first injection of vitamin B was given i.p.
15 minutes after the SCA, and then daily during next 14 days, while RBV was first
administered 3 days after the SCA, once daily until the end of the experimental
protocol. The effects of treatment on the recovery after SCA were followed by
behavioral tests (beam walking test - BW test), while molecular changes were detected
using RT-PCR, Western blot analysis and several immunohistochemical protocols.
We studied the effects of different combinations of B vitamins and RBV on
recovery of locomotor coordination after SCA. It is shown that administration of B
vitamins accelerates and improves coordination, with a treatment-specific efficacy: LC
≥ LC2>LC1, while the combination of B vitamins and RBV have the following
effectiveness: LC ≥ LCR> LR.
One of the goals of this study was to determine the effect of application of
different combinations of B vitamins and RBV on nerve cell responses and synaptic
plasticity. Plasticity and remodeling were monitored using specific markers for axon
and dendrites outgrowth and synapses formation (GAP43, MAP2, synaptophysin). The
complex of all 5 B vitamins (LC), and particularly its combination with RBV (LCR)
have the most pronounced effect. The LCR treatment induces sprouting of axons and
dendrites, dendritic branching and the formation of new synapses, which .probably
contributes to the establishment of new neural connections, and observed rapid
recovery of contralateral hind paw locomotor function. On the other hand, B vitamins
(especially the combination B2 + B3), their combination with RBV (LCR) and RBV
alone (LR) induced marked beneficial effects on nerve cells, as reflected both in their
morphology (immunostaining with antibodies against NF-H and SMI31), and reduced
number of degenerated neurons (Fluoro-Jade B labeling, stains only degenerated
neurons).
Additionally, the effect of vitamin B complex and RBV was investigated on the
process of reactive gliosis and glial scar formation in the region around the site of
injury. It is shown that application of the substances reduces the number of reactive
astrocytes and oligodendrocyte precursor cells and delays the formation of scar tissue
that is being formed 7 to 14 days after the SCA. GFAP, vimentin and S100B were used
as specific markers of reactive gliosis. At 14 days after the operation, in addition to the
increased number of reactive astrocytes, a marked increase in GFAP and vimentin
expression at both gene and protein level were observed in all experimental groups.
Administration of B vitamins (particularly LC) and RBV reduces the number of
reactive astrocytes and expression of S100B protein, with the following efficiency
LCR>>LR>LC. All treatments led to a significant reduction in the number of reactive
astrocytes and their transition to more resting-like morphology.
Furthermore, changes in the expression of ECM components after the injury
were studied by using the antibodies specific for chondroitin sulfate proteoglycans -
CSPG (CSPG 4S and 6S, phosphacan, neurocan and brevican), which are the main
elements of the ECM. Significant increase in the expression in a wide area around the
lesion was observed. Double immunofluorescence staining with GFAP revealed the
up-regulation of CSPG around the reactive astrocytes, which are known to secrete
them. The application of all B vitamins after SCA reduces the expression of
phosphacan, neurocan and brevican to a narrow zone surrounding the injury site,
thereby producing more permissive environment for neurite outgrowth and formation
of new synaptic connections that may contribute to the observed restoration of brain
functions.
The results obtained in this thesis show that the application of tested
substances, even though they act via different mechanisms, improves recovery after
SCA. This indicates that the cocktail of B (B1, B2, B3, B6, B12) vitamins in combination
with RBV could be an effective contribution to the existing therapeutic approaches in
the treatment of brain injuries, which certainly requires further clinical testing. | true |
Evoluciona ekofiziologija stresa : uloga enzimskih i neenzimskih antioksidanata u prirodnim populacijama Iris pumila L. (Iridaceae) | Evolutionary ecophysiology of stress: the role of enzymatic and non-enzymatic antioxidants in natural populations of Iris pumila L. (Iridaceae) | U evolucionoj biologiji sredinski stres se definiše kao mehanizam koji
dovodi do adaptacija i evolucije u promenljivoj sredini, i istovremeno uključuje
svojstva stresora, kao sredinsku komponentu i stresiranih, kao biološku
komponentu. Više biljke, kao modularni i sesilni organizmi, razvile su tokom
evolucije veliki broj biohemijskih, fizioloških i anatomsko-morfoloških
mehanizama koji im omogućuju povećanje individualne adaptivne vrednosti
pod nestabilnim uslovima životne sredine. Kratkoročno, odnosno u ekološkoj
vremenskoj skali, biljke se usaglašavaju sa variranjem biotičkih i abiotičkih
faktora u svojim staništima fleksibilnošću fenotipa, koja može da obuhvati sve
nivoe biološke organizacije - od molekularnog do morfološkog. Za prirodne
populacije biljaka, svetlost je jedan od prostorno i vremenski najvarijabilnijih
abiotičkih faktora životne sredine i, istovremeno, jedan od najvažnijih
sredinskih resursa. U tom kontekstu, od izuzetne važnosti su biohemijski
mehanizmi zaštite biljaka od oksidativnog stresa koje čine enzimski i
neenzimski antioksidanti.
Ciljevi ove doktorske disertacije su: (I) ispitivanje veličine i obrazaca
sezonske
fenotipske
plastičnosti
biohemijskih
(specifična
aktivnost
antioksidativnih enzima superoksid dismutaze – SOD, askorbat peroksidaze –
APX, katalaze – CAT, glutation reduktaze – GR, peroksidaza III klase - POD;
količina neenzimskih antioksidanata, antocijana – Anth i fenola - Phen),
morfološko-anatomskih (specifična površina lista - SLA i gustina stoma - SD) i
fizioloških osobina lista (sadržaj suve materije u listu – LDMC, sadržaj vode u
listu – LWC, relativni sadržaj vode u listu – RWC, sukulentnost – SU, količina
fotosintetičkih pigmenata: hlorofila a - Chl a, hlorofila b - Chl b i karotenoida -
Cars) biljaka I. pumila iz dve prirodne populacije, od kojih je jedna naseljavala
otvoreno (populacija „Dina“), a druga zasenčeno (populacija „Šuma“) stanište u
Deliblatskoj peščari; (II) testiranje hipoteze o lokalnim adaptacijama lista I.
pumila na abiotičke uslove otvorenog i zasenčenog staništa korišćenjem
recipročno-transplantacionog eksperimenta u prirodi; (III) utvrđivanje variranja
u stepenu (relativna varijansa sopstvene vrednosti, rVE) i obrascima fenotipske
integracije (CPC analiza) funkcionalno povezanih osobina lista I. pumila,
indukovanog heterogenošću životne sredine.
Obrazac sezonskog variranja funkcionalnih osobina lista kod biljaka
I. pumila iz populacija Dina i Šuma bio je specifičan za svaku osobinu, kao i za
svaku ispitivanu populaciju. Specifične aktivnosti većine antioksidativnih
enzima, kao i količina neenzimskih antioksidanata bile su najveće tokom
najstresnijeg, letnjeg perioda, koga karakterišu visok intenzitet svetlosti,
povišena temperatura vazduha i nedostatak vode u podlozi, u poređenju sa
drugim godišnjim dobima kao što su proleće i jesen. Tokom cele vegetacione
sezone biljke iz populacije Šuma su imale više aktivnosti antioksidativnih
enzima u odnosu na biljke iz populacije Dina, dok je suprotan trend utvrđen za
neenzimske antioksidante, naročito antocijane, čija je količina po pravilu bila
veća u listovima biljaka sa otvorenog staništa. Pored variranja u aktivnosti i
količini antioksidanata, u populaciji Dina uočeno je i smanjenje količine
hlorofila, kao jedan od mehanizama kojim biljke I. pumila sa otvorenog staništa
regulišu nastanak reaktivnih vrsta kiseonika u hloroplastima.
Testiranje
adaptivnosti
ispitivanih
biohemijskih,
fizioloških
i
morfološko-anatomskih osobina lista I. pumila pokazalo je da se srednje
vrednosti većine ovih osobina nisu statistički značajno razlikovale između
genotipova iz populacija Dina i Šuma ni na otvorenom kao ni na zasenčenom
staništu. Odsustvo statistički značajnih među-populacionih razlika kod I. pumila
na istom staništu ukazuje da se populacije Dina i Šuma nisu genetički
diferencirale pod uticajem lokalne selekcije u njihovim svetlosnim sredinama.
Dakle, pošto način variranja testiranih osobina lista (indukovanog različitim
intenzitetima ambijentalne svetlosti) povećava performansu biljaka I. pumila
pod datim sredinskim uslovima, ovaj tip promene fenotipa mogao bi se
definisati kao adaptivna plastičnost.
Veličina fenotipske plastičnosti u odnosu na svetlost, procenjena na
osnovu indeksa plastičnosti, bila je specifična za svaki tip analiziranih osobina
lista. Antioksidanti i fotosintetički pigmenti ispoljavali su veću fenotipsku
plastičnost u odnosu na morfološko-anatomske i fiziološke osobine.
Nepostojanje statistički značajnih razlika u plastičnosti većine analiziranih
osobina lista, između populacija sa otvorenog i zasenčenog staništa, ukazuje da
genotipovi I. pumila različitog porekla „reaguju“ na variranje intenziteta
svetlosti formiranjem sličnih, adaptivnih fenotipova, sprečevajući na taj način
delovanje lokalne selekcije na fenotipsku plastičnost ispitivanih osobina.
Poznato je iz literaturnih podataka da porast intenziteta sredinskog
stresa može da dovede do povećanja integrisanosti funkcionalno povezanih
osobina ili modula organizama, što je takođe potvrđeno i u slučaju I. pumila.
Najveći broj korelacija između analiziranih osobina lista konstatovan je u
najnepovoljnijem, letnjem periodu kod biljaka iz obe populacije, odnosno pod
uslovima povećanog intenziteta abiotičkog stresa. Broj i veličina koeficijenata
fenotipskih korelacija, bili su najveći u grupi osobina koje čine antioksidanti,
kao i u grupi osobina vezanih za fotosintetičke pigmente, što navodi na
zaključak da bi se ove dve grupe osobina mogle tretirati kao posebni
funkcionalni moduli unutar lista. | In evolutionary biology, environmental stress is defined as a mechanism
that leads to adaptation and evolution in a variable environment and includes
properties of stress, as an environmental component, and stressed, as the
biological component. During the evolution higher plants, as sessile and
modular organisms, have developed a number of biochemical, physiological
and morpho-anatomical mechanisms that enable them to increase individual
fitness under unstable environmental conditions. Short-term, in ecological time
scale, plants adjust to varying biotic and abiotic factors in their habitat by
phenotype flexibility, which may include all levels of biological organization -
from the molecular to the morphological. For natural populations of plants,
light is the most heterogeneous abiotic factor, both spatially and temporally,
and at the same time, one of the most important environmental resources. In
this context, antioxidative enzymes and non-enzymatic antioxidants, as specific
biochemical mechanisms, are of grat importance for plants protection against
oxidative stress.
The aims of this doctoral dissertation were: (I) to investigate the size and
patterns of seasonal phenotypic plasticity of biochemical (specific activities of
antioxidative enzymes superoxide dismutase - SOD; ascorbate peroxidase –
APX, catalase – CAT, gluthatione reductase - GR, class III peroxidase – POD;
content of non-enzymatic antioxidants, anthocyanins - Anth and phenolics -
Phen), morpho-anatomical (specific leaf area - SLA, stomatal density - SD) and
physiological leaf traits (leaf dry matter content - LDMC, leaf water content -
LWC, relative leaf water content - RWC, succulence – SU, concentration of
photosynthetic pigments: chlorophyll a - Chl a, chlorophyll b - Chl b and
carotenoids - Cars) in two I. pumila populations, one inhabiting open site
(“Dune” population) and the other one from woodland understory (“Wood”
population) in Deliblato sand; (II) testing the local adaptation hypothesis of
I.pumila to abiotic conditions at the open and shaded habitat, using reciprocal
transplant experiment in nature (III) to determine the variation of degree
(relative variance of eigen-value, rVE) and patterns of phenotypic integration
(CPC analysis) of functionally linked traits in the I.pumila leaves, induced by
heterogeneous environment.
The seasonal variation pattern of functional leaf traits of I.pumila from
two populations, Dune and Wood, was specific for each trait, as well as for the
population tested. Specific activities of antioxidative enzymes and content of
non-enzymatic antioxidants were the highest during the most stressful, summer
period, with high light intensities, elevated atmospheric temperature and water
deficit, in comparison to other seasons, such as spring or autumn. During the
whole vegetation period, plants from the Wood population had higher
antioxidative enzymes activities than those from the Dune population, while an
opposite trend was observed for non-enzymatic antioxidants, especially
anthocyanins, whose content was, in general, higher in the leaves of plants from
the open habitat. Besides the variation in the activities and contents of
antioxidants, a decrease in the chlorophyll concentration was observed in plants
from Dune population, which could be a means for regulating reactive oxygen
species generation in the chloroplasts of these plants.
Testing the adaptability of the biochemical, physiological and morpho-
anatomical leaf traits in the I. pumila showed that average values for most of the
traits investigated did not differ significantly between the Dune and Wood
population, neither in the open nor in the shaded habitat. No statistically
significant differences among I. pumila populations at the same habitat
implicate there was no genetic differentiation between Dune and Wood
populations due to local selection in their light environments. Therefore, as the
pattern of variation of tested leaf traits (induced by different intensities of
ambient light) increases the performance of I. pumila under the given
environmental conditions, this type of phenotypic changes could be defined as
adaptive plasticity.
The size of light-induced phenotypic plasticity, estimated by the
plasticity index, was specific for each type of leaf traits considered.
Antioxidants and photosynthetic pigments showed higher phenotypic plasticity
compared to morpho-anatomical and physiological traits. The absence of
statistical significance in the plasticity of the most of the leaf traits analyzed
indicates that I. pumila genotypes of different origin respond to light intensity
variation by forming similar, adaptive phenotypes, thus preventing the effect of
local selection on phenotypic plasticity of the investigated leaf trait.
According to the literature data, increased environmental stress intensity
can be followed by an increase in the integrity of functionally related traits or
organism modules, which was also confirmed in I.pumila. The highest number
of correlation between analyzed leaf traits was obtained in the least favourable,
summer period in the plants from both populations, that is, under increased
intensity of abiotic stress. The size and number of phenotypic correlation
coefficients were the highest in the group of antioxidants, as well as in the
group of traits related to photosynthetic pigments, leading to the conclusion
that these two groups of traits could be treated as separate leaf functional
modules. | true |
Uticaj propofola na signalni put neurotrofina u prednjem mozgu pacova starih 14 dana | Propofol-induced changes in neurotrophic signaling in 14-day-old rat forebrain | Neurotrofini čine familiju signalnih molekula sa brojnim uticajima na rast,
preživljavanje, diferencijaciju i sinaptičku plastičnost postmitotskih neurona, kako u
adultnom mozgu, tako i u mozgu tokom razvića. Neurotrofini (BDNF, NGF) se sa
određenim afinitetom vezuju za Trk receptore, iniciraju njihovu dimerizaciju i
autofosforilaciju i na taj način se pokreće aktivacija signalnih puteva kao što su
PI3K/Akt i MAPK/ERK. Akt i ERK kinaze imaju ključne regulatorne uloge u mozgu u
procesima kao što su neuronalna proliferacija, diferencijacija, razviće, migracija,
preživljavanje i dugotrajna sinaptička plastičnost. Aktivan (fosforilisan) Akt štiti ćeliju
od apoptoze tako što stimuliše ekspresiju proteina koji favorizuju preživljavanje ćelija i
sa druge strane inhibira egzekutorske kaspaze. Aktivacija ERK1/2 kinaze pomoviše
preživljavanje, međutim u određenim uslovima ERK1/2 može imati i pro-apoptotske
odlike. S obzirom da je uloga neurotrofina u neurotoksičnosti koja je indukovana
anestezijom tokom ranog postnatalnog razvića pokazana u nekoliko studija, cilj ove
doktorske disertacije je bio da se na molekulskom nivou ispitaju promene u signalnom
putu neurotrofina koje nastaju u kori i talamusu postnatalnih pacova nakon primene
anestetske doze propofola, kao i da se utvrdi potencijal tretmana da indukuje ćelijsku
smrt i/ili promene u sinaptičkoj plastičnosti.
Nakon jednokratne primene propofola (25 mg/kg i.p.) ispitana je vremenska i
prostorna ekspresija neurotrofina BDNF i NGF, njihovih aktiviranih receptora TrkB,
TrkA i p75 receptora, kao i nishodnih kinaza Akt i ERK kod 14 dana starih (PND14)
pacova. Ispitan je i potencijal propofolskog tretman da indukuje ćelijsku smrt,
praćenjem ekspresije TNF-α, TNFR1, aktivnog fragmenta kaspaze-3, njegovog
inhibitora XIAP proteina, kao i transkripcionog faktora NFκB. Finalno je ispitivan
potencijal propofolskog tretmana da indukuje promene u ekspresiji markera sinaptičke
plastičnosti (MAP-2, drebrina, GAP-43, sinaptofizina, sinukleina-1 i N-kadherina).
Promene su praćene u kori i talamusu kao primarnim ciljevima dejstva anestetskog
delovanja. Primenom sledećih metoda Western blot analize, RT- i PCR-a u realnom
vremenu ispitivane su promene u ekspresiji ciljnih proteina i iRNK, a primenom Fluoro-
žad B histološkog bojenja analizirana je pojava neurona u degeneraciji.
Dobijeni rezultati su pokazali da je propofolski tretman doveo do značajnih
promena u nivoima ispitivanih neurotrofina, njihovih receptora i nishodnih efektorskih
kinaza. Ove promene nisu povezane sa povećanom neurodegeneracijom ni u kori, ni u
talamusu, ali su detektovane značajne promene u zastupljenosti ispitivanih markera
sinaptičke plastičnosti. Naši podaci pokazuju da je ekspresija neurotrofina BDNF i NGF
različito regulisana u odgovoru na propofolski tretman i da se najznačajnije promene u
PND14 mozgu odigravaju preko BDNF signalnog puta i u znatno manjoj meri preko
NGF signalnog puta. Smatramo da prekomerna ekspresjia BDNF-a u našem
eksperimentalnom modelu nije isključivo ograničena na neuronalno preživljavanje, već
i na određene aspekte sinaptičke plastičnosti. Pored promene u ekspresiji neurotrofina,
propofolski tretman je indukovao promene u ekspresiji Akt i ERK kinaza na regionalno-
specifičan način. Fluoro-žad B histohemijsko bojenje nije pokazalo neuronalnu
degeneraciju, uprkos prolaznom povećanju u ekspresiji aktivnog fragmenta kaspaze-3.
Detektovane su značajne regionalno-specifične promene u ekspresiji markera sinaptičke
plastičnosti nakon propofolskog tretmana.
Možemo zaključiti da opšti anestetik propofol ispoljava kompleksne efekte na
neurotrofine i njihove nishodne signalne puteve u mozgu pacova tokom ranog
postnatalnog razvića. Dobijeni rezultati ukazuju da u mozgu PND14 životinja aktivacija
Akt i ERK signalnih puteva predstavlja važan aspekt endogenih odbrambenih
mehanizama kojim se mladi mozak štiti od potencijalnih neželjenih efekata izazvanih
jednokratnom primenom propofola. Balansirana ekspresija ove dve kinaze u istom
regionu mozga je važna za odgovarajući izlazni signal i povoljan fiziološki ishod.
Preciznije razjašnjenje molekulskih mehanizama koji se pokreću nakon izlaganja
mladih eksperimentalnih životinja opštoj anesteziji može pomoći u boljem razumevanju
starosno-zavisne komponente neurotoksičnosti i neuroplastičnosti koje izaziva primena
opštih anestetika u kliničkoj pedijatriji. | The neurotrophins are a family of secreted proteins that mediate numerous
functions in both the developing and mature nervous system, including growth, survival,
differentiation and synaptic plasticity of postmitotic neurons. The binding of
neurotrophins (BDNF, NGF) to Trk receptors induces their dimerization which is
followed by autophosphorylation of tyrosine residues within the intracellular kinase
domain, that leads to the activation of signaling pathways such as the PI3K/Akt and
MAPK/ERK pathways. Akt and ERK kinases play a crucial role in regulating various
processes in the brain, including neuronal proliferation, differentiation, development,
migration, survival and long-term synaptic plasticity. Phosphorylated Akt can protect
cells from apoptosis via stimulation of the expression of proteins that favor cell survival
and by inhibiting executor caspases. Activation of ERK1/2 generally promotes cell
survival, although under certain conditions, ERK1/2 can possess proapoptotic
properties. Since several studies have revealed a role for neurotrophins in anesthesia-
induced neurotoxicity in the developing brain, the goal of this study was to explore the
potential of anesthetic dose of propofol to influence neurotrophic signaling pathway in
the cortex and thalamus of 14-day-old (PND14) rats, and also to investigate whether
same treatment is able to induce neurodegeneration and/or changes in synaptic
plasticity.
In this study we monitored the spatial and temporal expression of neurotrophic
signaling molecules (BDNF, NGF, TrkA, TrkB, p75, Akt and ERK) in the brain of
PND14 Wistar rats after the application of a single propofol dose (25 mg/kg i.p). The
potential of propofol treatment to induce cell death was also examined, following the
expression of TNF-α, TNFR1, cleaved caspase-3 fragment, XIAP and NFκB. Finally,
we investigated the changes in the markers of synaptic plasticity (MAP-2, drebrin,
GAP-43, synaptophysin, synuclein-1 and N-cadherin) after the propofol treatment. The
structures of interest were the cortex and thalamus as the primary areas of anesthetic
actions. Changes of the levels of mRNA and proteins of interest were assessed by RT-
or Real Time PCR and Western immunoblot analysis at different time points during the
first 24 h after the treatment. Fluoro-Jade B staining was used to follow the appearance
of degenerating neurons.
The obtained results show that the treatment caused marked alterations in levels
of the examined neurotrophins, their receptors and downstream effector kinases. These
changes were not associated with increased neurodegeneration in either the cortex or the
thalamus, however significant changes in the expression of synaptic plasticity markers
was observed. Our results show that the expression of BDNF and NGF is differentially
regulated after propofol treatment. We consider that BDNF overexpression in our
experimental model is not strictly limited on neuronal survival, but also on some
specific aspects of synaptic plasticity. Propofol treatment induced region-specific
changes in the expression of Akt and ERK kinases. These results indicate that in the
brain of PND14 rats, the interaction between Akt/ERK signaling might be one of
important part of endogenous defense mechanisms which the developing brain utilizes
to protect itself from potential anesthesia-induced damage. Even though the expression
of cleaved caspase-3 fragment was detected in both examined structures, Fluoro-Jade B
staining did not reveal neurodegeneration. Nevertheless, significant region-specific
changes in the expression of synaptic plasticity markers was observed. We can conclude
that general anesthetic propofol exerts complex effects on neurotrophic signaling
pathways in the postnatal rat brain. Together, our results contribute towards an
improved understanding of the age-dependent component of anesthesia-induced
neurotoxicity and neuroplasticity; they also bare potential clinical implications. | true |
Invazivnost alohtonih vrsta makroinvertebrata i riba Dunava | Invasiveness of allochthonous macroinvertebrate and fish species of the Danube river | Istraživanje alohtone faune makroinvertebrata i riba Dunava rađeno je na osnovu
uzoraka prikupljenih tokom avgusta i septembra 2007. i 2013. godine. U radu je dat
detaljan pregled alohtone komponente sa tri aspekta: taksonomska i zoogeografska
analiza, testiranje indeksa za ocenu pritisaka i procena invazibilnosti područja, kao i
korelacija nivoa bioloških invazija i izabranih parametara pritiska. Posebno je obrađen
predlog mera za smanjenje stope unosa i širenja ranije već introdukovanih alohtonih
vrsta, kao i za smanjenje pritiska.
Analizom materijala zabeležena je 31 alohtona vrsta makroinvertebrata. Najveći
broj vrsta pripada rakovima – grupa Malacostraca, poreklom iz Ponto-kaspijskog
regiona. Analizom uzoraka ribljih zajednica ukupno je registrovano 13 alohtonih vrsta
poreklom iz tri biogeografske regije čiji su predstavnici podjednako zastupljeni: Ponto-
kaspijske, Severno-američke i Azijske.
Razmatranjem načina unosa i sredstava kojima su vrste raširene van granica
nativnog areala, možemo konstatovati da je za mali broj vrsta makroinvertebrata
dokazano da je u širenju areala bilo neposrednog uticaja antopogene aktivnosti, dok je
za disperziju riba antropogena aktivnost dominantna.
Prisustvo alohtone faune po sektorima Dunava je ujednačeno i za
makroinvertebrate i za ribe, sa značajnim opterećenjem alohtonom faunom u gornjem i
srednjem delu toka. Osam vrsta makroinvertebrata i sedam vrsta riba ima visok
potencijal za širenjem areala, uspostavljanjem populacija i visok potencijal da
prouzrokuje ekološke i negativne socio-ekonomske usled čega su stavljene na Crnu
listu.
Visok nivo biološkog zagađenja procenjen je za sedam vrsta makroinvertebrata i
tri vrste riba: Chelicorophium curvispinum, Corbicula fluminea, Dikerogammarus
villosus, Dreissena polymorpha, Dreissena bugensis, Pectinatella magnifica,
Sinanodonta woodiana, Carassius gibelio, Lepomis gibosus i Neogobius melanostomus.
Na osnovu ukupne ocene invazibilnosti područja glavnog toka Dunava u Srbiji
dobijene na osnovu izabranih parametara za koje se pretpostavlja da doprinose
intenzitetu bioloških invazija možemo zaključiti da je sektor od Beograda do brane
„Đerdap I“ najosetljiviji na biološko zagađenje, kao i lokalitet nizvodno od Novog
Sada. Na sektor od Beograda do brane „Đerdap I“ najveći uticaj na visoku klasu
invazibilnosti ima prvenstveno intezivan rečni saobraćaj, kao i nivo hidromorfoloških
promena.
Predložene aktivnosti u borbi protiv invazivnih vrsta obuhvataju: definisanje i
usvajanje odgovarajuće zakonske regulative, popis unešenih alohtonih vrsta, procena
stepena njihove invazivnosti, definisanje indikatora stanja i upoznavanje javnosti sa
problemom širenja invazivnih vrsta i podizanje javne svesti građana. | Investigation of allochthonous macroinvertebrates and fish species of the
Danube River was based on samples collected during August and September 2007 and
2013. Detailed overview of allochthonous species was presented regarding: taxonomic
and zoogeographic analyses, testing of indices and risk assessment procedure and
invasiveness of the area, as well as correlation of the level of biological invasions and
selected parameters of pressures. Further steps for reduction of new invasions,
spreading already established allochthonous species and reduction of pressures were
pointed separately.
In total, 31 allochthonous macroinvertebrate species were registered. The most
numerous were Crustaceans – group Malacostraca, originate from Ponto-caspian region.
Among fish, 11 allochthonous species were detected from three different biogeographic
region: Ponto-caspian, North American and Asian.
Regarding the mode of arrival and pathway we may conclude that the large
majority of non-indigenous fish species and only few macroinvertebrate species were
introduced as a direct effect of human intervention.
Analysis of allochthonous fauna showed that Upper and Middle sectors of the
Danube are most affected. Due to high potential to spread, high potential for
establishment in new environment and high potential to cause ecological and negative
socio-economic impacts, eight macroinvertebrate and seven fish species were specified
as Black list alien species.
For seven macroinvertebrate and three fish species high level of Biological
contamination Index was estimated: Chelicorophium curvispinum, Corbicula fluminea,
Dikerogammarus villosus, Dreissena polymorpha, Dreissena bugensis, Pectinatella
magnifica, Sinanodonta woodiana, Carassius gibelio, Lepomis gibosus i Neogobius
melanostomus.
Based on the overall analyses of selected parameters assumed that are important
for intensity of biological invasions, we may conclude that part of the Danube stretch
between Belgrade and Iron Gate I dam, as well as locality downstream Novi Sad are
most affected regarding biological contamination. High class of invisibility was
determined due to high intensity of water transport, as well as the level of
hydromorphological changes.
Activites regarding invasive species recommended in this thesis are: definition
of effective national legislation, creation of the National list of allochthonous species,
assessment of their invasiveness, list of indicators of current status of selected water
bodies, to increase public awareness about invasive species. | true |
Imunofenotipska karakterizacija ćelija akutne mijeloidne leukemije kod odraslih i njena uloga u dijagnozi, praćenju i prognozi bolesti | Immunophenotypic characterization of acute myeloid leukemia in adults and its role in the diagnosis,monitoring, and prognosis of the disease | U tezi su ispitivani savremeni aspekti primene imunofenotipizacije
multiparametarskom protočnom citofluorimetrijom (IMPC) u dijagnostici i
praćenju AML. Ispitivanjem je obuhvaćeno 320 odraslih bolesnika sa de novo
AML, od kojih je 294 uključeno u retrospektivno ispitivanje (dijagnoza,
klasifikacija i prognoza), dok je ostalih 26 bolesnika bilo uključeno u
prospektivno ispitivanje (praćenje minimalne rezidualne bolesti - MRB).
Bolesnici su klasifikovani kao AML-neklasifikovana (48%), AML sa
rekurentnim genetskim poremećajima (37,4%) i AML sa znacima
mijelodisplazije (14,6%). IPCM omogućava postavljanje dg AML analizom ks
kod 98% bolesnika. Ispitivanjem 44 različita hLDM utvrđeno je da je njihova
ekspresija na blastima deregulisana, o čemu govore aberacije u njihovoj
ekspresiji kod svakog bolesnika. Heterogen ćelijski sastav populacije
leukemijskih ćelija (leukemijski blasti i prekursori) utvrđen je kod 55%
bolesnika sa AML. Imunološka i citomorfološka klasfikacija AML su saglasne
kod 73% bolesnika, odnosno imunološka i SZO klasifikacija kod 68%
bolesnika. Ispitivanje MRB sprovedeno je primenom jedne (42%) ili dve
(58%) kombinacije IFSL po bolesniku. Primenom IMPC, pokazana je visoka
učestalost MRB kod naših bolesnika sa AML posle lečenja indukcionom
(69%) odnosno konsolidacionom terapijom (50%). Nivo MRB u ks bolesnika
≥0,1% NĆ posle indukcione terapije, svrstava bolesnike u grupu visokog
rizika za razvoj relapsa bolesti. Ispitivanje prognostičkog značaja hLDM kod
bolesnika sa AML, pokazalo je značajnu vezu između rane monocitne
diferencijacije leukemijskih ćelija, ekspresije CD22 molekula i pojave rane
smrti i (p<0,05), odnosno niže incidence kompletne remisije (p<0,05).
Multivarijantnom analizom je pokazano da je obrazac ekspresije molekula
CD45+low/med na blastima, povezan sa dužim trajanjem KR (p=0,002), dok je
ekspresija molekula CD7+CD22+CD34+ povezana sa kraćim preživljavanjem
bolesnika sa AML (p<0,05). | In
the
thesis
are
examined
modern
aspects
of
application
immunophenotyping and multiparameter flow cytometry (IMPC) in the
diagnosis and monitoring of AML. The study included 320 adult patients
with de novo AML, of which 294 included in the retrospective study
(diagnosis, classification and prognosis), while the other 26 patients were
included in a prospective study (minimal residual disease - MRD). Patients
were classified as AML-unclassified (48%), AML with recurrent genetic
abnormalities (37.4%) and AML with signs of myelodysplasia (14.6%). IPCM
allows setting dg AML by analysis of bone marrow (bm) in 98% of patients.
By examining of 44 different HLDM, it was found that its expression is
deregulated on the blasts, whereas at least one type of immunophenotypic
aberrations was found per patient. Heterogeneous cellular composition of
the population of leukemic cells (leukemic blasts and precursors) was found
in 55% of patients with AML. Immunological and cytomorphological
classification of AML agree with 73% of patients, respectively immunological
and WHO classification in 68% of patients. MRD trial was conducted by one
(42%) or two (58%) combination of IFSL per patient. By applying the IMPC,
a high incidence of MRD was detected in our patients with AML, after
induction (69%) and/or consolidation therapy (50%). The level of MRD in
bm of patients ≥0,1% NC after induction therapy, classified patients in the
high risk group for the development of relapse. The prognostic significance
of HLDM in patients with AML, showed a significant association between
early monocytic differentiation of leukemia cells, the expression of CD22
molecule and the appearance of early death (p<0.05), and lower incidence of
complete remission (p<0.05). Multivariate analysis showed that the
expression patterns of molecule CD45+low/med on the blasts, is associated
with a longer duration of the CR (p=0.002), while the expression of the
molecules CD22+CD7+CD34+ associated with shorter survival of patients
with AML (p<0.05 ). | true |
Izgradnja računovodstvenog sistema entiteta sektora Republike Srbije | Designing the accounting system of the public sector's entities in the Republic of Serbia | U savremenim uslovima, kada uloga javnog sektora postaje sve značajnija u
funkcionisanju nacionalnog sistema, neophodno je raspolagati pouzdanim
informacijama o njegovom poslovanju. S obzirom na veliku zainteresovanost
javnosti, a posebno poreskih obveznika, za upravljanje javnim poslovima i
upotrebu javnih resursa, potrebno je obezbediti transparentne izveštaje
neophodne za proces poslovnog odlučivanja i ocenu uspešnosti, efikasnosti,
efektivnosti, ekonomičnosti i odgovornosti javnog menadžmenta.
Adekvatno izgrađen računovodstveni sistem javnog sektora predstavlja osnovu
za kvalitetno finansijsko izveštavanje. U Republici Srbiji ne postoji
integrisan računovodstveni sistem javnog sektora koji bi obezbedio sveobuhvatne
informacije potrebne za ocenu finansijskog položaja, finansijske uspešnosti,
novčanih tokova i ostalih performansi entiteta javnog sektora.
Javni sektor Republike Srbije karakteriše institucionalna i funkcionalna
složenost, što je posledica postojanja velikog broja entiteta koji pripadaju
javnom sektoru. Uvažavajući specifičnosti i obuhvat javnog sektora Republike
Srbije, cilj je ukazivanje na nedostatke postojećeg sistema finansijskog
izveštavanja i predlaganje modela računovodstvenog sistema javnog sektora.
Uspešnost i dinamika izgradnje kvalitetnog računovodstvenog sistema javnog
sektora determinisana je brojnim faktorima, od kojih su najznačajniji politički,
društveni,
personalni
i
tehnološki.
Takođe,
izazovi
u
izgradnji
računovodstvenog sistema su brojni što pretpostavlja postojanje jake volje da se
sistem finansijskog izveštavanja unapredi i kvalitet postojećih finansijskih
izveštaja poboljša.
U težnji da se ukaže na nedostatke postojećeg sistema izveštavanja u
Republici Srbiji i da se predloži način za njegovo unapređenje, analizirana su
iskustva drugih zemalja, koja su odavno otpočela proces reformi u oblasti
računovodstva javnog sektora. Naša zemlja bi koristeći primere dobre prakse
mogla da otpočne proces reformi u računovodstvu javnog sektora na način koji će
6
omogućiti obelodanjivanje informacija o poslovanju svih entiteta javnog sektora.
Na ovaj način bi Republika Srbija ali i njeni građani konačno znali kojim
sredstvima raspolaže javni sektor, na koji način njima upravlja, koliko su buduće
generacije opterećene dugom, kakva je produktivnost u javnom sektoru, sa koliko
uspeha se obavljaju aktivnosti entiteta javnog sektora i sl.
S obzirom da svaka promena, pa čak i ona za koju se očekuju pozitivni efekti,
nailazi na podozrenje, važno je eliminisati apatiju među glavnim akterima
izgradnje računovodstvenog sistema, a to su političari i profesionalne
računovođe, i afirmisati ulogu i značaj kvaliteta finansijskog izveštavanja u
javnom sektoru, kako bi i proces odlučivanja i upravljanja bio kvalitetniji. | In contemporary conditions, when the public sector is becoming more important
in the functioning of the national system, accessing to reliable information is extremely
important for its business. Due to the great interest of the public, especially taxpayers, for
managing of public affairs and using of available resources, it is necessary to ensure
transparent reporting needed for the business decisions process and assessment of the
performance, efficiency, effectiveness, economy and accountability of public
management.
An adequate designed accounting system in the public sector is the basis for the
quality financial reporting. In the Republic of Serbia does not exist integrated accounting
system of the public sector which could provide comprehensive information needed for
assessing of the financial position, financial performance, cash flows and other
performance of the public sector's entities.
The public sector of the Republic of Serbia is characterized by institutional and
functional complexity, as a result of the large number of entities belonging to the public
sector. Respecting the specificity and scope of the public sector of the Republic of Serbia,
the goal is to point at the shortcomings of the current system of financial reporting and
propose model of public sector accounting system.
Many factors determine the success and dynamics of the quality designing of
accounting system of the public sector. The most significant are political, social, personal
and technological. Also, the challenges in the construction of the accounting system are
numerous and it presupposes the existence of a strong willingness to improve the financial
reporting system as well as the quality of existing financial statements.
In an effort to draw attention to the shortcomings of the existing system of reporting
in the Republic Serbia and suggest solution for its improvement, experiences of other
countries which have been started the process of reform many years ago are analyzed.
Using the examples of good practice, our country could start the process of reforms in the
public sector accounting in a way which will enable access of information about its
business for all public sector's entities. In this way, the Republic of Serbia and its citizens
8
could finally find out what kind of assets do they have, how do they operate, how many
are the future generations burdened with debt, what is the productivity level in the public
sector, how many successful are the activities carried out by public sector entities, etc.
Due the fact that every change, even that which could bring benefit, is the subject of
suspicion, It is very important to eliminate the apathy among the major actors of the
accounting system designing such as politicians and professional accountants as well as
affirm the role and importance of the quality financial reporting in the public sector in
order to achieve better decision-making and managing process. | true |
Holistički pristup oceni zdravstvenog stanja školske dece i omladine | Holistic approach to the evaluation of the health status of school children and adolescents | Istraživanje predstavlja studiju preseka na reprezentativnom uzorku od 672 deteta starosti od 7 do 19 godina u Vojvodini. Kao instrument istraživanja korišćeni su upitnici kojima su dobijeni podaci o demografskim i socijalno-ekonomskim karakteristikama, samoproceni zdravlja, zadovoljstvu životom, higijenskim navikama, navikama o ishrani, fizičkoj aktivnosti i slobodnom vremenu, mentalnom zdravlju, zastupljenosti rizika po zdravlje i korišćenju zdravstvene službe. Objektivni nalaz dobijen je merenjem telesne mase i visine, određivanjem indeksa telesne mase i merenjem krvnog pritiska. Osnovni ciljevi ovog rada bili su da se dobije ocena zdravstvenog stanja i utvrde vodeći zdravstveni rizici kao i razlike u odnosu na demografske i socijalne karakteristike, da se utvrde promene u zdravstvenom stanju školske dece i omladine u odnosu na retrospektivni deo istraživanja i da se testira validnost instrumenta. Rezultati su pokazali da 40,2% dece živi u domaćinstvima koja pripadaju grupi siromašnih, imaju jednog brata/sestru, 88% živi sa oba roditelja, a dve trećine ima svoju sobu. Redovne higijenske navike češće imaju devojčice, stariji uzast i deca iz bogatijih domaćinstava. Ishrana je neadekvatna kako sa aspekta redovnosti, tako i unosa pojedinih namirnica, nedovoljan je unos mleka i mlečnih proizvoda, voća i povrća i ribe, a prekomeran unos namirnica koje su izvor tzv. „praznih kalorija“, dok je 17% dece sa prekomernom telesnom masom i gojazno. Slobodno vreme karakterišu sedenterne aktivnosti i nedovoljna fizička aktivnost. Ponašanje u saobraćaju je rizično, većina ne koristi zaštitne mere. Povredu je imalo oko 15% dece, petina je bila izložena nekom obliku fizičkog ili psihičkog nasilja. Većina ocenjuje svoje zdravlje kao dobro, dve trećine svoju telesnu masu kao normalnu a tri četvrtine svoju fizičku aktivnost kao dobru. Higijenu i mogućnost rekreacije u školi petina ocenjuje kao lošu a svako deseto dete bezbesnost u školi kao lošu. Informacije o zdravlju najčešće dobijaju od roditelja. Zdravlje stavljaju na prvo mesto na lestvici životnih vrednosti i imaju visok nivo zadovoljstva životom. Većina ima dobru komunikaciju sa majkom a tri četvrtine sa ocem. Petina oseća napetost, stres i ima emocionalne probleme, lošije vrednosti na skali psihološkog distresa ima svaka šesta a na skali vitalnosti skoro svaka druga mlada osoba. Pušenje i konzumacija alkohola su visoko zastupljeni, a svaka deseta mlada osoba probala je neku od psihoaktivnih supstanci. Korišćenje zdravstvene zaštite je zadovoljavajuće osim u sferi reproduktivnog zdravlja adolescentinja, seksualno ponašanje karakteriše rano stupanje u seksualni odnos i nedovoljna zaštita. U odnosu na retrospektivni deo istraživanja značajne razlike dobijene su u higijenskim navikama, ishrani, fizičkoj aktivnosti i sedenternim aktivnostima, procentu gojaznih, oceni svog zdravlja, zadovoljstvu životom, mentalnom zdravlju, izloženosti duvanskom dimu, konzumaciji alkohola, poznavanju psihoaktivnih supstanci, seksualnom ponašanju, reproduktivnom zdravlju i korišćenju zdravstvene službe. | The research represents a cross-sectional study of the representative sample of 672 schoolchildren 7-19 of age in Vojvodina. Instrument was specially constructed questionnaire providing data about demographic and socio-economic characteristics, self assessment of the health, life satisfaction, hygiene, nutrition, physical activity, leisure time, mental health, social support, presence of risk factors, and use of health care. Anthropometric measures included body mass, height, blood pressure measurement and calculating body mass index. The aim of the study was to assess health status of the children, identify health risks and determine demographic and socio-economic differences, as well as to determine differences in relation to the retrospective part of the study, and to determine validity of the instrument. Results showed that 40 percents of children live in households with low level of socio-economic status (SES), 88 percents live with both parents, have 1 sibling, and two thirds have their own room. Good hygiene practice is more likely present in girls, older age and children with better SES. Nutrition habits are inadequate regarding regularity and different food intake, there is low consumption of milk and dairy products, fruits, vegetables and fish, high intake of sweets, snacks and sweet beverages, and there is 17 percents of overweight and obese children. Leisure time is characterized with sedentary activities and lack of physical activity. Road traffic behavior is risky with inadequate safety measures. Injury had 15 percents of children, and the fifth was object of physical and psychological violence. Most of the children assessed their health as good and very good, two thirds their body mass as normal and three quarters their physical activity as good. School hygiene and recreational conditions were poorly rated by fifth of the children, and one in ten assessed school safety as bed. Health information are mostly provided by parents. Health is highly rated among life values and life satisfaction is high. Most of them easy communicate with their mothers and three quoters with their fathers. Every fifth child is having emotional problems, anxiety and stress, lower values on Psychological Distress Scale has every sixth child and on Vitality Scale every second child. Prevalence of smoking and alcohol consumption is high, one in ten adolescents experimented or consumed drugs. Health care use is satisfying except in the sphere of reproductive health of female adolescents. Sexual behavior is characterized with early onset of sexual intercourse and inadequate protection against pregnancy and STD. Differences regarding retrospective part of the study exist in hygiene practice, nutrition, physical activity, sedentary activities, prevalence of overweight and obesity, self assessment of health, life satisfaction, mental health, exposure to tobacco, alcohol consumption, knowledge about drugs, sexual behavior and health care use. | false |
Analiza velikog konjugabilnog plazmida bakterije Lactococcus lactis subsp. lactis biovar. diacetylactis S50 | Analysis of large conjugative plasmid of Lactococcus lactis subsp. lactis biovar. diacetylactis S50 | Lactococcus lactis subsp. lactis biovar. diacetylactis S50 je prirodni izolat iz maslacne maje. Soj S50 sintetise bakteriocin uskog spektra delovanja i proteinazu PI tipa. Soj S50 poseduje tri vidijiva plazmida koji se mogu izolovati (pS50-7, pS50-10a i pS50-10b). Ciscenjem plazmida iz soja S50 istovremenim tretmanom subletalnom temperaturom i novobiocinom (10u:g/m!) dobijen je Bac- i Prt- derivat (S50-1). Analizom genoma soja S50 i derivata S50-1 na elektroforezi u pulsirajucem polju (PFGE) nakon digestije restrikcionim enzimima ustanovijeno je da se geni za sintezu proteinaze PI tipa i bakteriocina S50 nalaze na plazmidu velicine oko 290kb koji je oznacen kao plazmid pS50-290. Na osnovu razdvajanja secene DNK na PFGE i hibridizacije sa probama za proteinazni (Q; i Qo) i bakteriocinski gen (LenA) odredjena je restrikciona mapa plazmida pS50-290 i odredjen je polozaj gena na njemu. Plazmid pS50-290 pokazuje visok stepen retardacije na PFGE sto moze biti posledica vezivanja proteina za njega. Soj S50 je ukrstan sa vecim brojem recipijentnih sojeva (MG7284, 1L1403, VEL1122 i ocisceni derivati soja S50) u kojima su dobijani Bact konjugnti sa priblizno istom frekvencom. Na osnovu toga se moze tvrditi da je plazmid pS50-290 autokonjugabilan ili Tra+ plazmid, odnosno da na sebi poseduje sve neophodne gene za sopstveni transfer. Pored sinteze bakteriocina ustanovljeno je da svi dobijeni konjuganti sintetisu i proteinazu PI tipa istih karakteristika kao i soj S50. Analizom plazmidnog sastava konjuganata ustanovijeno je da i drugi plazmidi soja S50 (pS50-7 i pS50-10b) poseduju sposobnost konjugacionog transfera, najverovatnije zato sto poseduju gen za Mob protein i oriT sekvencu. Restrikcionom analizom plazmida izolovanih iz konjuganata, rekonjuganata i derivata S50- 20 (digestijia Smal restrikcionim enzimom i razdvajanje dobijenih fragmenata na PFGE) ustanovijeno je da je u njima plazmid pS50-290 (oznacen kao pS50-290A) skracen i da poseduje samo jedno Smal restrikciono mesto za razliku od plazmida pS50-290 izolovanog iz sojaS50 koji poseduje dva. Fragment koji se deletira je velicine nekoliko kilobaza i pokazuje homologiju sa ostalim plazmidima soja S50 (pS50-7, pS50-10a i pS50-10b). Hibridizacionim ekspeimentima u kojima je plazmid pS50-7 koriscen kao proba pokazano je da svi plazmidi soja S50 poseduju homologe sekvence cije prisustvo govori o zajednickom poreklu ovih plazmida. U procesu izolacije spektinomicin-rezistentnih derivata soja S50 izolovani su mutanti koji pokazuju visok nivo rezistencije na spektinomicin i koji ujedno poseduju inverziju u hromozomu velicine izmedju 180 i 790kb. Istu inverziju je posedovao i mutant derivata S50-20-62 rezistentan na visoku koncentraciju spektinomicina. | Lactococcus lactis subsp. lactis biovar. diacetylactis S50 was isolated from butter starter culture. Strain S50 produced bacteriocin which has narrow antibacterial spectrum, In addition, strain S50 synthesized extracellular cell wall-associated proteinase of Pl-type. The strain S50 has three plasmids detectable by conventional procedures for isolation of plasmids (pS50-7, pS50-10a and pS50-10b). Curing experiments of strain S50 with novobiocin (10u1g/ml) and sublethal temperature (41°C) resulted in obtaining a Prt. Bac- derivative (S50-1). The analysis of original strain S50 and Prt-. Bac- derivative S50-1 by restriction enzymes on PFGE revealed that prt and bac genes are located on the large plasmid (approximately 290 kb) named pS50-290. Restriction map of plasmid pS50-290 was done by concomitant using PFGE and hybridization experiments with prt (Q; and Qg2) and bac (LenA) probes. The plasmid pS50-290 shows significant retardation on PFGE. DNA sequence which is target for binding protein(s) is tocated between Smal restriction site (at position 80 kb) and Ncol (148 kb) on restriction map. The plasmid pS50-290 is self-transmissible (Tra+) plasmid. Strain S50 was used as a donor in conjugation experiments for transfer of plasmid pS50-290 to the other lactococcal strains (MG7284, 1L1403 and VEL1122). All conjugations gave high number of conjugants. The obtained transconjugants (frequency of conjugation were about 10-7) produced bacteriocin S50 and proteinase of PI type. These data show that ability for conjugal transfer depends only on genetic elements located on pS50-290 plasmid. d Conjugants MG10 and SIL102, besides plasmid pS50-290 acquired smaller plasmids of strain S50 (MG10 has plasmid pS50-7 and SVEL102 pS50-10b). Transfer of plasmids pS50-7 and pS50-10b (they are non-self-transmissible plasmids) appeared to be dependent on cotransfer of the conjugative plasmid pS50-290. The analysis of conjugants and derivative S50-20 (derivative of the strain S50 cured of plasmids pS50-7 and pS50-10b) by restriction enzyme Smal and PFGE showed that the plasmid pSS50-290 (designated pS50-290A) present in them has only one Smal site. Hybridization experiments (labelled pS50-7 was used as a probe) revealed that plasmids pS50- 290, pS50-10a and pS50-10b contain homologous sequences to the particular region of plasmid pS50-7. Plasmid pS50-290A which is present in conjugants and derivative S50-20 hasn't shown homology because it has deleted that region. During the isolation of spectinomycin-resistant mutants of the derivative S50-1 as well as S50-20-62 to Sbe used as a recipients in conjugation crosses an inversion within chromosomal DNA has occurred in them. | false |
Izolacija i funkcionalna karakterizacija gena uključenih u sintezu karnozinske kiseline | Isolation and functional characterization of the genes involved in the synthesis of carnosic acid. | Karnozinska kiselina, fenolni diterpen, poseduje brojne biološke
aktivnosti zbog čega je potencijalno veoma značajna za farmaceutsku
industriju. Grčka žalfija (Salvia fruticosa Mill.) i ruzmarin (Rosmarinus
officinalis L.) bogati su prirodni izvori ovog jedinjenja. Iako je mnogo toga
poznato u vezi strukture i bioloških aktivnosti karnozinske kiseline,
veoma malo se ne zna o njenom biosintetskom putu u biljkama. Osnovni cilj
disertacije bio je izučavanje biosinteze karnozinske kiseline na
molekularnom nivou, izolacijom i funkcionalnom karakterizacijom gena
uključenih u ovaj proces. U tu svrhu, izvršena je analiza postojeće cDNK
biblioteke žlezdanih dlaka grčke žalfije, što je za rezultat imalo
identifikaciju dva gena, nazvanih SfCPS i SfKSL, koji potencijalno
kodiraju diterpen sintaze. Geni kandidati su izolovani i funkcionalno
okarakterisani u bakteriji Escherichia coli Mig., kvascima (Saccharomyces
cerevisiae Meyen ex E. C. Hansen) i duvanu (Nicotiana benthamiana Domin).
Heterologa ekpresija gena SfCPS i SfKSL rezultirala je sintezom
miltiradiena, što je utvrđeno GC-MS analizom i 1D i 2D NMR
spektroskopijom (1H, 13C, DEPT, COSY H-H, HMQC i HMBC). Za pronalaženje
preostalih gena odgovornih za biosintezu karnozinske kiseline, obavljeno
je sekvencioniranje transkriptoma grčke žalfije i ruzmarina, uz
korišćenje 454 GS FLX Titanium platforme. Na osnovu rezultata
fitohemijske analize LC-PDA-LTQ-Orbitrap FTMS metodom, žlezdane dlake
mladih listova blago stresiranih biljaka genotipa “Kavusi” grčke žalfije
i žlezdane dlake mlađih stadijuma razvoja komercijalnog genotipa
ruzmarina (B & T World Seeds) izabrane su kao biljni materijal za analizu
transkriptoma. Rezultat sekvencioniranja bio je anotacija velikog broja
novih sekvenci, a posebno značajna bila je identifikacija gena
metaboličkih puteva povezanih sa sekundarnim metabolizmom. Izolovano je
ukupno 24 gena koji su kodirali za enzime familije citohrom P450 (CYP)
monooksigenaze. Funkcija enzima CYP76Sf2 grčke žalfije, i CYP76Ro3 i
CYP76Ro4
ruzmarina,
kao
feruginol
sintaza,
utvrđena
je
esejima
funkcionalne karakterizacije u kvascima i duvanu. Kloniranje i
funkcionalna karakterizacija gena koji svojom zajedničkom aktivnošću
dovode do sinteze feruginola kod grčke žalfije i ruzmarina, pruža osnovu
budućim istraživanjima biosintetskog puta karnozinske kiseline. Uz to,
izvršena je lokalizacija biosintetskog puta karnozinske kiseline u
žlezdanim dlakama grčke žalfije i ruzmarina. Priloženi rezultati
doprinose boljem razumevanju metabolizma karnozinske kiseline, i time
omogućavaju primenu najsavremenijih biotehnoloških metoda za proizvodnju
ovog farmaceutski značajnog jedinjenja. | Carnosic acid is a phenolic diterpene potentially highly significant for the
pharmaceutical industry, due to its numerous biological activities. Cretan sage
(Salvia fruticosa Mill.) and Rosemary (Rosmarinus officinalis L.) are rich natural
sources of this compound. Although the structure and biological activities of
carnosic acid are widely known, its biosynthetic pathway in plants remains
unexplored. The main aim of the dissertation was to investigate the carnosic acid
biosynthesis at the molecular level, by isolating and functionally characterizing the
genes involved in this process. For that purpose, the existing Cretan sage trichome
cDNA library has been analyzed, which has resulted in the identification of two
putative diterpene synthase genes, named SfCPS and SfKSL. The candidate genes
were isolated and functionally characterized in Escherichia coli Mig., yeast
(Saccharomyces cerevisiae Meyen ex E. C. Hansen) and Nicotiana benthamiana
Domin. Heterologous expression of SfCPS and SfKSL genes has resulted in the
synthesis of miltiradiene, which has been confirmed by GC-MS analysis and 1D and
2D NMR spectroscopy (1H, 13C, DEPT, COSY H-H, HMQC и HMBC). In order to
retrieve the remaining genes responsible for the biosynthesis of carnosic acid, the
glandular trichomes of Cretan sage and Rosemary have been sequenced using 454
GS FLX Titanium platform. Based on the results of the LC-PDA-LTQ-Orbitrap
FTMS phytochemical analysis, glandular trichomes of Cretan sage young leaves
(genotype Kavoussi) exposed to mild stress, and glandular trichomes on the
Rosemary leaves of the younger developmental stage (commercial genotype B&T
World Seeds), were chosen for the transcriptome analysis.The sequencing has
resulted in the annotation of a large number of new sequences. The identification
of the genes belonging to the secondary metabolism pathways was especially
significant. A total of 24 genes coding for cytochrome P450 monooxygenases was
isolated. Using the functional characterisation assays in yeast and Nicotiana
benthamiana it has been determined that the Cretan sage enzyme CYP76Sf2 and
Rosemary enzymes CYP76Ro3 and CYP76Ro4 have the activity of ferruginol
synthases. Molecular cloning and functional characterization of the genes that play
a role in the synthesis of ferruginol in Cretan sage and Rosemary, forms a basis for
the future investigation of the carnosic acid biosynthetic pathway. Additionaly, this
pathway has been localized in the glandular trichomes of Cretan sage and
Rosemary. The results of this dissertation help better understanding of the carnosic
acid metabolism, which make production of this pharmaceutically valuable
compound possible, using the advanced biotechnological methods. | true |
Detekcija i karakterizacija genomske nestabilnosti tumora mozga glijalnog porekla | Detection and characterization of genomic instability in patients with malignant glioma | Glioblastomi (GBM) predstavljaju najčešći i najmaligniji tip tumora mozga. Iako imaju istu
histopatološku sliku, primarni i sekundarni glioblastomi se razlikuju po mehanizmu nastanka
i setu karakterističnih genetičkih promena. Iako je tokom poslednjih godina došlo do
određenog poboljšanja u sagledavanju njihove kompleksne prirode, glioblastomi i dalje
predstavljaju neizlečivu bolest. Osnovni cilj ove doktorske disertacije bio je detekcija i
kvantifikacija genomske nestabilnosti, kao i identifikacija specifičnih genetičkih promena
odgovornih za promociju i progresiju malignih glioma, a sa dugoročnim ciljem da se definišu
potencijalni molekularni markeri za dijagnozu i prognozu bolesti. Sa tim u vezi, izvršena je i
analiza najčešćih poznatih genetičkih promena u gliomima: detekcija inaktivacije ključnih
tumor supresor gena (p53, PTEN i p16) i amplifikacije EGFR onkogena. Promene dva ili više
navedenih gena su prisutne u većini analiziranih uzoraka, što potvrđuje značaj višestrukih
genskih alteracija u patogenezi glioma.
Upoređivanjem AP-PCR DNK profila tumorskog i zdravog tkiva pacijenata uočena su dva
tipa razlika: kvalitativne razlike koje nastaju usled promena u sekvenci DNK molekula i
predstavljaju manifestaciju mikrosatelitske nestabilnosti i nestabilnosti pojedinačnih
nukleotida (MIN-PIN), kao i kvantitativne razlike nastale usled amplifikacija ili delecija
većih hromozomskih regiona, pokazatelj hromozomske nestabilnosti (CIN). Oba tipa
promena su prisutna u svim analiziranim uzorcima, doprinoseći podjednako visokom stepenu
ukupne genomske nestabilnosti u oba histološka podtipa GBM. Detaljnija analiza DNK
profila je omogućila identifikaciju specifičnih promena 11 novih gena, koji do sada nisu
povezani sa progresijom glioma: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2,
KCNG2, PDE4D, KIR3DL3 i INPP5A. Većina identifikovanih gena ima značajnu ulogu u
procesima signalne transdukcije i ćelijske adhezije, koji su veoma značajni za nastanak i
progresiju kancera. Prisustvo promena u identifikovanim genima je, dalje, korelisano sa
kliničko-patološkim parametrima, stepenom genomske nestabilnosti i preživljavanjem
pacijenata, kao i sa statusom p53, PTEN, p16 i EGFR gena. Pokazane su statistički značajne
ko-alteracije nekih od identifikovanih gena sa p53, p16 i EGFR genima, ali nije pokazana
statistički značajna povezanost između inaktiviranog PTEN-a i bilo kojeg novog
identifikovanog gena. Svi rezultati ove studije potvrđuju da se u osnovi glioma nalaze
izuzetno složeni genetički mehanizmi, pri čemu je naročito značajan visok stepen genomske
nestabilnosti koji potencijalno predstavlja jednu od osnovnih karakteristika ovog tipa
tumora. Osim toga, ova studiija je prva koja je ukazala na potencijalni značaj
identifikovanih gena u patogenezi glioma, kao i na njihovu primenu kao potencijalnih
biomarkera za dijagnostiku i prognozu pacijenata sa primarnim ili sekundarnim
glioblastomima. | Glioblastoma is the most frequent and the most malignant human brain tumor. Despite a similar
histological appearance, primary and secondary glioblastomas are distinct tumor entities with
different genetic alterations but none being specific enough to distinguish them. Despite better
insight in its complex genetic nature, glioblastoma is still incurable disease, with extremely
short median survival. The purpose of this study was to detect specific genetic changes, as well
as to quantify overall level of genomic instability in samples of malignant glioma patients.
Besides, we analyzed genetic alterations of key tumor suppressors (p53, PTEN and p16) and
EGFR oncogene, commonly aberrant in glioma samples. Alterations of two or more genes were
present in majority of analyzed samples indicating importance of multiple changes for
gliomagenesis.
AP-PCR DNA profiling revealed two types of genetic differences between tumor and normal
tissue: qualitative changes which represent accumulation of changes in DNA sequence and are
the manifestation of microsatellite and point mutation instability (MIN-PIN) and quantitative
changes which represent amplifications or deletions of existing chromosomal material and are
the manifestation of chromosomal instability (CIN). Both types of alterations were present in
all analyzed samples contributing almost equally to the total level of genomic instability, and
showing no differences between histological subtypes. Further investigation of alterations in
DNA profiles revealed specific changes in the following 11 genes that were not previously
associated with glioma pathogenesis: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2,
KCNG2, PDE4D, KIR3DL3, and INPP5A. Most of identified genes have significant role in
signal transduction or cell adhesion, which are important processes for cancer development and
progression.
The frequencies of observed alterations were correlated with clinicopathologic parameters, the
level of genomic instability and patient survival, as well as with presence of alterations in p53,
PTEN, p16 and EGFR genes. Some of the identified genes showed significant association with
p53 and p16 tumor suppressors, as well as with EGFR, but there was no significant correlation
between loss of PTEN and any of identified genes.
In conclusion, our results confirmed complexity of glioma genetic nature, emphasizing high
level of genomic instability as hallmark of this tumor type. Identified novel genes could be used
as potential biomarkers for diagnosis of primary and secondary glioblastoma, as well as
predictors of patients’ outcome. | true |
Regulacija ekspresije gena za metalotionein tipa 3 heljde i uloga proteina MT3 u odbrani biljnih tkiva od stresa izazvanog teškim metalima | Regulation of gene expression of metallothionein type 3 and buckwheat protein MT3 role in defending the plant tissues from stress induced by heavy metals | Iz biblioteke cDNK semena heljde u srednjoj fazi sazrevanja prethodno su izolovana dva klona cDNK koja kodiraju metalotioneinu-sličan protein. Izolovan je i odgovarajući genomski klon koji obuhvata deo kodirajućeg regiona kao i 71 bp 5`UTR i 569 bp promotorskog regiona. U ovom radu, izolovani promotorski region gena FeMT3, kao i dve njegove 5` delecione varijante, funkcionalno su analizirani u stabilno transformisanom duvanu. Histohemijskim esejem GUS je detektovana intenzivna aktivnost promotorskih fragmenata u vaskulatnim elementima lista i u polenu, a slabija aktivnost uočena je i u tkivu korena. Kvantitativnim esejem GUS je pokazano značajno povećanje aktivnosti sva tri promotorska fragmenta (proporcionalno njihovim dužinama) u odgovoru na uslove kompleksnog stresa, odnosno u listovima potopljenim u MS medijum sa saharozom. Jaka indukcija promotorske aktivnosti je uočena i pod dejstvom jona Cu2+ i Cd2+. Navedeni rezultati sugerišu postojanje kompleksne transkripcione regulacije gena FeMT3 i učešće većeg broja različitih faktora. Povećanje nivoa transkripta FeMT3 u listu heljde pod uticajem jona Cu2+ i Cd2+ potvrđeno je metodom "Real-time RT-PCR". U cilju približavanja ulozi koju FeMT3 ima u biljnom tkivu, testirane su protektivne sposobnosti proteina kodiranog ovim genom u živim sistemima izloženim dejstvu teških metala. Odbrambene sposobnobnosti FeMT3 su potvrđene u prokariotskom sistemu E. coli i u tranzijentno transformisanim listovima duvana N. debneyii. Izlaganje visokim koncentracijama CdCl2 ili CuSO4, izazivalo je značajno sporiji razvoj oštećenja u tkivu koje je eksprimiralo FeMT3 u poređenju sa kontrolom. Unutarćelijska lokalizacija FeMT3 je praćena u fuziji sa fluorescentnim proteinom (YFP) u transformisanim ćelijama duvana. Proteinska fuzija YFP-FeMT3 je uočena u citoplazmi, a odsustvovala je u vakuoli i hloroplastima. Lokalizacija se nije promenila ni nakon tretmana teškim metalima, što sugeriše drugačiji mehanizam odbrambenog delovanja metalotioneina u odnosu na fitohelatine. Svojstva promotora FeMT3 i proteina kodiranog ovim genom koja su pokazana u ovom radu, ukazuju na potencijal gena FeMT3 za primenu u fitoremedijaciji. | Two cDNA clones coding for metallothionein-like protein (FeMT3) were isolated previously from the cDNA library of developing buckwheat seeds. The corresponding genomic clone, containing part of the coding region, as well as 71 bp of the 5' UTR and 569 bp of the promoter region, was also isolated. To shed light on expression regulation of the metallothionein gene from buckwheat (FeMT3), herein functional promoter analysis was performed with a complete 5’ regulatory region and two deletion variants, employing stably transformed tobacco plants. Histochemical GUS assay of transgenic tobacco lines showed the strong signals in vascular elements of leaves and in pollen grains, while somewhat weaker staining was observed in the roots of mature plants. Quantitative GUS assay showed strong up-regulation of all three promoter constructs (proportional to the length of the regulatory region) in leaves submerged in liquid MS medium containing sucrose, after a prolonged time period, representing a complex stress situation composed of several synergistically related stress stimuli. Functional analysis of the promoter region also revealed extremely high inducibility upon Cu2+ and Cd2+ treatments. These findings suggest complex transcriptional regulation of FeMT3, requiring interaction of a number of different factors. FeMT3 transcription level increase under the influence of Cd2+ and Cu2+, in buckwheat leaves, was confirmed by Real-time RT-PCR. The protective role in vivo of FeMT3 during metal stress was examined. Increased tolerance to heavy metals of FeMT3 producing E. coli cells was detected. The defensive ability of buckwheat MT3 during Cd2+ and Cu2+ stresses was also demonstrated in Nicotiana debneyii leaves transiently expressing FeMT3. When exposed to CdCl2 or CuSO4, FeMT3 overexpressing leaf regions showed much slower development of tissue damage, compared with mock transformed regions. To reveal intracellular localization of FeMT3, a protein fusion with yellow fluorescent protein (YFP) was used. YFP-FeMT3 was localized in cytoplasm and was absent from vacuole and chloroplasts. In contrast to phytochelatins, the cytoplasmatic localization of FeMT3 was not altered under heavy metal stress. The confirmed defense ability of FeMT3 protein in vivo and the great responsiveness of its promoter during heavy metal exposure, make this gene a suitable candidate for biotechnological applications. | false |
Nosivost zavrtnjeva kao sredstva za sprezanje u prefabrikovanim spregnutim konstrukcijama od čelika i betona | Resistance of bolted shear connectors in prefabricated steel-concrete composite decks | Primenom prefabrikovanih betonskih ploča vreme izgradnje spregnutih
nosača mostova ili u zgradarstvu se značajno smanjuje. Ukoliko se kao sredstva za
sprezanje koriste zavrnjevi ubetonirani u prefabrikovanu betonsku ploču,
moguće je postići veći stepen prefabrikacije u poređenju sa rešenjem sa
grupisanim moždanicima sa glavom i otvorima u betonskim pločama. Takođe,
zavrtnjevi kao sredstva za sprezanje nude nesumnjivu prednost po pitanju uticaja
konstrukcije na životnu okolinu, time što omogućuju lako uklanjanje
konstrukcije ili zamene njenih delova. Ipak, zavrtnjevi se retko koriste kao
sredstva za sprezanje, uglavnom zbog nedostatka potrebnih istraživanja i
pravila za projektovanje. Cilj istraživanja prikazanog u ovoj doktorskoj
disertaciji je omogućavanje šire upotrebe zavrtnjeva kao sredstva za sprezanje
kroz detaljnu analizu njihovog ponašanja. Prvo je analizirana opravdanost
primene zavrtnjeva kao sredstva za sprezanje u podužnom smičućem spoju
spregnutih konstrukcija, uzimajući u obzir tehničke zahteve, analizu
isplativosti i uticaj na životnu okolinu. Zavrtnjevi sa jednom ubetoniranom
navrtkom su se pokazali kao najpogodniji za praktičnu primenu. Primer
uporednog proračuna spregnutog nosača raspona dvanaest metara, sa zavrtnjevima
i moždanicima kao sredstvima za sprezanje pokazao je da usled nepotpune
interakcije (usled početnog klizanja zavrtnja u rupi) u slučaju sprezanja pomoću
zavrtnjeva dolazi do uvećanja ugiba nosača i napona u čeličnoj gredi (10-20%).
Ovakvo ponašanje se smatra prihvatljivim, pogotovo ako se tokom gradnje
primene privremeni oslonci i nadvišenje čeličnog nosača. Dalja detaljna
analiza ponašanja zvrtnjeva kao sredstva za sprezanje izvršena je poređenjem sa
zavarenim moždnicima sa glavom u testu smicanja (push-out test). Eksperimenti
su vršeni koristeći zavrtnjeve M16 i M24 (klasa 8.8). Poređene su osnovne
karakteristike sredstva za sprezanje: nosivost na smicanje, krutost na smicanje i
duktilnost. Zavrtnjevi su dostigli skoro jednaku nosivost kao i moždanici sa
v
glavom dok su krutost i duktilnost redukovane. Napredni modeli na bazi metode
konačnih elemenata su napravljeni da odgovaraju testu smicanja i kalibrisani
koristeći eksperimentalne rezultate. Korišćena je kvazi-statička analiza
primenom eksplicitnog dinamičkog solvera softverskog paketa Abaqus.
Zavrtnjevi i navrtke su modelirani sa stvarnom geometrijom, a korišćeni su i
modeli loma čeličnog i betonskog materijala što je omogućilo realno
analiziranje loma u testu smicanja. Ovako kalibrisani modeli na bazi MKE
korišćeni su dalje za parametarsku analizu. Prvo je sprovedena inicijalna
parametarska analiza sa ciljem da identifikuje uticaj pojedinih parametara na
nosivost i duktilnost zavrtnjeva kao sredstva za sprezanje. Razmatrani su
parametri: sila prednaprezanja u zavrtnju, broj ubetoniranih navrtki, podužno
rastojanje između zavrtnjeva i visina zavrtnjeva. Nakon toga izvršena je glavna
parametarska analiza sa parametrima koji najviše utiču na nosivost i
duktilnost. To su: prečnik zavrtnja, čvrstoća betona i visina zavrtnja. Ovi
parametri su analizirani u korelaciji jednih sa drugim. Na osnovu rezultata
eksperimenata i numeričke analize, prepoznata su dva osnovna modela loma
zavrtnja kao sredstva za sprezanje: lom zavrtnja na spoju čelične nožice i
betonske ploče i lom betona čupanjem zavrtnja (pryout failure).Ovi modeli loma su
razjašnjeni do detalja i analitički modeli njihovog ponašanja su razvijeni i
potvrđeni koristeći eksperimentalne i rezultate MKE. Nakon toga, određeni su
izrazi za određivanje nosivosti i duktilnosti zavrtnjeva kao sredstva za
sprezanje sa jednom ubetoniranom navrtkom pogodni za primenu u Evrokodu. Ovi
izrazi su potvrđeni poređenjem sa rezultatima predstavljenog eksperimentalnog
istraživanja i eksperimentalnih istraživanja drugih autora. | Prefabrication of concrete slabs reduces construction time for composite steel-
concrete buildings and bridge decks. Casting of bolted shear connectors in prefabricated
concrete slabs offers the higher level of prefabrication when compared to a technique
with grouped headed studs in envisaged pockets of concrete slabs. Additionaly, bolted
shear connectors offer some sustainability advantages, such as ability of the structure to
be easily removed or replaced. However, bolted shear connectors are rarely used in
composite structures due to the lack of detailed research and design rules. The aim of
the research presented in this thesis is to promote the application of bolted shear
connectors through detailed examination of their behaviour. Firstly, feasibility of their
application in the longitudinal shear connection of composite decks is examined
considering requirements for application, technical aspects, cost effectiveness and
environmental impacts. Bolted shear connectors with single embedded nut was found to
be the most appropriate for practical application. A case study considering incomplete
interaction on a 12 m span composite beam with bolted shear connectors and welded
headed studs showed that deflections and stresses in steel are increased (10-20%) in
case of bolted shear connectors due to initial slip in hole. This is acceptable, especialy in
the case where propped construction and propper camber are used. Further, detailed
examination of their behaviour is made through comparison to welded headed studs in
push-out tests on M16 and M24 bolted shear connectors (grade 8.8). Basic shear
connector properties are observed: shear resistance, stiffness and ductility. Bolted shear
connectors achieved similar shear resistance, while stiffness and ductility are reduced
when compared to the headed studs. Advanced FE models of push-out tests are built
and calibrated with regards to the experimental results. Abaqus/Explicit dynamic solver
and damage material models for steel and concrete were used with realistic bolt and nut
geometry, allowing the beyond-state-of-the-art failure analysis of push-out tests. Initial
slip in hole is analysed and quantified with use of experimental and FEA results.
Previously calibrated verification FEA models are used for the parametric study. Firstly,
initial parametric study is conducted in order to analyse the significance of the influence
iii
of certain parameters on resistance and ductility of the bolted shear connection.
Parameters considered in the initial parametric study are: bolt preloading force, number
of embedded nuts, longitudinal spacing between shear connectors and shear connector
height. Later, the most significant parameters influencing shear resistance and ductility
of bolted shear connectors; bolt diameter, concrete strength and shear connector height
are coupled in main parametric study. Failure modes of bolted shear connectors with
single embedded nut are recognized as failure of the bolt at the flange-concrete interface
and pryout failure of the concrete both in experimental and FEA results. Those failure
modes are explained in details and analytic models are developed and validated with use
of FEA. Shear resistance and ductility prediction models are proposed on the basis of
the FEA parametric study and validated with regards to experimental results of present
and previously published research. Good agreement is found. Design rules are proposed
in form suitable for inclusion in Eurocode 4. | true |
Karakterizacija osmotski aktiviranih jonskih struja u membrani citoplazmatskih kapi izolovanih iz sporangiofora gljive Phycomyces blakesleeanus Burgeff | Characterization of osmotically activated ion currents in the membrane of cytoplasmic droplets from Phycomyces blakesleeanus Burgeff sporangiophores. | Membrana citoplazmatskih kapi dobijenih iz rastućih sporangiofora gljive Phycomyces
blakesleeanus Burgeff je jedan od samo dva za sada poznata model sistema u kojima se mogu
ispitivati elektrofiziološka svojstva plazma membrane filamentoznih gljiva (drugi model
sistem je sluzavi mutant gljive Neurospora crassa). Pokazano je da bar 20% citoplazmatskih
kapi nakon 2 h formira ćelijski zid što ukazuje da njihova membrana odgovara plazma
membrani hife. Eksperimentima prikazanim u disertaciji je po prvi put izvršeno registrovanje
struja metodom nametnute voltaže u konfiguraciji cela ćelija na model sistem membrane
citoplazmatskih kapi. Ovaj način snimanja jonskih struja daje uvid u tipove i osobine
dominantnih ukupnih struja prisutnih u ispitivanoj membrani. Okarakterisan je odgovor
membrane na hipoosmotske uslove sredine, izazvane dijalizom čitave kapi hiperosmotskim
rastvorom. Za razliku od familije struja u izoosmotskim uslovima, kojima dominiraju pasivna
svojstva membrane, male konduktivnosti, u hipoosmotskim uslovima je karakteristično
dominantno prisustvo izlazno ispravljene brzoinaktivirajuće trenutne struje (IRIS), aktivirane
na depolarišućim potencijalima. Pored IRIS, prisutne u svakoj ispitivanoj membrani
citoplazmatskih kapi čija je maksimalna gustina struje na +70 mV iznosila 129 ± 14 pA/pF
(n=30), u 73% registrovanih odgovora je bila primetna i sporoaktivirajuća ulazna struja,
gustine 99 ± 11 pA/pF na -150 mV (n=22) koja nije dalje ispitivana.
IRIS je prava osmotski aktivirana struja jer: 1. ima dozno zavisnu osetljivost na osmotski
stimulus; 2. promena sredine iz hipoosmotskih u hiperosmotske trenutno gasi IRIS. Prema
svojim osobinama IRIS se jasno razlikuje od svih do sada opisanih jonskih struja kod gljiva,
ali pokazuje brojne sličnosti sa zapreminom regulisanom anjonskom strujom (VRAC-
volume regulated anionic channel) kod kičmenjaka. Ove sličnosti su sledeće: 1. aktivacija u
uslovima osmotski izazvanog povećanja zapremine; 2. umereno izlazno ispravljanje, sa
naelektrisanjem vratnica od zg = 0,82 ± 0,1; 3. voltažno i vremenski zavisna inaktivacija na
pozitivnim potencijalima i oporavak od inaktivacije na negativnim potencijalima; 4. izražena
selektivnost za anjone u odnosu na katjone sa karakterističnom sekvencom provodljivosti
koja odgovara Ajsmanovoj seriji I (I-: Cl- : HCO3-: glukonat- : glutamat- = 1,4: 1: 0,25: 0,01:
0,088); 5. progresivno smanjenje amplitude struje u vremenu, koje usporava unutarćelijski
ATP; 6. aktivacija nehidrolizujućim analogom GTP u izoosmotskim uslovima, kao i kod
VRAC, ukazujući da se IRIS aktivira putem nekog GTP-zavisnog signalnog puta. 7.
smanjenje struje u prisustvu jona magnezijuma sa unutarćelijske strane.
Farmakološka karakterizacija IRIS je pokazala da DIDS, poznati blokator anjonskih kanala,
nema efekta na IRIS. IRIS inhibiraju niflumična kiselina (NFK) (koncentracija 0,5 mM
izaziva blok maksimalne struje od 79 ± 5% (n=4)) i antracen-9-karboksilna kiselina (A9C)
(koncentracija 1 mM blokira 68 ± 5% maksimalne struje (n=5)).
Praćenjem promena veličine citoplazmatskih kapi pri hipoosmotskom stimulusu od 45 mOsm
pokazano je da dolazi do povećanja prečnika kapi za 8 ± 4 % (n = 4), ali nije primećeno
merljivo regulisano opadanje zapremine koje bi bilo očekivano da sledi, kao način adaptacije,
nakon povećanja zapremine u hipoosmotskim uslovima, moguće usled nedovoljno
intenzivnog stimulusa.
S obzirom da su citoplazmatske kapi na čijoj membrani je prisutan IRIS poreklom od rastućih
sporangiofora, ispitan je efekat blokatora IRIS na rast micelijuma P. blakesleeanus i
utvrdjeno je da ga blokatori IRIS usporavaju ili zaustavljaju. Niflumična kiselina je efikasniji
inhibitor sa vrednostima IC50 za rast od 23 μM dok je IC50 za A9C 130 μM. Merenje
respiracije u prisustvu NFK i A9C je pokazalo da oba blokatora snažno inhibiraju disanje
micelijuma (IC50(NFK) = 50 μM a IC50(A9C) = 142 μM). Oba blokatora dovode do značajnog
smanjenja koncentracije ATP, koja u slučaju dodavanja 2 mM A9C pada za čak 91%. A9C,
ali ne i NFK, dovodi do zakišeljavanja kako citoplazme, tako i vakuole za oko 0,3 pH
jedinice. Mehanizmi ovih procesa nisu sa sigurnošću utvrđeni, ali je pokazano da postoji
čvrsta veza između energetskog metabolizma ćelije i regulacije aktivnosti IRIS. Dalja
ispitivanja mehanizama aktivacije i regulacije IRIS, kao i njihovo poredjenje sa procesima
aktivacije VRAC kod kičmenjaka, trebalo bi da omoguće bolje razumevanje uloge IRIS u
fiziologiji filamentoznih gljiva. | Cytoplasmic droplet membrane obtained from growing sporangiophore of fungus
Phycomyces blakesleeanus Burgeff is one out of two known exerimental models for
electrophysiological exploration of plasma membrane properties of filamentous fungi (the
other being Neurospora crassa slime mutant). At least 20 % of cytoplasmic droplets forms
cell wall after 2 h, confirming that their membrane coresponds functionally to hyphal plasma
membrane. This is the first patch-clamp registration in whole cell configuration obtained on
cytoplasmic droplet membrane model sistem. This configuration gives insight into types and
properties of dominant ion currents present on membrane under investigation. The membrane
response to hypoosmotic stimuli, performed by whole droplet dialysis by hyperosmotic
solution, is characterised in detail. In contrast to familly of currents under isoosmotic
conditions, that are dominated by passive membrane properties and small conductances,
under hypoosmotic conditions there is prominent outwardly rectified fast-inactivating
instantenous current (ORIC) that is activated at depolarisied potentials. In addition to ORIC
that was present in every doplet examined (with average maximal current density at +70 mV
= 129 ± 14 pA/pF (n=30)), in 73% of obtained responses there was noticable an additional
current, slowly activating inward current, current density at -150 mV = 99 ± 11 pA/pF
(n=22). The inward current was not examined in more detail.
IRIS is true osmoticaly activated current according to following properties: 1. Its osmotic
sensitivity is dose-dependent; 2. It shuts down immidiately upon change from hypoosmotic to
hyperosmotic enviroment. IRIS is clearly different from other known fungal ion channels, but
shares a number of features with vertebrate VRAC (volume regulated anionic channel): 1.
activation under conditions of osmotially induced volume increase; 2. moderate outward
rectification with gate charge zg = 0.82 ± 0.1; 3. time- and voltage-dependent inactivation at
positive potentials and recovery from inactivation at negative potentials; 4. distinct selectivity
for anions over cations, with characteristic permeability sequence corresponding to Aismann
series I (I-: Cl- : HCO3-: glukonat- : glutamat- = 1.4: 1: 0.25: 0.01: 0.088); 5. time dependent
current shut down that is slowed down by intracellular ATP; 6. activation by unhydrolizable
GTP analogs under isoosmotic conditions, pointing to ORIC being activated through GTP-
dependent cascade; 7. decrease of current in the presence of intracellular magnesium ions.
Pharmacological characterization shows that DIDS, known anion channel blocker, is not
effective on ORIC. ORIC is inhibited by niflumic acid (0.5 mM concentration induces 79 ±
5% block of maximal current (n=4)) and by A9C (1 mM A9C blocks 68 ± 5% maximal
current (n=5)).
We have recorded changes of cytoplasmic droplet diameter under 45 mOsm hipoosmotic
chalenge and found that droplets diameter increased by 8 ± 4 % (n = 4). No measurable
regulatory volume decrease has been found, probably due to ineffective hipoosmotic
stimulation.
Taking into consideration that ORIC is present on the membrane of cytoplasmic droplets
obtained from growing sporangiophores, the effect of ORIC blockers on micelia growth was
tested. It was found that blockers inhibit growth: niflumic acid was more efficient inhibitor
(IC50 = 23 μM) than A9C (IC50 = 130 μM). Respiration measurements have shown that both
blockers have strong inhibitory effect on micelia respiration (niflumic acid IC50 = 50 μM;
A9C IC50 = 142 μM). Both blockers induced a decrease in ATP concentration (with 2 mM
A9C, there was 91% decrease). Additionaly, A9C induced a pH shift (in cytosol and in
vacuole as well), and that was not observed with nif lumic acid application. Mechanisms of
these processes have not been elucidated yet, but it seems that there is a clear connection
between energetic methabolism and ORIC activity. Further studies of ORIC activation and
regulation mechanisms, as well as their comparison with VRAC regulation, should be
enabling for better understanding of ORIC role in filamentous fungus physiology. | true |
Biološka aktivnost fermentisanih mlečnih napitaka dobijenih primenom kombuhe i konvencionalnih starter kultura | Biological activity of fermented milk beverages obtained using kombucha and conventional starter culture | Proizvodnja fermentisanih mliječnih napitaka unapreijeđenih funkcionalnih karakteristika postala je jedan od glavnih fokusa u industriji prerade mlijeka. Cilj doktorske disertacije je ispitivanje biološke aktivnosti fermentisanih mliječnih napitaka dobijenih primjenom nekonvencionalne starter kulture, kombuhe (kultivisane na crnom čaju zaslađenim saharozom u koncentraciji od 10%) i poređenje sa karakteristikma proizvoda dobijenih primenom konvencionalnih starter kultura, jogurtne odnosno probiotske, tokom skladištenja. Za fermentaciju je korišćeno mlijeko sa 2,8% mliječne masti na temperatura 42°C. Promjene tokom fermentacije mlijeka primjenom kombuhe i konvencionalnih starter kultura praćene su određivanjem stepena proteolize, sadržaja laktoze, D– galaktoze, D–glukoze i D–fruktoze i masnih kiselina pri sledećim pH vrijednostima: 6,4; 6,0; 5,5; 5,0 i 4,6. Promjene antihipertenzivne aktivnosti (AKE inhibitorna aktivnost), antioksidativne aktivnosti (ABTS i DPPH metod) kao i promjene stepena proteolize, reoloških i senzornih karakteristika sve tri vrste fermentisanih mliječnih napitaka praćene su tokom 21-og dana skladištenja. Osim toga praćene su i promjene sadržaja šećera, masnih kiselina, minerala (kalcijuma, natrijuma i kalijuma), vitamina C i biogenih amina. Tokom procesa fermentacije mlijeka primjenom različitih starter kultura može se zaključiti da postoji razlika u promjenama udijela pojedinačnih proteinskih frakcija analiziranih metodom kapilarne elektroforeze. Različite starter kulture utiču na različitu AKE inhibitornu aktivnost tokom skladištenja, što ukazuje na različitu proteolitičku aktivnost korišćenih starter kultura. Utvrđeno je da AKE inhibitorna aktivnost raste tokom skladištenja, pri čemu uzorci proizvedeni primjenom kombuhe imaju najveću AKE inhibitornu aktivnost na kraju 14 dana skladištenja i ona iznosi 79,4%, dok su u jogurtu i probiotskom jogurtu te vrijednsoti 63,4 i 64,6% redom. Takođe, tokom skladištenja stepen proteolize raste u svim uzorcima sa značajnim međusobnim varijacijama. Antiksidativna aktivnost svih uzoraka opada tokom skladištenja ali je u svim uzorcima zabilježena veća aktivnost na ABTS nego na DPPH slobodne radikale. Nakon 21-og dana skladištenja najveći antioksidativni potencijal određen metodom stabilizacije ABTS.+ katjona imali su uzorci sa jogurtnom starter kulturom (TEAC vrijednost 8,922 mmolmg-1). U pogledu sastava masnih kiselina, tokom 14 dana skladištenja u kombuha fermentisanim mliječnim napicima kao i napicima dobijenim sa jogurtnom i probiotskom starter kulturom dolazi do porasta udjela zasićenih (SFA) i opadanje mononezasićenih (MUFA) i polinezasićenih masnih kiselina (PUFA). Nakon 21-og dana skladištenja sadržaj SFA; MUFA i PUFA u kombuha fermentisanom mliječnom napitku iznosio je 65,94; 30,73 i 3,33% redom, dok su te vrijednosti kod jogurta iznosile 66,02; 30,77 i 3,21% i probiotskog jogurta 66,04; 30,66 i 3,30 % redom. Najveći sadržaj vitamina C nakon proizvodnje i 14 dana skladištenja imali su uzorci sa kombuha starter kulturom (0,5457 ± 0,017 mg100g-1). Uzorci dobijeni upotrebom konvencionalnih startera pokazali su bolje reološke osobine pri ispitivanim uslovima tokom 21 dana skladištenja. Kombuha fermentisani mlečni proizvod imao je karakterističan, blago kiseli, osvežavajući ukus i naglašenu aromu. Na osnovu dobijenih rezultata biološke aktivnosti i promjena kvaliteta kombuha fermentisanog mliječnog napitka tokom skladištenja, u odnosu na karakteristike proizvoda dobijenih upotrebom konvencionalnih starter kultura može se objasniti opravdanost upotrebe kombuha starter kulture u fermentaciji mlijeka sa ciljem dobijanja novog funkcionalnog fermentisanog mliječnog proizvoda. | Nowadays, production of fermented dairy products with elevated benefits on human health has become one of the major focuse in dairy industry. The aim of the PhD thesis is to examine the biological activity of fermented milk products obtained using non-conventional starter culture kombucha (cultivated on black tea with 10% of sucrose) and comparision with products obtained by conventional starter cultures, probiotic/yoghurt during storage. Milk with 2.8% of milk fat was used for the samples production at temperature of 42 °C. The changes of components content during the milk fermentation by kombucha and conventional starter cultures were monitored at the following pH values: 6.4; 6.0; 5.5; 5.0 and 4.6., by determining the degree of proteolysis, lactose, D-galactose, D-glucose and D-fructose, fatty acids. The antihypertensive activity (ACE inhibitory activity), antioxidant activity (ABTS and DPPH tests) and the degree of proteolysis, sensory and rheological characteristics of all three types of fermented milk products were observed during 21 days of storage. Moreover, the chemical qualities of samples were monitored analyzing the contents of sugars, fatty acids, minerals (calcium, sodium and potassium), vitamin C and biogenic amines. There were differences in protein fractions (analyzed by capillary electrophoresis) of products obtained by using different starter cultures during the milk fermentation. Different starter cultures affect different ACE inhibitory activity during the storage, which implies different proteolytic activity of used starter cultures. It has been found that the ACE inhibitory activity was increased during the storage; wherein the samples obtained using kombucha starter culture have the highest ACE inhibitory activity at the 14th day of storage, 79,4%, while in yogurt and probiotic yoghurt it was 63.4 and 64.6% respectively. Also, the degree of proteolysis during the storage was increased in all samples with significant mutual variations. In all products, higher ABTS than 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined, while both activities slightly decreased during the storage. The antioxidant activity of all samples decreases during storage. After 21 days of storage, the highest antioxidant potential, determined by the ABTS. + method had a yoghurt samples (TEAC value of 8.922 mmolmg-1). In terms of the fatty acids composition during 14 days of storage in all type of fermented dairy products relative content of SFA (saturated fatty acids - SFA) increased, while relative contents of MUFA (monounsaturated fatty acids) and PUFA (polyunsaturated fatty acids) decreased during that period of storage. After 21 days of storage the content of SFA; MUFA and PUFA in kombucha fermented milk product was 65.94; 30.73 and 3.33% respectively. In yogurt sample their content was 66.02, 30.77 and 3.21%, while in probiotic 66.04; 30.66 and 3.30% respectively. In all fermented milk products, long chain fatty acids were dominant with a total share of about 45% in all varieties of fermented dairy products. The highest content of vitamin C after production and 14 days of storage was in samples obtained by kombucha starter culture (0.5457 ± 0.017 mg100g-1). Samples obtained by conventional starter showed better overall rheological properties at the tested conditions for 21 days of storage. Kombucha fermented milk product had a characteristic, distinctive mild sour, refreshing taste and conspicuous aroma. The obtained results of biological activity and the quality of kombucha fermented milk products during storage in comparison to the same characteristics of the products obtained using conventional starter culture, could explain that kombucha is convenient starter for milk fermentation with the aim of obtaining new functional fermented milk products with pronounced bioactive characteristics and distinctive sensory and rheology properties. | false |
Diferencijacija vrsta iz sekcije Juniperus roda Juniperus L. (Cupressaceae) sa Balkanskog poluostrva, korišćenjem fitohemijskih i molekularnih karaktera | Differentation of species within Section Juniperus of Genus Juniperus L. (Cupressaceae) from the Balkan peninsula using phytochemical & molecular characters | U ovom radu analizirana je fitohemijska i molekularna varijabilnost i diferencijacija prirodnih populacija taksona sekcije Juniperus roda Juniperus (Cupressaceae) sa Balkanskog poluostrva... | This thesis deals with phytochemical & molecular variability variability & differentation of natural population of taxa belonging to section Juniperus of the genus Juniperus (Cupressaceae) from the Balkan peninsula... | false |
Effects of anthocyanins and their metabolites on the function of human endothelial cells and platelets in vitro | Uticaj antocijana i njihovih metabolita na funkciju endotelnih ćelija i trombocita čoveka in vitro | Kardiovaskularne bolesti predstavljaju oboljenja koja zahvataju srce i krvne sudove i najčešći su uzrok obolevanja i umiranja u svetu. Prema mestu javljanja mogu se podeliti na bolesti srca, cerebrovaskularne bolesti i bolesti perifernih krvnih sudova. Osnovni uzročnik kardiovaskularnih bolesti je ateroskleroza, hronično zapaljensko oboljenje velikih i srednje velikih arterijskih krvnih sudova koje se karakteriše aterosklerotičnim lezijama koje nastaju nakupljanjem lipida, ćelija i proteina vezivnog tkiva unutar zida krvnog suda. Ove patološke promene krvnih sudova ometaju normalan protok krvi, a u kasnijim stadijumima bolesti mogu dovesti i do pucanja plaka i aterotromboze, što za posledicu može imati infarkt miokarda ili šlog. Sam nastanak ateroskleroze povezan je sa poremećenom funkcijom endotela krvnog suda koja podstiče adheziju leukocita za aktivirane endotelne ćelije i njihovu transendotelnu migraciju. Unutar zida krvnog suda monociti se diferenciraju u makrofage, vrše ingestiju oksidovanog LDL holesterola i formiraju penušave ćelije čije nakupljanje vremenom dovodi do formiranja aterosklerotičnih lezija. Važnu ulogu u nastanku ateroskleroze imaju i trombociti. Interakcija aktiviranih trombocita sa leukocitima i endotelnim ćelijama podstiče adheziju leukocita i njihovu transendotelnu migraciju i time dodatno podstiče zapaljenski proces i razvoj ateroskleroze. Stoga, ispitivanje uzajamnog delovanja endotelnih ćelija, leukocita i trombocita predstavlja značajno područje istraživanja u cilju prevencije i kontrole kardiovaskularnih bolesti. Ishrana ima važnu ulogu, kako u nastanku i razvoju kardiovaskularnih bolest, tako i u njihovoj prevenciji i lečenju. Rezultati brojnih epidemioloških i kliničkih studija pokazuju povoljno delovanje ishrane bogate voćem i povrćem na kardiovaskularno zdravlje. Ovo pozitivno delovanje namirnica biljnog porekla pripisuje se delom njihovom niskom energetskom vrednošću, visokom sadržaju vlakana i esencijalnih mikronutrijenata, ali pre svega nenutritivnim, biološki aktivnim sastojcima, poput polifenola... | Increasing number of scientific evidence suggests the beneficial role of dietary anthocyanins, phytochemicals mainly present in berries and derived products, in cardiovascular health. These anthocyanin health benefits may be attributed to their effect on endothelial cells or platelets that represent the key players in the development of cardiovascular diseases (CVD). However, the exact molecular mechanisms underlying anthocyanin cardioprotective effects are not fully understood. The aim of this thesis was to investigate the effect of anthocyanins and their metabolites in vitro on endothelial and platelet function and identify the underlying mechanisms of their action using physiologically relevant conditions. Results from this thesis showed that the pretreatment of endothelial cells with physiologically relevant concentrations of circulating anthocyanins and their metabolites attenuated monocyte adhesion to activated endothelial cells as well as their transendothelial migration, which are the initial steps in the development of atherosclerosis that precede CVD. In agreement with these results, gene expression analysis revealed that the treatment of endothelial cells with these compounds modulated the expression of genes involved in regulation of cell-cell adhesion, actin cytoskeleton reorganisation, focal adhesion and leukocyte transmigration. Bioinformatics analyses of gene expression data allowed the identification of potential transcription factors involved in the observed nutrigenomic effects and cell signalling proteins regulating their activity. Molecular docking analyses further revealed cell signalling proteins to which these bioactives may bind to and potentially affect their activity and the activation of downstream signalling proteins and transcription factors, effects that were in agreement with the results of Western blot analyses. Anthocyanins and their metabolites also modulated the expression of microRNAs, especially those involved in regulation of endothelial cell permeability, contributing to the observed changes in endothelial cell function... | false |
Uklanjanje fenola iz otpadnih voda sortama zelene salate (Lactuca sativa L.) | Removal of phenol from wastewater using different verieties of lettuce (Lactuca sativa L.) | U ovoj doktorskoj disertaciji ispitivana je mogućnost uklanjanja fenola iz rastvora,
korišćenjem različitih sorti zelene salate, sa ciljem njihove upotrebe u procesu
fitoremedijacije. Ispitivano je 11 različitih sorti zelene salate, čija semena su klijala na
podlogama sa različitim koncentracijama fenola (0-400 mgL-1) tokom 20 dana. Pokazano je
da dve sorte zelene salate mogu klijati na visokim koncentracijama fenola. Sorta Ljubljanska
ledenka (LJL) klija na koncentraciji fenola do 350 mgL-1 i sorta Nansen (N) podnosi
koncentraciju fenola do 300 mgL-1. Dalja istraživanja aktivnosti antioksidativnih enzima
peroksidaza (POX), katalaza (CAT), kao polifenol oksidaza (PPO) rađena su na klijancima
koji su gajeni na inhibitronim koncentracijama fenola (IK50), odnosno onim koncentracijama
fenola, specifičnim za svaku sortu, na kojima klija 50% semena. Rezultati su prikazani za
četiri sorte zelene salate i pokazano je da je aktivnost POX relativno niska, dok je aktivnost
CAT i PPO viša u odnosu na klijance koji su gajeni bez prisustva fenola. Uporedo je praćena i
morfologija klijanaca koji su gajeni na IK50 fenola. Praćen je tempo uklanjanja fenola iz
otpadne vode i analiziran je tokom gajenja odrasle zelene salate u hidroponičnim uslovima.
Odabrane su dve sorte zelene salate (LJL i N) koje su pokazale najveću otpornost na prisustvo
fenola, i gajene su na koncentraciji fenola u rastvoru od 200 mgL-1. Ova koncentracija fenola
je najviša koncentracija ispod IK50 na kojoj klija 100% semena. Pomenuta koncentracija
fenola dodavana je u hidroponični rastvor na početku eksperimenta (početna koncentracija
fenola) ili svaka dva dana tokom trajanja eksperimenta (konstantna koncentracija fenola).
Praćena je promena koncentracije fenola u rastvoru posle 2, 4, 6, 8 i 10 dana gajenja, kao i
aktivnost enzima (POX, CAT, SOD i PPO) u korenu i listovima zelene salate. Obe sorte
zelene salate uklanjaju fenol iz rastvora za osam dana, a brzina uklanjanja fenola je veća kod
sorte LJL u odnosu na sortu N. Fenol konstantne koncentracije biljke obe sorte nisu u
mogućnosti da u potpunosti uklone, a kapacitet za uklanjanje fenola kontantne koncentracije
gubi se posle osam dana gajenja. Kod svih ispitivanih enzima, osim POX, primećen je porast
aktivnosti tokom gajenja biljaka na fenolu. Pored promena u enzimskoj aktivnosti, primećena
je promena u koncentraciji prolina, hlorofila, ukupnih pigmenata, kao i anatomiji korena
zelene salate koja je gajena na fenolu. Uspešno je dobijena kultura hairy roots zelene salate
genetičkom transformacijom pomoću Agrobacterium rhizogenes A4M70GUS, a najveći
prinos biomase imala je linija Ljubljanska ledenka 18 (LJL18), koja je dalje gajena na
različitim koncentracijama fenola u rastvoru, da bi se ispitala mogućnost fitoremedijacije
pomoću kontinualne kulture korenova. Pokazano je da linija LJL18 uklanja fenol početne
koncentracije od 100 mgL-1 iz rastvora za deset dana, dok kultura netransformisanih korenova
iste sorte može ukoliniti najviše 50 mgL-1 fenola. Pored praćenja morfologije transformisanih
korenova gajenih na fenolu, analizirana je i aktivnost enzima (POX, CAT, SOD i PPO) posle
2, 4, 6, 8 i 10 dana gajenja. Primećena je značajna promena u aktivnosti enzima tokom gajenja
hairy roots na početnoj ili konstantnoj koncentraciji, kao i razlika u odnosu na
netransformisane korenove podvrgunte istom tretmanu. Rezultati ove doktorske disetracije
dokazuju da se zelena salata može uspešno koristiti za uklanjanje fenola iz otpadnih voda
korišćenjem celih biljaka ili hairy roots kulture.
Naučna oblast: Biologija
Uža naučna oblast: Fiziologija biljaka
UDK broj: 581.1:635.52]:502.175:547.56(043.3) | In this doctoral dissertation, the possibilities of use of various cultivars of lettuce have been
investigated for removal of dissolved phenol in the process of phytoremediation. Seeds of 11
different cultivars of lettuce germinated on media with different phenol concentrations (0-400
mgL-1) over 20 days. It has been shown that seeds of two cultivars of lettuce can germinate
even on high phenol concentrations: Seeds of Ljubljanska ledenka (LJL) can germinate on
350 mgL-1, while Nansen (N) tolerates as high as 300 mgL-1. Further investigation of
antioxidant enzyme proxidases (POX), cataleses (CAT) and polyphenol oxidase (PPO)
activity was carried out on seedlings grown on inhibitory concentrations of phenol (IC50),
that is, the cultivar-specific concentration of phenol reducing the germination efficiency to
50% for the seeds of a given cultivar. Results are shown for four lettuce cultivars, revealing
that POX activity is relatively low, while CAT and PPO activity show higher values
compared to seedlings grown without phenol. Morphological features of seedlings grown on
IC50 of phenol were also recorded. Dynamics of phenol removal from waste water were
recorded in adult lettuce plants grown hydroponically. Two lettuce cultivars that showed the
highest tolerance to phenol, were chosen for hydroponic growth at 200 mgL-1 phenol, which
is the highest concentration on which 100% germination has been recorded for these cultivars.
This concentration of phenol was either established in the hydroponic solution once, at the
beginning of the experiment (this was called the "starting phenol concentration") or
maintained by supplying additional phenol to the solution every two days (which was called
the "constant phenol concentration"). Changes in phenol concentration in the solution, as well
as enzyme activity (POX, CAT, SOD and PPO) in lettuce roots and leaves were recorded
after 2, 4, 6, 8 and 10 days of growth. Both lettuce cultivars are able to completely remove the
starting concentration of phenol from the solution within eight days, the cultivar LJL being
more efficient than N. Plants of both cultivars were unable to completely remove the phenol
at constant concentration. Furthermore, they lost the capacity for removing constant
concentrations of phenol after eight days. All the investigated enzymes except POX show a
trend of activity increase over the course of growth on media containing phenol. Beside
changes in enzyme activity, changes in endogenous concentrations of proline, chlorophyll,
total pigments, as well as changes in root anatomy of lettuce grown on phenol, were also
recorded. A culture of lettuce hairy roots was successfully established with the use of
Agrobacterium rhizogenes A4M70GUS, with the highest value for biomass yield being
recorded for the clone Ljubljanska ledenka 18 (LJL 18), which was further cultivated on
different concentrations of phenol in the solution, in order to investigate the potential of use
of continuous root cultures for phytoremediation purposes. It has been shown that LJL18 can
remove the 100 mgL-1 starting concentration of phenol from the solution within 10 days,
while the culture of non-transformed roots of the same cultivar can remove only 50 mgL-1
phenol. Significant changes in POX, CAT, SOD and PPO activity were recorded over the
course of hairy roots growth on starting or constant concentrations of phenol, as well as a
difference in comparison to non-transformed roots growing in the same conditions. The
results of this doctoral dissertation show that lettuce, either in the form of intact plants or
hairy roots cultures, can be successfully used for removing phenol from waste waters.
Scientific field: Biology
Specific scientific field: Plant Physiology
UDC number: 581.1:635.52]:502.175:547.56(043.3) | true |
Fiziološki i biohemijski aspekti propagacije endemičnih vrsta Micromeria pulegium (Rochel) Benth. i Micromeria croatica (Pers.) Schott in vitro | Physiological and biochemical aspects of propagation in endemic species Micromeria pulegium (Rochel) Benth. and Micromeria croatica (Pers.) Schoot in vitro | aromatičnih biljaka koje se koriste u tradicionalnoj medicini, hortikulturi, hemijskoj,
prehrambenoj i kozmetičkoj industriji. Retke, endemične i/ili ugrožene vrste familije
Lamiaceae predstavljaju poseban izazov za proučavanje, prevashodno u cilju očuvanja
specijskog diverziteta i njihovih populacija u prirodi, ali i zato što su izvor važnih
bioaktivnih molekula raznovrsnog dejstva (antimikrobna, antvirusna, fungicidna,
antioksidantna, citotoksična, alelohemijska, insekticidna aktivnost, itd.) i potencijalno
široke primene.
Usled izrazite heterogenosti, predstavnici roda Micromeria su bili često predmet
taksonomskih diskusija. Pripadnike sekcije Pseudomelissa gde spada i M. pulegium,
nakon molekularno genetičkih analiza hloroplastne DNK, Brauchler (2005) premešta u
rod Clinopodium. Ipak, u ovoj tezi je primenjen tradicionalni taksonomski koncept,
predložen od strane Harley-a i sar. (2004), koji podržava homogenost roda Micromeria,
pri čemu je sect. Pseudomelissa njegova sastavna jedinica. U skladu sa primenjenim
sistemom dve istraživane vrste su klasifikovane u idividualne sekcije roda: Micromeria
sect. Micromeria (M. croatica), odnosno Micromeria sect. Pseudomelissa (M.
pulegium).
Micromeria pulegium predstavlja endemičnu vrstu Južnih Karpata u Rumuniji,
sa enklavom u istočnoj Srbiji, a Micromeria croatica balkansku endemičnu vrstu čiji se
areal vezuje pre svega za planinski venac Dinarida. Njihove prirodne populacije imaju
mali broj jedinki i nastanjuju staništa koja su pod negativnim uticajem antropogenih
faktora. Imajući u vidu značaj roda Micromeria, kao i činjenicu da su pomenute vrste
retke i ugrožene, javila se potreba da se one gaje zarad očuvanja i istraživanja bez
pritiska na prirodne populacije i bez remećenja prirodnog genofonda. Iz pomenutih
razloga obe vrste roda Micromeria uvedene su kulturu biljnih tkiva in vitro.
U ovoj disertaciji vršeno je ispitivanje efekata regulatora rastenja na
morfogenezu i produkciju etarskih ulja endemičnih vrsta M. pulegium i M. croatica in
vitro. Kultura in vitro M. pulegium uspostavljena je korišćenjem segmenata stabla sa
nodusom i dva listića. Uslovi pod kojima se ostvaruje najbolje razviće eksplantata su
bili sledeći: najveći broj pupoljaka formiran je na eksplantatima koji su rasli na MS
hranljivoj podlozi sa 3 µM BA. Dužina aksilarnih izdanaka bila je najveća na podlozi sa
0,3 µM BA i 0,57 µM IAA. Najveći prinos sveže i suve mase ostvaren je gajenjem
izdanaka na podlozi sa 10 µM BA. Ožiljavanje izdanaka bilo je uspešno na MS/2
hranljivoj podlozi sa 1 mg/L IAA. Dobijene biljke uspešno su se aklimatizovale i time
je regeneracija biljaka M. pulegium bila potpuna.
Kultura in vitro M. croatica uspostavljena je aseptičnim isklijavanjem semena, a
zatim sa dobijenih biljaka su izolovani segmenti stabla sa nodusom i parom listića.
Najveći broj pupoljaka formiran je na eksplantatima M. croatica gajenim na MS
hranljivoj podlozi sa 0,3 µM kinetina. Na istoj podlozi, dužina aksilarnih izdanaka je
bila najveća i postignut je najveći prinos sveže mase izdanaka. Najbolji prinos suve
mase imali su izdanci gajeni na MS podlozi sa 10 µM BA i 0,57 µM IAA. Ožiljavanje
aksilarnih izdanaka bilo je uspešno na MS/2 hranljivoj podlozi sa 0,3 mg/L IAA.
Uspešnom aklimatizacijom, postignuta je potpuna regeneracija M. croatica.
Kvantitativnom i kvalitativnom analizom biohemijskih parametara regenerisanih
izdanaka vršeno je upoređenje sastava etarskih ulja biljaka iz prirode i in vitro gajenih
izdanaka na MS hranljivoj podlozi bez fitohormona i podlozi na kojoj je dobijen najveći
prinos biomase. To je za M. pulegium podloga sa 10 µM BA, a za M. croatica podloga
sa 0,3 µM kinetina. U svim uljima M. pulegium dominiraju pulegon i menton. Pulegon
je najzastupljeniji u ulju samoniklih biljaka, a menton u ulju poreklom od izdanaka
gajenih na MS podlozi bez fitohormona. U ulju biljaka M. croatica iz prirode
dominantne komponente su borneol, α-kadinen i β-vetivenen. U ulju izdanaka
propagiranih in vitro na MS podlozi bez fitohormona dominantne komponente su
borneol, geranial, cis-p-menta-1(7),8-dien-2-ol i β-vetivenen, a u ulju izdanaka gajenih
na podlozi sa kinetinom dominantne komponente su geranial, cis-p-menta-1(7),8-dien-
2-ol i α-kadinen.
Analiziran je hemijski sastav metanolnih, etil-acetatnih i heksanskih ekstrakata,
biljaka iz prirode i izdanaka gajenih in vitro na MS podlozi bez prisustva fitohormona.
U ekstraktima obe vrsta Micromeria identifikovana su 53 jedinjenja, od kojih je 42,22%
zajedničko za ekstrakte M. pulegium iz prirode i kulture in vitro, odnosno 67,65%
zajedničko za ekstrakte M. croatica iz prirode i kulture in vitro.
Analizom antioksidativnih i antimikrobnih svojstava metanolnih ekstrakata
utvrđena je veća vrednost ukupne redukcione moći, kao i sadržaja ukupnih polifenola
kod izdanaka gajenih u uslovima in vitro (bez fitohormona) u odnosu na biljke iz
prirode. Metanolni ekstrakti M. pulegium i M. croatica delovali su na sve testirane
bakterijske sojeve, ali znatno slabije u odnosu na referentne antibiotike. | Plants from Lamiaceae family are prominent representatives of the edible, medicinal
or aromatic herbs used in traditional medicine, horticulture, chemical, cosmetic and food
industry. The rare, endemic and/or threatened species from family Lamiaceae pose a special
challenge for studying, primarily in order to preserve species diversity and their populations
in the wild, but also as they are source material for important bioactive molecules with
diverse activity (antimicrobial, antiviral, fungicidal, antioxidant, cytotoxic, allelochemical,
insecticidal and other types of activity) and potential broad spectrum of use.
Due to the pronounced heterogeneity, representatives of genus Micromeria used to be
a common topic of taxonomic discussions. After the molecular-genetic analyses of
chloroplast DNA, Brauchler (2005) has transferred the representatives of section
Pseudomelissa, which includes М. pulegium, into the genus Clinopodium. However, this
thesis is using the traditional taxonomic concept suggested by Harley et al. (2004),
supporting the homogeneity of genus Micromeria, where sect. Pseudomelissa remains its
integral unit. According to this system the two studied species were classified within the
individual sections of the genus: Micromeria sect. Micromeria (M. croatica) and Micromeria
sect. Pseudomelissa (M. pulegium).
Micromeria pulegium represents an endemic species of Southern Carpathians in
Romania, with an enclave in Eastern Serbia, while Micromeria croatica is an endemic
Balkan species primarily connected with the mountain chain of Dinarides. Their natural
populations are characterized by a small number of individuals inhabiting habitats under the
negative influence of anthropogenic factors. Due to the importance of genus Micromeria and
the fact that these two species are rare and threatened, a need arose to grow them in captivity
so they may be preserved and studied without additional pressure on natural populations and
without disturbance of the natural gene pool. For these reasons both species of genus
Micromeria were introduced in plant tissue culture in vitro.
This dissertation is focusing on studying the effects of growth regulators on
morphogenesis and production of essential oils in endemic species M. pulegium and M.
croatica in vitro.
Culture in vitro of М. pulegium was established by using stem segments with a node
and two leaflets. The conditions providing the best development of explants were as
following: the greatest number of buds was formed in explants grown on MS medium with 3
µM BA. The length of axillary stems was the greatest at the medium with 0.3 µM BA and
0.57 µM IAA. The greatest yield of fresh and dry biomass was recorded in stems grown on
medium with 10 µM BA. The stems have successfully formed roots on MS/2 nutritive
medium with 1 mg/L IAA. The new plants have acclimatized successfully and regeneration
of M. pulegium was completed.
Culture in vitro of M. croatica was established by aseptic germination of seeds, and
the resulting plants were used to isolate stem segments with a node and a pair of leaflets. The
greatest number of buds, as well as the longest buds was formed on explants of М. croatica
grown on MS nutritive medium with 0.3 µM kinetin. The highest yield of fresh biomass of
stems was achieved on the same medium. The highest yield of dry mass was achieved by
stems grown on MS medium with 10 µM BA and 0.57 µM IAA. The axillary shoots have
successfully formed roots on MS/2 nutritive medium with 0.3 mg/L IAA. After the successful
acclimatization the regeneration of М. croatica was complete.
Quantitative and qualitative analyses of biochemical parameters of regenerated stems
were used for comparison of composition of essential oils in plants taken from nature and
stems grown in vitro on MS nutritive medium without phytohormones and on the medium
with the highest yield of biomass. For M. pulegium this was the medium with 10 µM BA,
while for M. croatica it was the medium with 0.3 µM of kinetin. All oils of M. pulegium are
dominated by pulegone and menthone. Pulegone has shown the highest levels in oil from
plants collected in the wild, while menthone was most prominent in oil from stems grown on
MS substrate without phytohormones. The oil of M. croatica taken from the wild was
dominated by borneol, α-cadinene and β-vetivenen. The dominant components in oil of stems
propagated in vitro on MS substrate without phytohormones were borneol, geranial, cis-p-
menta-1(7),8-dien-2-ol and β-vetivenen, while in the oil from stems grown on substrate with
kinetin the dominant components were geranial, cis-p-menta-1(7),8-dien-2-ol and α-cadinene.
Analysis was also performed on the chemical composition of methanol, ethyl-acetate
and hexane extracts in plants collected in the wild and stems grown in vitro on MS medium
without phytohormones. The extracts of both species of Micromeria included a total of 53
compounds, 42,22% were shared by extracts of M. pulegium in wild populations and culture
in vitro, while 67,75% were shared by extracts of M. croatica in wild populations and culture
in vitro.
The analysis of antioxidant and antimicrobial activity of methanol extracts has shown
higher values of total reductive power as well as the content of total polyphenols in stems
grown in vitro (without phytohormones) when compared to plants in the wild. The methanol
extracts of M. pulegium and M. croatica have shown effect on all tested bacterial strains, but
much weaker than the referent antibiotics. | true |
Molekularni markeri poremećaja koagulacije, inflamacije, apoptoze, formiranja i remodelovanja koštanog tkiva kod Leg-Kalve-Pertesove bolesti | Molecular markers of impaired coagulation, inflammation, apoptosis, formation and bone remodeling processes in Legg-Calve-Perthes disease | Leg-Kalve-Pertesova (LKP) bolest je idiopatska avaskularna osteonekroza epifize femura. Etiologija ove bolesti je nepoznata. Uprkos 100 godina istraživanja mehanizma njene patofiziologije i detaljnog opisivanja kliniĉkih i radioloških karakteristika, LKP bolest i dalje predstavlja jednu od najkontroverznijih bolesti u oblasti pedijatrijske ortopedije. Smatra se da je ova bolest multifaktorijalna, izazvana kombinacijom sredinskih i genetiĉkih faktora, ali se o genetiĉkim faktorima koji doprinose njenom razvoju vrlo malo zna. Glavni cilj ovog istraživanja je bio da se analiziraju molekularni markeri procesa koagulacije, inflamacije, apoptoze, formiranja i remodelovanja koštanog tkiva u LKP bolesti. Rezultati ovog istraživanja mogu doprineti identifikovanju prediktivnih genetiĉkih i molekularnih markera, što bi pomoglo u postavljanju dijagnoze i razvoju novih terapijskih pristupa, koji bi omogućili skraćenje perioda tokom kojeg je glava femura podložna deformitetu. Koagulacioni faktori su bili prvi genetiĉki faktori za koje se pretpostavilo da bi mogli doprineti razvoju LKP bolesti, meĊutim dobijeni su kontroverzni rezultati. Zbog toga je jedan od ciljeva ovog istraživanja bio analiza asocijacije varijanti gena ĉiji produkti uĉestvuju u procesu koagulacije: Faktor V G1691A (Leiden mutacija) (rs6025), Faktor II G20210A (rs1799963), MTHFR C677T (rs1801133) i PAI-1 4G/5G (rs1799889) sa pojavom LKP bolesti kod pacijenata iz Srbije. Navedene genske varijante su analizirane PCR-RFLP metodom i direktnim sekvenciranjem PCR fragmenta. PoreĊenjem uĉestalosti genotipova i alela navedenih genskih varijanti izmeĊu grupe pacijenata i kontrolne grupe nije pronaĊena statistiĉki znaĉajna razlika... | Legg-Calve-Perthes (LCP) disease is the idiopathic avascular osteonecrosis of the hip in children, with an unknown etiology. Despite nearly 100 years of detailed characterization of its clinical and radiological features, as well as a research devoted to the pathophysiology of this disease, LCP disease still remains one of the most controversial conditions in pediatric orthopedics. The prevailing view is that LCP disease is a multifactorial, caused by a combination of environmental and genetic factors, but the contribution of genetic factors remains largely unknown. The main objective of this study was to analyse the molecular markers of coagulation, inflammation, and apoptosis processes, as well as formation and bone remodeling in LCP disease. The obtained knowledge could contribute to the identification of the predictive genetic and molecular markers that would help in the diagnosis and development of new therapeutic approaches that would shorten the period during which the femoral head is susceptible to deformation. Coagulation factors were the first genetic factors suspected to have a role in the pathogenesis of this disease, but studies have shown inconsistent results. For this reason, one of the aims of this study was to analyse the association of variants of genes involved in coagulation: FV G1691A (Leiden mutation) (rs6025), FII G20210A (rs1799963), MTHFR C677T (rs1801133) and PAI-1 4G/5G (rs1799889), with LCP disease, in a patient group from Serbia. These genetic variants were determined by PCR-RFLP and direct sequencing methods. When genotype and allele frequencies of these genetic variants were compared among patient and control groups, no significant differences were observed... | false |
Unapređenje bezbednosti saobraćaja na putno-pružnim prelazima | Safety improvement of traffic at railway crossings | U ovom radu su istraženi statistički modeli za procenu frekvencije nesreća, težine
posledica
nesreća
i
empirijskog
rizika
koji
najviše
odgovaraju
lokalnim
karakteristikama putnih prelaza Srbiji u cilju identifikacije mesta visokog rizika na
mreži. Uzorak putnih prelaza lociranih na železničkoj mreži u Srbiji je korišćen za
istraživanje sa istorijskim podacima o nesrećama od pet godina. Korišćeni su modeli
regresione analize (Puasonova regresija, Puasonov model sa viškom nula – ZIP,
negativni binomni model – NB, NB model sa viškom nula – ZINB). Za modeliranje
frekvencije nesreća najbolje se pokazao ZIP model. Kod modeliranja i procene težine
posledica nesreća korišćen je multinomni logit model. Za modeliranje empirijskog
rizika podacima najviše odgovara ZINB model. Identifikovane su promenljive koje su
značajno povezane sa brojem i posledicama nesreća. Konačno, kalibrisani modeli
zajedno sa dva kriterijuma za rangiranje koji su primenjeni u ovom radu su poslužili za
identifikaciju putnih prelaza visokog rizika na mreži Železnica Srbije. Prvi kriterijum je
srednji totalni rizik na putnom prelazu, a drugi kriterijum je procena rizika koja je
zasnovana na srednjem empirijskom riziku.
Prihvaćeni modeli frekvencije i posledica nesreća su poslužili za procenu redukcije
nesreća na putnim prelazima primenom odgovarajućih tehničkih mera za podizanje
nivoa bezbednosti. U tu svrhu urađena je sveobuhvatna analiza tehnologija i mera iz
drugih regiona, a koje se danas primenjuju ili se ispituje njihova efikasnost. Koristeći
njihova iskustva data je ocena efikasnosti koju bi te mere imale na putnim prelazima u
Srbiji.
U ovom radu vršeno je i neposredno istraživanje ponašanja vozača na putnom
prelazu osiguranom drumskom signalizacijom, kao i nakon osiguranja istog putnog
prelaza aktivnim sistemom. Ovaj prelazak sa jednog sistema osiguranja na drugi sistem
bio je pogodan da bi se sprovelo istraživanje u cilju sagledavanja ponašanja vozača u
dva različita sistema, kao i bezbednosnih efekata koji su proizašli iz toga. | In this paper there were developed statistical models for estimation of accident
frequency, accident severity and empirical risk, that correspond to local characteristics
of crossings in Serbia. The goal was to identify high risk locations. The data used were
from five year period. Several regression models were tried (Poisson regression, Zero-
inflated Poisson – ZIP, negative binomial model – NB, Zero-inflated NB model –
ZINB). The most suitable model for modeling accident frequency was ZIP model. For
modeling accident severity multinomial logit model was used, whereas the best model
for empirical risk was ZINB model. The variables significantly linked to accident
frequency and severity were identified. Finally, the calibrated models together with two
ranking criteria were used in order to indentify high risk crossings in Serbian railway
network. The first criterion was mean total risk at a crossing, and the second one was
based on mean empirical risk.
The accepted models for accident frequency and severity were used for estimating
the reduction of accidents at railway crossings applying the corresponding technical
procedures for safety improvement. In order to accomplish this, there has been
performed a complete analysis of technologies and procedures from various regions,
which are used nowadays or are in phase of examining there efficency. Their results and
experience were used to estimate the efficency that those procedures would have in
Serbia.
In this paper the drivers' behaviour on railway crossing was also studied, both with
road signs and with active warining devices. This upgrade from one system to another
was useful for examining drivers' behaviour in two different systems, as well as the
safety effects that concequently appeared. | true |
Značaj polimorfizama u genima za glukokortikoidni i adrenokortikotropni receptor u nastanku adrenalnih incidentaloma | Importance of polymorphisms in glucocorticoid receptor and adrenocorticotropic receptor genes in development of adrenal incidentalomas | UVOD: Glukokortikoidni hormoni (GC) ostvaruju svoje efekte vezivanjem za
glukokortikoidni receptor (GR). Adrenokortikotropni hormon (ACTH) reguliše sintezu
GC vezivanjem za ACTH receptor (ACTHR). Prisustvo polimorfizama u genu za GR
(BclI, N363S, ER22/23EK and A3669G) i promotoru ACTHR može uticati na efekte
glukokortikoida i predispoziciju za nastanak unilateralnih adrenalnih incidentaloma.
CILJ RADA: Utvrđivanje mogućeg uticaja funkcionalnih polimorfizama u genima za
GR i ACTHR na predispoziciju za nastanak adrenalnih incidentaloma i osetljivost na GC
i ispitivanje ekspresije GR u tumorskom, peritumorskom i zdravom adrenokortikalnom
tkivu.
METODE: U ispitivanje je bilo uključeno 112 pacijenata i 100 zdravih dobrovoljaca,
koji su podvrgnuti metaboličkom, genetičkom, biohemijskom i antropometrijskom
testiranju. DNK je dobijena iz leukocita periferne krvi. Prisustvo polimorfizama je
detektovano metodama PCR, RFLP i sekvenciranja DNK. Uzorci tkiva su analizirani
imunohistohemijskom metodom.
REZULTATI: Prisustvo dužeg C alela BclI (p<0.001) polimorfizma i kraćeg G alela
A3669G (p<0.001) polimorfizma GR gena su bili nezavisni prediktori adrenalnih
incidentaloma. Pacijenti sa prisutnim C alelom BclI su imali veće tumore (p=0.002), a oni
sa G alelom A3669G više vrednosti postdeksametazonskog kortizola (p=0.025).
Istovremeno prisustvo oba alela je koreliralo sa manjim obimom struka (p=0.002), a
višim baznim i postdeksametazonskog kortizolom (p=0.024). Smanjena ekspresija GRα i
GRβ izoformi zapažena je u tumorskom, a GRα u peritumorskom tkivu. Lokalizacija
GRβ je bila dominantno nukleusna.
ZAKLJUČAK: Prisustvo C alela BclI i G alela A3669G polimorfizmama gena za GR se
nalaze u vezi sa nastankom unilateralnih adrenalnim incidentalomima, a njihovo
zajedničko prisustvo dovodi do smanjene osetljivosti na GC. Stečena intraadenomatozna
glukokortikoidna rezistencija može da dovede do disregulacije produkcije kortizola i
rasta tumora u isto vreme, dok prirodna osetljivost na glukokortikoide najverovatnije
modifikuje ove efekte. | INTRODUCTION: Glucocorticoid hormones (GCs) accomplish their effects through
binding to glucocorticoid receptor (GR). Adrenocorticotropic hormone (ACTH) regulates
synthesis of GCs through binding to ACTH receptor (ACTHR). Presence of common GR
gene (BclI, N363S, ER22/23EK and A3669G) and ACTHR promoter polymorphisms can
modulate GCs sensitivity.
OBJECTIVE: The aim of present study was to determine weather functional
polymorphisms in GR and ACTHR genes influence susceptibility for unilateral adrenal
incidentalomas and GC sensitivity, and to investigate GR expression in tumorous,
peritumorous and normal adrenocortical tissue samples.
METHODS: The study included 112 patients with adrenal incidentalomas and 100
population-matched controls. All subjects underwent metabolic, genetic, biochemical and
anthropometric testing. DNA was obtained from peripheral blood leucocytes. The
polymorphisms were detected using PCR, RFLP and DNA sequencing. Tissue samples
were studied by immunohistochemistry.
RESULTS: GR gene variant, C allele of BclI (p<0.001) and G allele of A3669G
(p<0.001) polymorphisms were independent predictors of adrenal incidentalomas.
Patients with present C allele had larger tumors (p=0.002), but those with G allele had
higher postdexamethasone serum cortisol (p=0.025). Both allele carriers had lesser waist
circumference (p=0.002), higher basal (p=0.024) and postdexamethasone cortisol
concentrations. In tumorous tissues GRα and GRβ isoforms had lower expression, but
only GRα in peritumorous tissue. Localization of GRβ was dominantly nuclear.
CONCLUSION: GR gene variants, larger C allele of BclI and minor 3669G allele are
associated with adrenal incidentalomas. Their concurrent presence in patients reduces GC
sensitivity. The acquired tumorous GC resistance probably promote dysregulated cortisol
production and tumor growth, but natural sensitivity to glucocorticoides maybe modifies
these effects. | true |
Strukturirano učenje nad velikim podacima zasnovano na verovatnosnim grafovskim modelima | Structured learning from big data based on probabilistic graphical models. | Struktuirano ucenje je oblast koja se bavi ucenjem složenih objekata iz složenih (struktuiranih) podataka... | Structured learning is an area that deals with the learning of complex objects from complex (structured) data. Structured data, in this context, represent data that consists of several interdependent parts. Structured learning is an important segment of machine learning, and is an active research topic with numerous unsolved problems. Applications that heavily utilize this kind of learning include textual, signal processing (audio, image and video), social, biomedical (genetic, healthcare), and many other... | false |
Odlike životne istorije i distribucije bele kanje Neophron percnopterus u Republici Makedoniji | Characteristics of the life history and distribution of the Egyptian vulture Neophron percnopterus in the Republic of Macedonia. | Bela kanja Neophron percnopterus (Aves: Accipitridae) je globalno
ugroženi, mali, oportunistički, monogamni, uglavnom solitarni i dugoživeći
lešinar, rasprostranjen u južnim delovima Evrope, Bliskog Istoka, Kavkaza,
centralne Azije, Indije, i severne Afrike, sa svetskom populacijom od 13000-
41000 adultnih jedinki. Populacija u Evropi broji oko 3300-5050 parova.
Globalna populacija je zabeležila dramatični pad, intenzivan i u Evropi, i već
je potpuno nestala sa nekih delova Balkana. U Evropi se antropogeni faktori,
naročito
trovanje,
uništavanje
staništa,
redukcija
izvora
hrane
i
uznemiravanje, smatraju za najozbiljnije faktore ugrožavanja.
Makedonska populacija belih kanja je pala za oko 86% u zadnje tri
dekade, i za bezmalo 50% tokom zadnjih 10 godina, da bi brojala samo oko 21 par
na početku 2012. godine. Zato, ciljevi ovog rada su: da se prikaže recentna
distribucija bele kanje u Republici Makedoniji, da se analizira trend
populacije i promene u prostornoj distribuciji parova i njihova gustina
gnežđenja, da uvid u reproduktivne parametre vrste, identifikuju najznačajniji
faktori za pad populacije uz analizu faktora koji utiču na opstanak teritorija
i na prametre gnežđenja. Isto tako, u cilju efetivne zaštite vrste, određuje se
najverovatna sudbine populacije bele kanje u Makedoniji, utvrđuje se njen
nacionalni status ugroženosti, i identifikuju se najbolje mere zaštite za
očuvanje ove vrste.
Terenska istraživanja su vršena u periodu 2003. - 2011. godine,
uzimajući teritoriju kao jedinicu posmatranja da bi se odredila veličina i
trend populacije. Prostorne analize su rađenje u GIS-u, korišćenjem χ2 za
određivanje frekfencije distribucije, G-statistike kao meru ravnomernosti
distribucije gnezda i Mann-Whitney U-test za određivanje značajnosti tih
promena,
uz
proračunavanje
gustine
gnežđenja.
Gnezdeći
parametri
(produktivnost populacije, uspeh gnežđenja i stopa proletavanja) su bili
viii
praćeni u periodu 2006. – 2011., a poređenja između parova i godina su urađena
jednofaktorskom ANOVA-om.
Da bi se odredio uticaj sredinskih faktora na opstanak teritorije
uzorak je proširen sa još 27 teritorija koje su nestale u periodu 1983.-2002. Za
sve teritorije opisano je ukupno 157 varijabli koje se odnose na gnezdo, stenu i
predeo, na tri prostorna radijusa oko gnezda (1 km – centar aktivnosti, 2,1 km –
polovina prosečne distance do najbližeg susednog para u 2003. godini, i 8 km –
prosečni radijus kretanja belih kanja oko gnezda). Uticaj faktora je analiziran
regresijskim stablima, koristeći RandomForest metodologiju. Da bi se odredio
uticaj sredinskih faktora na parametre gnežđenja (produktivnost i uspešnost),
145 varijabli je bilo analizirano metodom pojačanih regresijskih stabala.
Analiza vijabilnosti populacije je urađena programom VORTEX 9.99,
kao ulazne parametre koristeći rezultate terenskih posmatranja i literaturne
podatke. Pored osnovnog scenarija, razvijeno je još 20 dopunskih scenarija koji
testiraju senzitivnost analize na promene u pojedinim parametrima, kao i
različite strategije za zaštitu vrste.
Rezultati ukazuju da su za gnežđenje bele kanje značajno češće odabirale
toplija područja zemlje. U periodu 2003.-2011. brojnost populacije belih kanja u
Makedoniji pala je sa 38 na 22 parova. Obim prisustva se smanjio za oko 32,4%, a
naseljeno područje se smanjilo za 31,0%. Od nestalih parova, pet njih se nije
vratilo sa migracije. U poznatim slučajevima nestanka oko gnezdećih teritorija,
trovanje je bio dokazani ili verovatni uzrok smrti.
Distribucija belih kanja u Makedoniji nije bila ravnomerna, i na
osnovu preklapanja teritorija, većina parova su se mogli grupisati u klastere
(jedan u 2003. i dva u 2011.). Unutar klastera distribucija takođe nije bila
ravnomerna - vrednosti G-statistike se kreću od 0,31 do 0,43. Postojale su
značajne promene u prosečnoj distanci do najbliže susedne teritorije između
2003. godine (4149,87±2927,95 m,) i u većem klasteru u 2011. godini (7231,74±
3994,20 m). Gustina gnežđenja u klasteru u 2003. godini je bila 0,76 parova/100
km2, i pala je na 0,60 parova/100 km2 u većem i 0,72 parova/100 km2 u manjem
klasteru u 2011. godini.
ix
U periodu 2006.-2011. registrovano je 16 slučajeva u kojima parovi nisu
gnezdili, i praćeno je 122 pokušaja gnežđenja. Srednji procenat parova koji
počinje inkubaciju bio je 0.90±0.06. Produktivnost populacije u ovom periodu je
iznosila 0,84±0,20 mladunca po paru, uspeh gnežđenja 0,93±0,20 mladunaca po
gnezdećem paru i stopa proletanja 1,19±0,16 mladunaca po uspešnom paru. Sve
teritorije nisu imale podjednak uspeh gnežđenja, i razlika između parova je
značajna. Ne postoji značajna razlika između srednjih godišnjih vrednosti
uspeha gnežđenja. Dobijeni rezultati o parametrima razmnožavanja i gustini
populacije su u saglasnosti sa literaturnim podacima.
Modeliranje opstanka teritorija je pokazalo da je model koji je
razmatrao samo aditivne sredinske efekte bio najbolji. Selekcija stabilnosti je
izabrala devet varijabli koje se sa najvišim udelom konstantno pojavljuju kao
dominantni faktori u opstanku teritorija: orijentacija gnezda, pokrovnost
najbliže okoline gnezda žbunastom vegetacijom, ukupna dužina stena unutar 1
km radijusa, pokrovost listopadnim šumama u radijusu od 1 km oko gnezda,
visina stene na kojoj je gnezdo, distanca do najbližeg susednog para, grupa
parova (uže geografsko područje), indeks neregularnosti terena u 2,1 km
radijusa, i distanca do najbližeg ljudskog objekta. Modeli slabo pokazuju uticaj
stohastičkih faktora na populaciju (stradanje zbog trovanja ili tokom migracije
i prezimljavanja), ali efekti determinističkih faktora se uglavnom slažu sa
rezultatima ranijih istraživanja ove vrste u Evropi. Smatramo da je korišćenje
otrovnih mamaca glavni razlog mortaliteta belih kanja oko teritorija gnežđenja
u Makedoniji tokom zadnjih 30 godina.
Najznačajnije varijable koje su uticale na produktivnost su bili srednji
nagib terena unutar 2,1 km radijusa od gnezda, diverzitet staništa u najbližem
okruženju gnezda i indeks primarne produkcije u istom radijusu.
Najznačajnije varijable koje su uticale na uspeh gnežđenja su bile
srednji nagib terena u radijusu od 2,1 km oko gnezda, diverzitet staništa u
centralnom području oko gnezda, i površina stena u 8 km radijusa.
Analiza vijabilnosti populacije je pokazala da ni u jednom od 21
scenarija nije dobijena pozitivna stopa ni determinističkog ni stohastičkog
rasta populacije. Veličine populacija su pokazale kontinuirani pad u 17
x
slučajeva, a u četiri preostala scenarija (suplementacije sa po osam mladunaca
godišnje) populacija je imala pozitivan trend samo za vreme trajanja
potencijalnog programa suplementacije. Osnovni scenario ukazuje na sigurno
nestajanje bele kanje iz Makedonije u periodu od narednih 50 godina, sa
medijalnim vremenom do prve ekstinkcije od 25,0 godina i srednjim vremenom od
26,1 godine. Promene u produktivnosti populacije veoma malo utiču na trend
populacije, dok promene u mortalitetu imaju znatno veći uticaj.
Rezultati koji ukazuju na pad brojnosti populacije, smanjenje teritorije
koju ona naseljava u Makedoniji, kao i rezultati analize vijabilnosti
populacije, zajedno sa negativnim trendom vrste širom Balkanskog poluostrva,
ukazuju na to da se bela kanja treba smatrati kritično ugroženom vrstom u
Makedoniji.
Postoji velika praznina u poznavanju migratornih puteva i mesta
prezimljavanja belih kanja iz Makedonije, kao i o faktorima ugrožavanja duž
migracije i u Africi. I pored toga, na osnovu raspoloživih literaturnih
podataka može se pretpostaviti da su oni visoki. Takođe, hitno su potrebna
istraživanja o genetičkoj strukturi i inbridingu populacije, kao i o njenoj
povezanosti sa ostalim jezgrima vrste na Balkanskom poluostrvu. Mere zaštite
u Makedoniji bi morale uključivati suzbijanje korišćenja otrovnih mamaca,
uspostavljanje zaštićenih područja u najvažnijim delovima areala, redukciju
uznemiravanja, izbegavanje konverzije zemljišta, i povećanje dostupnosti hrane
za većinu parova, ili preko održavanih hranilišta, ili preko očuvanja sistema
ekstenzivnog stočarstva. Hitno je potrebno započeti planiranje programa
suplementacije populacije u Makedoniji, kao i na nivou Balkanskog poluostrva. | The Egyptian Vulture Neophron percnopterus (Aves: Accipitridae) is a
globally endangered, small, opportunistic, monogamous, mostly solitary and long-lived
vulture, distributed in the southern parts of Europe, the Middle East, Caucasus, Central
Asia and north Africa, with global population of 13000-41000 adult individuals. The
European population is estimated to 3300-5050 pairs. The global population
experienced dramatic decline, intensive also in Europe, and has completely disappeared
from some parts of the Balkan Peninsula. In Europe the anthropogenic factors,
particularly poisoning, destruction of the habitats, food source reduction and
disturbance are considered to be the most important factors leading to the decline.
The Macedonian population of the Egyptian Vulture has declined for about
86% in the last three decades, and for almost 50% in the last 10 years, counting only
about 21 pairs in the beginning of the 2012. Therefore, the goals of the present work are
to show the recent distribution of the Egyptian Vulture in Macedonia, to analyse the
population trend and changes in the spatial distribution of the breeding pairs and their
breeding densities, to show the breeding parameters of the species, identify the most
important factors leading to population decrease with analysis of the factors affecting
territory persistence and breeding parameters. Aiming at more effective conservation of
the species, this works points out the most probable destiny of the Egyptian Vulture in
Macedonia, establishes its national conservation status and identifies the best
conservation measures for its preservation.
The field work was performed in the period 2003 - 2011, using territory as the
observation unit in order to establish the size and the trend of the population. The spatial
analyses were performed in GIS, using χ2 to determine the frequencies of the
distribution, G-statistics as a measure of regularity of the nest distribution, and Mann-
Whitney U-test to check for significance in those changes, also calculating the breeding
densities. The breeding parameters (productivity of the population, breeding success
and the fledging rate) were studied in the period 2006-2011 and comparisons among the
pairs and among the years were done with One-way ANOVA.
xii
In order to study the effect of the environmental variables on territory survival
the sample was expanded with randomly selected 27 territories disappeared in the
period 1983-2002. For all territories total of 157 variables were used, describing the
nest, breeding cliff and the landscape, on three spatial scales around the nest (1 km – the
core area, 2.1 km – half of the average nearest neighbour distance in 2003, and 8 km –
the average radius of the home range). Effects of the factors were analyzed by
regression trees, using RandomForest approach. In order to study the effect of the
environmental factors on the breeding parameters (productivity and breeding success),
145 variables were analyzed using boosted regression trees.
Population viability analysis was done using the program VORTEX 9.99,
where the results of the field observations were used in parallel with the literature data
as entry parameters. Besides the basic scenario, 20 additional scenarios testing the
sensitivity of the analyses on changes in some parameters were used, as well as for
controlling of different conservation strategies to save the species.
Results indicate that for their breeding the Egyptian Vultures significantly
favoured the warmer parts of the country. In the period 2003-2011 the population size
of the Egyptian Vultures declined from 38 to 22 pairs. The extent of occurrence
declined for about 32.4%, and the area of occurrence for 31.0%. From the cases where
pair losses were observed, at least five are owing to losses during migration or
wintering. In the known cases of losses on the breeding territories, poisoning was the
proven or probable cause of death.
The distribution of the Egyptian Vultures in Macedonia was not even, and
basing on home range overlap, most of the territories were grouped into clusters (one in
2003 and two in 2011). Inside the clusters the distribution of the territories was not
random – G-values were in range of 0.31 to 0.43. There were significant changes in the
average nearest neighbour distance between 2003 (4149.87±2927.95 m) and the larger
cluster from 2011 (7231.74± 3994.20 m). The breeding density in the cluster from 2003
was 0.76 pairs/100 km2, and declined to 0.60 pairs/100 km2 in the larger and 0.72
pairs/100 km2 in the smaller cluster in 2011.
In the period 2006-2011 there were 16 cases in which the pairs did not attempt
breeding, and 122 breeding attempts were monitored. The average share of the
population starting breeding was 0.90±0.06. The productivity of the population in this
xiii
period was 0.84±0.20 fledglings per pair, the breeding success was 0.93±0.20 fledglings
per breeding pair and the fledgling rate was 1.19±0.16 fledglings per successful pair.
Not all territories had equal breeding success and the differences are significant. There
is no significant difference among the annual values of the productivity. The results on
breeding parameters and breeding densities are in accordance with published data.
Modelling territory survival has shown that the model which considered only
additive environmental effects was the best one. Stability selection identified nine
variables which appear as dominant factors in the territory survival with highest share:
nest orientation, cover of the core area with transitional woodlands, total cliff length in
the core area, cover of the core area with broadleaved forests, height of the breeding
cliff, distance to the nearest neighbour, the group of pairs (the restricted geographical
area), the irregularity index in the 2.1 km radius and distance to the nearest man-made
object. The models poorly show the effect of the stochastic factors (losses due to poison
baits or during migration or wintering in Africa), but the effects of the deterministic
factors are mostly in accordance with the literature data from the earlier studies of this
species in Europe. We consider the use of poisonous baits is the primary reason for the
mortality for the Egyptian Vultures at the breeding territories in Macedonia during the
last 30 years.
The most important variables affecting the productivity were the mean slope of
the terrain within 2.1 km of the nest, habitat diversity in the core area, and the primary
productivity index in the same radius.
The most important variables affecting the breeding outcome were the mean
slope of the terrain within 2.1 km of the nest, habitat diversity in the core area, and the
coverage with rocks in the home range (8 km).
Population viability analysis showed that in none of the 21 scenarios a positive
growth rate was obtained, neither stochastic nor deterministic. The population sizes
were showing continuous declining trend in 17 of the cases, and the remaining four
cases were those with supplementation of eight fledglings per year, for as long as the
supplementation was continued. The basic scenario indicates certain extinction of the
Egyptian Vulture from Macedonia in the forthcoming 50 years, with the median time to
first extinction of 25 years and the mean time to first extinction of 26.1 years. The
xiv
changes in the fecundity rates have a weak impact on the population trend, while the
changes in the mortality rates have significantly better effects.
The results on population decline, reduction of the extent and area of
occurrence and the results of population viability analysis, together with the declining
trend of the species throughout the Balkan Peninsula, indicate that the species should be
considered as critically endangered in Macedonia.
There is great gap in the knowledge on the migratory routes and wintering
areas of the Egyptian Vultures from Macedonia, as well as on the threats they face in
Africa. Besides this, based on the available literature data, it can be presumed that those
threats are high. Urgent further research on the genetic structure and inbreeding in the
population are also needed, along with study of its connectivity with the other core areas
of the species on the Balkans. Conservation measures in Macedonia must include
elimination of poisonous baits, establishment of protected areas in the most important
parts of the Egyptian Vulture’s range, reduction of the disturbance, avoidance of habitat
conversion and increase in the amount of the available food, either through feeding
place maintenance or via preservation of the extensive livestock-breeding system. There
is urgent need to start planning of supplementation program in Macedonia and on
Balkan level. | true |
Uloga glukokortikoidnih hormona u regulaciji energetskog metabolizma i inflamacije u jetri pacova nakon ishrane obogaćene fruktozom | The role of glucocorticoid hormones in regulation of energy metabolism and inflammation in the liver of fructose-fed rat | Jedan od najznaĉajnijih uzroka razvoja metaboliĉkog sindroma je moderan naĉin ishrane
bogate fruktozom koja u jetri pokreće de novo lipogenezu, glukoneogenezu i hroniĉnu
inflamaciju, što kumulativno vodi razvoju insulinske rezistencije. Glukokortikoidni hormoni su
vaţni regulatori energetskog metabolizma, koji deluju antagonistiĉki u odnosu na insulin i mogu
da doprinesu patofiziologiji metaboliĉkog sindroma. Metaboliĉki efekti glukokortikoida u jetri
najĉešće se pripisuju njihovoj povećanoj lokalnoj regeneraciji što ukazuje na znaĉaj enzima koji
uĉestvuju
u
njihovom
prereceptorskom
metabolizmu.
Ipak,
za
regulaciju
procesa
glukoneogeneze, sinteze triglicerida i metaboliĉke inflamacije, neophodna je aktivacija
glukokortikoidnog receptora i regulacija ekspresije ciljnih gena. Polazeći od tih saznanja, cilj ove
doktorske disertacije je bio da se ispita uloga signalnog puta glukokortikoida u razvoju simptoma
metaboliĉkog sindroma, deregulaciji energetskog metabolizma i razvoju inflamacije povezane sa
metaboliĉkim sindromom u jetri pacova nakon ishrane obogaćene fruktozom.
Tokom izrade ove studije primenjena su dva reţima ishrane, što je bio preduslov za
ispitivanje vrste i stepena metaboliĉkih promena izazvanih rastvorima fruktoze razliĉite
koncentracije (10% i 60%). Kombinovanjem molekularno-bioloških i biohemijskih metoda
analizirani su efekti devetonedeljne ishrane obogaćene fruktozom na morfološke karakteristike i
biohemijske parametre muţjaka Wistar pacova. Glukokortikoidna signalizacija u jetri je praćena
na nivou prereceptorskog metabolizma, kao i na nivou ekspresije i aktivacije glukokortikoidnog
receptora. Posebna paţnja je posvećena ispitivanju lipidnog metabolizma, metaboliĉke
inflamacije i stepena insulinske osetljivosti.
Rezultati ove doktorske disertacije su pokazali da dugotrajna ishrana obogaćena
fruktozom dovodi do povećanja koncentracije triglicerida u krvi pacova, ali ne i do
hiperglikemije, hiperinsulinemije, niti povećanja telesne mase. Ipak, ishrana 60% rastvorom
fruktoze dovodi do razvoja visceralne gojaznosti i povećanja relativne mase jetre. Ishrana
obogaćena fruktozom, bez obzira na koncentraciju, utiĉe na prereceptorski metabolizam
glukokortikoida u jetri pacova, ali ne i na koncentraciju tkivnog kortikosterona, što je u skladu sa
odsustvom promena u aktivaciji glukokortikoidnog receptora i transkripciji njegovih ciljnih
gena. Nakon ishrane 10% rastvorom fruktoze stimulisana je sinteza triglicerida, ali se paralelno
odigrava i mitohondrijalna β-oksidacija kao vrsta adaptivnog mehanizma na povećan nivo
slobodnih masnih kiselina u cirkulaciji. Sa druge strane, 60% fruktoza je veliko energetsko
opterećenje za metaboliĉke procese u jetri, što rezultira stimulisanom de novo lipogenezom i
inhibiranom β-oksidacijom. Primenjene koncentracije fruktoze u jetri pacova dovode do razvoja
metaboliĉke inflamacije i poslediĉnog slabljenja insulinske signalne kaskade.
Na osnovu rezultata prikazanih u ovom radu moţe se zakljuĉiti da nepromenjen signalni
put glukokortikoida predstavlja deo regulatornog mehanizma u jetri koji teţi da ograniĉi
posledice disbalansa izmeĊu povećanog kalorijskog unosa izazvanog ishranom obogaćenom
fruktozom i energetskih potreba organizma. Razlike u koliĉini fruktoze prisutne u ishrani najviše
se odraţavaju na metabolizam lipida u jetri, ali taj efekat nije posredovan glukokortikoidima.
Rezultati ove disertacije daju doprinos boljem razumevanju kompleksne veze izmeĊu
molekularnih mehanizama delovanja glukokortikoidnih hormona i kliniĉkih manifestacija
metaboliĉkog sindroma. | One of the major causes of metabolic syndrome development is modern diet rich in
fructose. Fructose-enriched diet initiates de novo lipogenesis, gluconeogenesis and chronic
inflammation in the liver, which cumulatively lead to progress of insulin resistance.
Glucocorticoid hormones are important regulators of energy metabolism that act antagonistically
to insulin and may contribute to metabolic syndrome pathophysiology. Metabolic effects of
glucocorticoids in the liver are usually attributed to their increased local regeneration, pointing to
the importance of enzymes involved in prereceptor glucocorticoid metabolism. However,
glucocorticoid receptor activation and modulation of its target genes expression are required for
the regulation of gluconeogenesis, triglyceride synthesis and metabolic inflammation. The aim of
this doctoral dissertation was to investigate the role of glucocorticoid signaling pathway in
development of the metabolic syndrome symptoms, deregulation of energy metabolism and
development of metabolic syndrome-associated inflammation in the liver of fructose-fed rat.
In this study, two diet regimes were applied as a prerequisite for testing the type and
degree of metabolic changes induced by different concentrations of fructose solution (10% and
60%). Morphological characteristics and biochemical alterations in the liver of male Wistar rats
upon the nine-week fructose-rich diets were analyzed by combining standard experimental
methods of biochemistry and molecular biology. Glucocorticoid signaling in the liver was
examined at the level of prereceptor metabolism and the level of expression and activation of
glucocorticoid receptor. Special attention was paid at investigation of lipid metabolism,
metabolic inflammation and changes in insulin sensitivity.
The results of this study showed that the long-term diets enriched in fructose (10% and
60%) led to increased circulating triglyceride concentration, but not to hyperglycemia,
hyperinsulinemia or increase in body weight. However, only consumption of 60% fructose
solution led to visceral obesity and an increase of relative liver mass. Fructose-enriched diet,
regardless of concentration, affected prereceptor glucocorticoid metabolism in the rat liver,
without changes in the concentration of corticosterone within the tissue, which is consistent with
the absence of alterations in the glucocorticoid receptor activation and transcription of its target
genes. Upon consumption of 10% fructose solution, both triglyceride synthesis and
mitochondrial β-oxidation were stimulated in the liver, probably as an adaptive mechanism to
increased levels of free fatty acids in the circulation. On the other hand, consumption of 60%
fructose solution appeared to be a great burden for the metabolic processes in the liver, resulting
in a stimulation of de novo lipogenesis and inhibition of β-oxidation. Both applied
fructose-enriched diets led to development of metabolic inflammation and subsequent
attenuation of insulin signaling in the rat liver.
According to the results presented in this study it can be concluded that unaltered
glucocorticoid signaling pathway is a part of hepatic regulatory mechanism that tends to limit the
consequences of the imbalance between increased energy intake caused by fructose-enriched diet
and energy requirements of the body. Differences in the amount of dietary fructose are mainly
reflected in the hepatic lipid metabolism, but the effects of fructose are not mediated by
glucocorticoids. The results of this dissertation contribute to better understanding of the complex
relationship between molecular mechanisms of glucocorticoid actions and clinical manifestations
of metabolic syndrome. | true |
Efekat ekspresije gena za protein-kinazu 1 duvana (NPK1) kod transformisanih biljaka karfiola (Brassica oleracea L. var. botrytis) na razvoj tolerancije prema povišenim koncentracijama NaCI in vitro | Effect of tobacco protein kinase 1 (NPK1) gene expression in transformed cauliflower (Brassica oleracea L. var. botrytis) plants on the development of tolerance to increased concentrations of NaCl in vitro | Biljke su neprekidno izloţene razliĉitim vrstama stresa iz spoljašnje sredine, koji nepovoljno utiĉu na njihov rast i razviće. Povećan salinitet zemljišta je jedan od faktora spoljašnje sredine, koji u velikoj meri ograniĉava produktivnost gajenih biljaka. Karfiol (Brassica oleracea L. var. botrytis), je znaĉajna povrtarska kultura koja je, kao i ostale povrtarske kupusnjaĉe, podloţna uticaju razliĉitih stresnih faktora, pa i povećanom salinitetu usled intenzivnog zalivanja. Uz klasiĉne metode oplemenjivanja, biotehnološke metode mogu biti efikasan pristup za povećanje tolerancije biljaka prema stresu. Mitogenom aktivirane protein-kinaze (MAPK) igraju kljuĉnu ulogu u rastu i razviću biljaka i njihovom odgovoru na biotiĉke i abiotiĉke faktore spoljašnje sredine. Introdukcija gena za komponente MAPK kaskada moţe dovesti do razvoja transgenih biljaka sa povećanom tolerancijom prema stresu, pri ĉemu su MAPKKK najpogodnije za genetiĉki inţenjering jer funkcionišu na poĉetku kaskade koja vodi aktivaciji više transkripcionih faktora i stres inducibilnih gena. Preduslov za primenu ovih metoda u oplemenjivanju biljaka jeste uspostavljanje efikasnog sistema za regeneraciju biljaka in vitro. Kod ĉetiri varijeteta B. oleracea (karfiola, kupusa, brokolija i kelja) uspostavljen je sistem za regeneraciju pupoljaka in vitro putem organogeneze. Eksplantati hipokotila su kod skoro svih testiranih varijeteta imali najbolji regenerativni potencijal (od 75,0-91,2% sa produkcijom 3,5-7,4 pupoljaka po eksplantatu). Podloge koje su sadrţale N6-benziladenin (BA) su bile optimalne, kako za regeneraciju pupoljaka, tako i za njihovu kasniju multiplikaciju. Regenerisani izdanci su uspešno oţiljeni i aklimatizovani. Kod kupusa i karfiola uspostavljen je i jednostavan protokol za direktnu somatsku embriogenezu iz nezrelih zigotskih embriona gajenih na B5 podlozi bez regulatora rastenja (B5-0), koji je omogućio efikasnu transformaciju karfiola kao, u tom pogledu, problematiĉnog varijeteta. Kod oba varijeteta, zigotski embrioni u kotiledonarnom stadijumu razvoja imali su najveći kapacitet za formiranje somatskih embriona (11,84 kod kupusa i 11,95 kod karfiola)... | Plants are continuously exposed to various types of stress from the environment that adversely affect their growth and development. The increased salinity of the soil is one of the environmetal factors that greatly limits the productivity of the cultivated plants. Cauliflower (Brassica oleracea L. var. botrytis), is an important vegetable crop, which, like other Brassica vegetable, is grown under the influence of various biotic and abiotic stresses, including the increased salinity due to intensive watering. In addition to classical breeding methods, biotechnology methods may be an effective approach to increase plants stress tolerance. The mitogen-activated protein kinases (MAPK) play a key role in the growth and development of plants and their response to biotic and abiotic stresses. Introduction of genes for components of MAPK cascade may lead to the development of transgenic plants with the increased tolerance to stress, wherein MAPKKK are most suitable for genetic engineering, because they act at the beginning of the cascade that leads to activation of several transcription factors and stress inducible genes. A prerequisite for the application of these methods in plant breeding is establishing an efficient plant regeneration system in vitro. A regenerative system via organogenesis was established in four B. oleracea varieties (cauliflower, cabbage, broccoli, kale). The hypocotyl explants in almost all the tested varieties have the best regenerative potential (from 75.0-91.2% with the production of 3.5-7.4 shoots per explant). The media containing N6-benzyladenine (BA) were optimal both for shoot regeneration and their subsequent multiplication. Regenerated shoots were succesfully rooted and acclimatized. In cabbage and cauliflower a simple protocol was established for the direct somatic embryogenesis from immature zigotic embryos grown on B5 medium without growth regulators (B5-0), which enabled the efficient transformation of cauliflower, in this regard, as a problematic variety. In both varieties, zygotic embryos at the cotyledonary stage of development had the highest embryo-forming capacity (11.84 in cabbage and 11.95 in cauliflower)... | false |
Analiza ekspresije celokupnog genoma u cilju identifikacije ključnih gena i mikroRNK za nastanak urođenih anomalija bubrega i urinarnog trakta čoveka | Analysis of whole genome expression in order to indentify the key genes and microRNAs for the occurence of congenital anomalies of the kidney and urinary tract in humans | UNIVERZITET U BEOGRADU
BIOLOŠKI FAKULTET
Ivan G. Jovanović
Analiza ekspresije celokupnog genoma u
cilju identifikacije ključnih gena i mikro RNK
za nastanak urođenih anomalija bubrega i
urinarnog trakta čoveka
doktorska disertacija
Beograd, 2016
UNIVERSITY OF BELGRADE
FACULTY OF BIOLOGY
Ivan G. Jovanović
Analysis of whole genome expression in
order to identify the key genes and
microRNAs for the occurrence of congenital
anomalies of the kidney and urinary tract in
humans
Doctoral Dissertation
Belgrade, 2016
Mentori:
dr Aleksandra Stanković, naučni savetnik, Univerzitet u Beogradu, Institut za
nuklearne nauke „Vinča“
dr Katarina Zeljić, docent, Univerzitet u Beogradu, Biološki fakultet
Članovi komisije:
dr Marina Stamenković-Radak, redovni profesor, Univerzitet u Beogradu, Biološki
fakultet
Datum odbrane doktorske disertacije:
__________________________
Zahvalnica
Ova doktorska disertacija je osmišljena i urađena u Laboratoriji za
radiobiologiju i molekularnu genetiku Instituta za nuklearne nauke „Vinča“,
Univerziteta u Beogradu u okviru projekata: „Genetska osnova humanih vaskularnih
i inflamatornih bolesti“ (OI175085) i „Integralna studija identifikacije regionalnih
genetskih faktora rizika i faktora rizika životne sredine za masovne nezarazne
bolesti humane populacije u Srbiji“ (III41028). Rukovodiocu projekta OI175085 i
glavnom mentoru ove doktorske disertacije, dr Aleksandri Stanković, naučnom
savetniku, se iskreno zahvaljujem što mi je pružila priliku da se bavim genetičkim
istraživanjima, što me je sve vreme usmeravala da kombinujem molekularnu
biologiju i informatiku, vodila stručnim savetima, na energiji i pomoći koju je uložila
tokom realizacije ovog rada.
Mentoru, docentu, dr Katarini Zeljić se zahvaljujem na velikoj pomoći tokom
pregleda doktorske disertacije.
Prof. dr Marini Stamenković-Radak se zahvaljujem na pregledu i oceni rada i
korisnim sugestijama.
Dr Draganu Alavantiću, naučnom savetniku, hvala na stručnoj pomoći i
pruženoj podršci kao i na viziji koja je omogućila postojanje i opremanje
najsavremenije laboratorije iz oblasti molekularne genetike u Srbiji, bez koje
eksperimenti na kojima je zasnovana ova disertacija ne bi bili mogući.
Dr Maji Živković, naučnom savetniku i dr Tamari Đurić-Delić, višem naučnom
saradniku, hvala na pomoći u analizi rezultata i stručnim savetima i sugestijama koje
su značajno unapredile kvalitet ove disertacije. Dr Tamari, velikom gurmanu, se
posebno zahvaljujem na večitoj spremnosti da u 11h „zveknemo“ neki jači doručak.
Dr Maji se posebno zahvaljujem na angažovanju oko održavanja moje zanatske
svestranosti i fizičke spremnosti, kao i oko odlazaka na stručnu praksu i timske
pripreme.
Zahvaljujem se kolegama sa Univerzitetske Dečje Klinike „Tiršova“, a posebno
prof. dr Mirjani Kostić i prof. dr Zoranu Krstiću, na dugogodišnjoj saradnji i podršci, i
u prikupljanju i kliničkoj karakterizaciji uzoraka koji su analizirani u ovoj studiji, a
bez kojih ova disertacija ne bi bila moguća.
Svim kolegama iz Laboratorije za radiobiologiju i molekularnu genetiku se
zahvaljujem na velikoj podršci, uspešnom timskom radu i neprestanoj dominaciji.
Vinča’s blues will play forever!
Prijateljima hvala što su uvek bili uz mene.
Mojoj porodici hvala na svemu što su mi pružili. Više od svih svojih
dosadašnjih uspeha, ponosan sam što imam vas.
Evi hvala što postoji.
Mojoj Jaci se najtoplije zahvaljujem što je i u najlepšim i u najtežim trenucima
bila pored mene i nesebično mi davala svoju ljubav.
Analiza ekspresije celokupnog genoma u cilju identifikacije ključnih gena i
mikro RNK za nastanak urođenih anomalija bubrega i urinarnog trakta
čoveka
Sažetak
Urođene anomalije bubrega i urinarnog trakta (engl. Congenital Anomalies
of the Kidney and Urinary Tract – CAKUT) su razlog nastanka čak polovine
slučajeva terminalne bubrežne insuficijencije ali patofiziološka osnova ove bolesti
još uvek nije poznata u potpunosti pa je samim tim otežana prevencija i terapija. U
ovoj studiji je, upoređivanjem ekspresije celokupnog genoma u tkivu uretera
pacijenata sa CAKUT-om i zdravih kontrola, identifikovano 78 diferencijalno
eksprimiranih gena. Bioinformatičkom analizom je po prvi put zaključeno da
postoji visok nivo kompleksnosti bioloških procesa i molekularnih puteva u tkivu
čoveka koji su u CAKUT-u diferencijalno regulisani i čije važne interakcije se mogu
predstaviti sa sedam molekularnih mreža, od kojih četiri sadrže gene sa funkcijama
koje se direktno mogu asocirati sa CAKUT-om. Eksperimentalnom validacijom
ključnih mreža qRT-PCR metodom utvrđen je povišen nivo iRNK za gene LCN2 (7,6
puta), PROM1 (8 puta) i SOSTDC1 (5,6 puta) i snižen nivo iRNK za gene RASD1 (4,5
puta), INA (3,3 puta) i TAC3 (6,6 puta) u tkivu uretera pacijenata u poređenju sa
kontrolnim tkivom, koji mogu biti molekularni markeri za CAKUT. Ove mreže su
ujedno i prikaz interakcija ključnih molekula u CAKUT-u i predstavljaju temelj
budućih funkcionalnih studija. Primenom metode koja povezuje podatke sa
genskog ekspresionog čipa i predikcije vezujućih mesta različitih miRNK iz više
predikcionih algoritama, sa unapređenom rezolucijom, identifikovane su miRNK sa
potencijalno povišenom ekspresijom u CAKUT-u u odnosu na zdrave kontrole.
Validacijom ključnih miRNK qRT-PCR metodom je identifikovan povišen nivo
miR-144 (5,7 puta) u tkivu uretera pacijenata u odnosu na kontrole, koji može biti
molekularni marker za CAKUT. | Urođene anomalije bubrega i urinarnog trakta (engl. Congenital Anomalies
of the Kidney and Urinary Tract – CAKUT) su razlog nastanka čak polovine
slučajeva terminalne bubrežne insuficijencije ali patofiziološka osnova ove bolesti
još uvek nije poznata u potpunosti pa je samim tim otežana prevencija i terapija. U
ovoj studiji je, upoređivanjem ekspresije celokupnog genoma u tkivu uretera
pacijenata sa CAKUT-om i zdravih kontrola, identifikovano 78 diferencijalno
eksprimiranih gena. Bioinformatičkom analizom je po prvi put zaključeno da
postoji visok nivo kompleksnosti bioloških procesa i molekularnih puteva u tkivu
čoveka koji su u CAKUT-u diferencijalno regulisani i čije važne interakcije se mogu
predstaviti sa sedam molekularnih mreža, od kojih četiri sadrže gene sa funkcijama
koje se direktno mogu asocirati sa CAKUT-om. Eksperimentalnom validacijom
ključnih mreža qRT-PCR metodom utvrđen je povišen nivo iRNK za gene LCN2 (7,6
puta), PROM1 (8 puta) i SOSTDC1 (5,6 puta) i snižen nivo iRNK za gene RASD1 (4,5
puta), INA (3,3 puta) i TAC3 (6,6 puta) u tkivu uretera pacijenata u poređenju sa
kontrolnim tkivom, koji mogu biti molekularni markeri za CAKUT. Ove mreže su
ujedno i prikaz interakcija ključnih molekula u CAKUT-u i predstavljaju temelj
budućih funkcionalnih studija. Primenom metode koja povezuje podatke sa
genskog ekspresionog čipa i predikcije vezujućih mesta različitih miRNK iz više
predikcionih algoritama, sa unapređenom rezolucijom, identifikovane su miRNK sa
potencijalno povišenom ekspresijom u CAKUT-u u odnosu na zdrave kontrole.
Validacijom ključnih miRNK qRT-PCR metodom je identifikovan povišen nivo
miR-144 (5,7 puta) u tkivu uretera pacijenata u odnosu na kontrole, koji može biti
molekularni marker za CAKUT.
Ključne reči
CAKUT, Bioinformatika, Integrativni pristup, Mikro RNK, Pedijatrija, Biomarker,
Razviće, Ekspresija gena, Genski ekspresioni čip.
Naučna oblast: Biologija
Uža naučna oblast: Genetika
UDK broj: [575.113:616.6]:57.087 (043.3)
Analysis of whole genome expression in order to identify the key genes and
microRNAs for the occurrence of congenital anomalies of the kidney and
urinary tract in humans
Abstract
Congenital anomalies of the kidney and urinary tract (CAKUT) are the cause
for half of the cases with renal failure, but pathophysiological basis of this disease
is not fully known. Therefore, the therapy and prevention of the disease is difficult.
In this study, by comparing the whole-genome expression in the ureter tissue of
patients with CAKUT and healthy controls, 78 differentially expressed genes were
identified. By employing bioinformatical analysis it was concluded that there is a
high level of complexity of biological processes and molecular pathways,
differentialy regulated in CAKUT, whose important interactions can be represented
with seven molecular networks, of which four contain genes with functions that
can be directly associated with CAKUT. Experimental validation of key networks by
qRT-PCR method identified elevated mRNA levels of LCN2 (7.6 fold), PROM1 (8
fold) and SOSTDC1 (5.6 fold), and decreased mRNA levels of RASD1 (4.5 fold), INA
(3.3 fold) and TAC3 (6.6 fold) in the ureter tissue of the patients compared to
control tissue, which may be molecular markers for CAKUT. These networks
display the interactions of key molecules in CAKUT and provide the basis for future
functional studies, as well. By applying the method that links microarray gene
expression data and prediction of miRNA binding sites from several prediction
algorithms, with enhanced resolution, miRNAs with potentialy upregulated
expression in CAKUT compared to healthy controls were identified. Validation of
key miRNAs by qRT-PCR method identified increased level of miR-144 (5.7 fold) in
the ureter tissue of the patients compared to controls, which may be a molecular
marker for CAKUT. | true |
Prostorna distribucija i ekologija orhideja (Orchidaceae) zapadne Srbije | Spatial distribution and ecology of orchids (Orchidaceae) of western Serbia | Orhideje su poznate po bogatstvu taksona, kompleksnoj ekologiji i velikom broju
ugroženih vrsta. Osnovni ciljevi ove disertacije su utvrđivanje prostorne distribucije i
diverziteta orhideja zapadne Srbije, kao i utvrđivanje ekoloških preferencija i
konzervacionog statusa orhideja. Za svaki takson izrađene su karte rasprostranjenja u
zapadnoj Srbiji. Za ispitivanje uticaja nadmorske visine, 19 bioklimatskih faktora i
heterogenosti staništa na bogatstvo taksona u pojedinim UTM kvadratima 10 × 10 km,
kao i analizu bogatstva taksona orhideja duž gradijenta nadmorske visine korišćena je
regresiona analiza. OMI (eng. outlying mean index) analiza je korišćena za analizu
ekoloških niša i faktora koji utiču na brojnost i distribuciju orhideja. Utvrđeno je
prisustvo 57 vrsta i podvrsta orhideja, među kojima je 7 novih taksona za floru Srbije.
Utvrđeno je da planina Tara predstavlja najvažniji centar diverziteta orhideja u zapadnoj
Srbiji, sa 34 registrovana taksona u UTM kvadratu CP76. Rezultati ovog istraživanja su
pokazali da nadmorska visina i heterogenost staništa značajno utiču na bogatstvo
taksona u UTM kvadratima 10 × 10 km, dok maksimalne vrednosti nadmorskih visina i
minimalne vrednosti temperaturnih varijabli najsnažnije utiču na bogatstvo prstasto-
tuberoidnih orhideja. Utvrđeno je da se najviše taksona orhideja javlja u područjima
srednjih nadmorskih visina (1000-1100 m). Najveći broj taksona orhideja je registrovan
na krečnjacima, u zajednicama klasa Festuco-Brometea, Quercetea pubescentis i
Molinio-Arrhenatheretea. Definisano je 6 ekoloških grupa šumskih i 8 ekoloških grupa
orhideja zeljastih tipova vegetacije. Određeni su IUCN statusi ugroženosti, kao i faktori
ugrožavanja orhideja zapadne Srbije, sa predlogom mera zaštite i konzervacionim
prioritetima. | Orchids are known for their taxa richness, complex ecology and a large number of
endangered species. The main objectives of this dissertation are the determination of
spatial distribution and diversity of orchids in western Serbia, and the determination of
ecological preferences and conservation status of orchids. Distribution maps were made
for each taxon in western Serbia. Regression analysis was used to explore the influence
of elevation, 19 bioclimatic factors and the habitat heterogeneity on the taxa richness
within individual UTM 10 × 10 km grid cells, as well as to analyse the taxa richness
along the elevational gradient. The OMI (outlying mean index) analysis was used to
analyse ecological niches and factors affecting the abundance and distribution of
orchids. The presence of 57 orchid species and subspecies was recorded, among which
7 new taxa to the flora of Serbia. Mt. Tara represents the most important center of
diversity of orchids in western Serbia, with 34 taxa recorded in CP76 UTM grid cell.
Results of this study showed that elevation and habitat heterogeneity significantly affect
the taxa richness in UTM 10 × 10 km grid cells, whereas the maximum values of
elevations and minimum values of temperature variables most significantly affect the
richness of orchids with palmate and fusiform tubers. It has been found that most of
orchid taxa occur in the middle elevations (1000-1100 m). The largest number of orchid
taxa was recorded on limestone, in the communities of the classes Festuco-Brometea,
Quercetea pubescentis and Molinio-Arrhenatheretea. Six ecological groups of forest
orchids and eight ecological groups of orchids of herbaceous vegetation types were
defined. IUCN threatened status, as well as the threats to orchids of western Serbia were
determined, with a proposal for protection measures and conservation priorities. | true |
Studija genetičke osnove Parkinsonove bolesti kod stanovništva Srbije | Study of the genetic basis of Parkinson's disease in Serbian population | Parkinsonova bolest je drugo najčešće neurodegenerativno oboljenje koje pogaĎa od 1% do
2% stanovništva starijeg od 65 godina i više od 4% stanovništva starijeg od 85 godina.
Klinička prezentacija obuhvata četiri kardinalna znaka bolesti: bradikineziju, tremor u miru,
rigor i posturalnu nestabilnost, kao i druge motorne i nemotorne simptome. Parkinsonova
bolest se karakteriše kao multifaktorijalno oboljenje u čijem nastanku najverovatniju ulogu
ima interakcija genetičkih i sredinskih faktora. Genetička osnova je veoma kompleksna i do
danas je otkriveno preko 28 hromozomskih regiona koji su sa većom ili manjom sigurnošću
povezani sa nastankom bolesti. Uzrok autozomno-dominantne forme bolesti najčešće su
mutacije u LRRK2 genu, dok je VPS35 najskorije potvrĎen gen, asociran sa ovakvim
načinom nasleĎivanja. Kod autozomno-recesivne forme su najčešće mutacije u Parkin
genu, a najznačajniji faktor rizika za razvoj Parkinsonove bolesti su mutacije u GBA genu
koje dovode do autozomno-recesivnog oboljenja, Gošeove bolesti.
Cilj:
Cilj ovog istraživanja je karakterizacija genetičke osnove Parkinsonove bolesti kod
stanovništva Srbije i formiranje preporuka za genetičko testiranje bolesnika. U tom cilju je
vršeno odreĎivanja učestalosti i spektra mutacija kod obolelih od Parkinsonove bolesti u
genima LRRK2, Parkin, VPS35 i GBA, kao i utvrĎivanje potencijalnih korelacija genotipa
sa fenotipom.
Materijal i metode:
U ovo istraživanje je uključeno ukupno 496 bolesnika (305 muškog i 191 ženskog pola)
kojima je klinička dijagnoza Parkinsonove bolesti postavljena na Klinici za neurologiju,
Kliničkog centra Srbije. Svi bolesnici su testirani na prisustvo mutacija u odabranim
regionima LRRK2 i VPS35 gena metodom direktnog sekvenciranja. Detektovane promene
koje do sada nisu opisane u literaturi su analizirane i kod 143 kontrolnih, neurološki
zdravih subjekata, straijih od 50 godina. U subgrupi od 225 bolesnika sa ranim početkom
bolesti i/ili pozitivnom porodičnom anamnezom je vršena sveobuhvatna analiza Parkin
gena direktnim sekvenciranjem i MLPA metodom. Analiza odabranih regiona GBA gena
direktnim sekvenciranjem je primenjena kod 481 bolesnika, 15 srodnika obolelih od
Gošeove bolesti i 348 zdravih kontrola. Potencijalna patogenost do sada neopisanih
mutacija je testirana in silico analizom primenom dostupnih predikcionih softvera.
Rezultati i diskusija:
U kodirajućim sekvencama LRRK2 gena su detektovane četiri različite mutacije
(c.6055G>A, c.4111A>G, c.4522A>G i c.5971A>G) kod četiri nesrodna bolesnika. Dve
mutacije, c.4522A>G i c.5971A>G, nisu do sada opisane u literaturi i nisu detektovane kod
286 kontrolnih hromozoma. Detektovana je i jedna promena koja potencijalno utiče na
mesto iskrajanja egzona, c.4536+3A>G, kod dva nesrodna bolesnika, ali je ova promena
detektovana i kod 3 kontrona ispitanika. Klinička prezentacija nosilaca mutacija u LRRK2
genu se nije značajno razlikovala u odnosu na bolesnike bez mutacija. Jedino je odnos
polova bio značajno drugačiji u odnosu na čitavu ispitivanu grupu bolesnika.
Analizom VPS35 gena nije detektovana jedina do sada opisana patogena mutacija
(c.1858G>A) ni kod jednog od testiranih bolesnika. TakoĎe, u sekvenciranom egzonu 15
nije detektovana ni jedna promena sekvence, kao ni u egzon-intron granicama obuhvaćenim
analizom.
U Parkin genu je direktnim sekvenciranjem detektovano 13 različitih promena, od čega se
8 može smatrati patogenim (c.101_102delAG, c.167T>A, c.245C>A, c.398_399insACC,
c.758G>A, c.823C>T, c.1180G>A i c.1310C>T), dok su MLPA metodom detektovane
promene kod 6 bolesnika (delecija egzona 2, delecija egzona 2-3, duplikacija egzona 3,
duplikacija egzona 4, duplikacija egzona 4-7, duplikacija egzona 5-7, kao i triplikacija
egzona 3). Kod ukupno četiri bolesnika koji su bili složeni heterozigoti zapažene su ranije
godine početka bolesti u odnosu na testirane bolesnike bez mutacije, dok kod osam
bolesnika koji su bili heterozigotni nosioci mutacija ovakav trend nije primećen.
Selektivnom analizom GBA gena, detektovano je ukupno 58 heterozigotnih promena kod
bolesnika sa Parkinsonovom bolešću, 3 složenih heterozigotnih i 1 homozigotna promena
kod bolesnika sa Parkinsonovom i Gošeovom bolešću, 12 heterozigotnih promena kod
srodnika bolesnika sa Gošeovom bolešću i 17 heterozigotnih promena u grupi kontrola.
Detektovane su sledeće promene pojedinačnog nukleotida: c.1103G>A, c.1223C>T,
c.1226A>G, c.1256A>T, c.1289C>T, c.1292A>G, c.1342G>C, c.1448T>C, c.1504C>T i
c.1505G>A, kao i tri kompleksna rekombinantna alela: RecΔ5 (c.1263–1317del55),
[D409H;H255Q]
(c.1342G>C,
c.882T>G)
i
RecNcil
(c.1448T>C,
c.1483G>C,
c.1497G>C). Učestalost mutacija u GBA genu je značajno veća kod bolesnika sa PB u
odnosu na učestalost u kontrolnoj grupi. Klinička prezentacija bolesti kod nosilaca mutacije
u GBA genu se u većini simptoma nije razlikovala od sporadične Parkinsonove bolesti,
osim što je veći broj noslaca mutacije imao bol kao inicijalni simptom.
Zaključak:
U ovom istraživanju su utvrĎene učestalosti i spektar mutacija u analiziranim genima i
korelacije genotipa sa fenotipom bolesnika, koje su bile u korelaciji sa dostupnim podacima
iz literature. Na osnovu dobijenih rezultata su formirane preporuke za protokol
dijagnostičkog genetičkog testiranja. Genetičke analize LRRK2, VPS35, Parkin i GBA gena
su uvedene u rutinsku kliničku dijagnostiku Parkinsonove bolesti. Tokom ovog istraživanja
oformljena je banka uzoraka krvi i DNK bolesnika sa Parkinsonovom bolešću, koja trentno
obuhvata više od 500 uzoraka. | Parkinson’s disease is the second most common neurodegenerative disorder that affects 1%
to 2% of the population older than 65 years and over 4% of the population older than 85
years. Clinical presentation includes four cardinal signs of disease: bradykinesia, resting
tremor, rigidity an postural instability, as well as other motor and non-motor symptoms.
Parkinson’s disease is characterized as a multifactorial disorder with probable interaction of
genetic and environmental factors. The genetic basis is very complex and more than 28
chromosomal regions, associated with the disease, are discovered so far. Mutations in the
LRRK2 gene are the most common cause of autosomal dominant Parkinson’s disease, while
the mutations in VPS35 gene are recently discovered as a new cause of dominantly
inherited form of the disease. Autosomal recessive Parkinson’s disease is the most
frequently linked to mutations in the Parkin gene. The strong risk factor for the
development of Parkinson's disease are heterozygous mutations in the GBA gene.
Homozygous and compound heterozygous mutations in GBA gene are cause of autosomal
recessive Gaucher’s disease.
Aim:
The aim of this study was to determine genetic basis of Parkinson’s disease in Serbian
population and to establish recommandations for genetic testing of patients with this
disorder. It was planed to determine the frequency of mutations and the mutation spectrum
in LRRK2, Parkin, VPS35 i GBA genes in Serbian patients with Parkinson’s disease and to
examine correlations of genotype and phenotype.
Material and methods:
In this study were included 496 patients with Parkinson’s disease (305 male and 196
female) diagnosed in Neurology Clinic, Clinical Center of Serbia. The presence of
mutations in selected exons of LRRK2 and VPS35 genes was analyzed by direct
sequencing. Detected variants which are not described in the literature yet are analyzed in
143 healthy controls older than 50 years. In subgroup of 225 patients with early onset of the
disease and/or positive family history of Parkinson’s disease analysis included direct
sequencing of all coding regions of Parkin gene and duplication and deletion detection
using MLPA method. Analysis of selected exons of the GBA gene was performed in 481
patients with Parkinson’s disease, 15 relatives of patients with Gaucher’s disease and 348
healthy controls. Potential pathogenicity of novel variants was tested in silico using
available prediction softwares.
Results and discussion:
Four different mutations (c.6055G>A, c.4111A>G, c.4522A>G and c.5971A>G) were
deteced in coding regions of LRRK2 gene in four unrelated patients. Mutations c.4522A>G
and c.5971A>G were not described in the literature yet and they were not detected in 286
control chromosemes. One detected intronic variant c.4536+3A>G, that is potentialy
affecting splicing, was detected in two unrelated patients and in 3 controls. Clinical
presentations of the disease in mutation carriers were not different compared to non-
carreiers. Only the sex ratio was significantly different compared to the entire study group
of patients.
The proven pathogenic mutation c.1858G>A in VPS35 gene was not detected in tested
patients. Also, analysis of exon 15 and exon-intron borders of VPS35 gene did not revealed
any sequence change.
In Parkin gene was detected 13 different variants by direct sequencing. Eight of these
changes can be considered as pathogenic (c.101_102delAG, c.167T>A, c.245C>A,
c.398_399insACC, c.758G>A, c.823C>T, c.1180G>A i c.1310C>T). MLPA method
revealed dosage changes in six patients (exon 2 deletion, exons 2-3 deletion, exon 3
duplication and triplication, exon 4 duplication, exons 4-7 duplications and exons 5-7
duplications). Four patients with homozygous or compound heterozygous mutations had
earlier onset of the disease compared to non-carriers or heterozygotes.
In selected exons of the GBA gene was detected 58 heterozygotes in patients with
Parkinson’s disease, three compund heterozygotes and one homozygote in patients with
Parkinson’s and Gaucher’s disease, 12 heterozygotes in relatives of patients with Gaucher’s
disease, and 17 heterozygotes among healthy controls. Single nucleotide changes were:
c.1103G>A, c.1223C>T, c.1226A>G, c.1256A>T, c.1289C>T, c.1292A>G, c.1342G>C,
c.1448T>C, c.1504C>T i c.1505G>A, and complex, recombinant alleles were RecΔ5
(c.1263–1317del55), [D409H;H255Q] (c.1342G>C, c.882T>G) i RecNcil (c.1448T>C,
c.1483G>C, c.1497G>C). Mutation frequency in GBA gene was higher in patients with
Parkinson’s disease compared to controls. Clinical presentations of the disease in mutation
carriers were not different compared to non-carreiers, exept pain that was frequently
presenting symptom in mutation carriers.
Conclusion:
Mutation frequencies and spectrum in analyized genes and genotype-fenotype correlations
in patients with Parkinson's disease were in accordance with available data from the
literature. Based on the obtained results, recommendations for diagnostic genetic testing
were proposed. Genetic analysis of LRRK2, VPS35, Parkin and GBA genes were introduced
into routine clinical diagnosis of Parkinson's disease. During this research, a bank of blood
and DNA samples of patients with Parkinson's disease has been established, which
currently includes more than 500 samples. | true |
Filogenetski odnosi rodova subtribusa Lysiphlebina i taksonomski status vrsta roda Adialytus (Foerster) (Hymenoptera, Braconidae, Aphidiinae) | Phylogenetic relationships among genera of subtribe Lysiphlebina and taxonomic status of Adialytus (Foerster) species (Hymenoptera, Braconidae, Aphindiinae) | Rod Adialytus predstavlja jedan od tri roda subtribusa Lysiphlebina. Pored ovog,
ovde spadaju rodovi Lysiphlebus i Lysiphlebia. Ukupno je opisano sedam vrsta roda
Adialytus, koje su rasprostranjene širom Palearktika. Adialytus je dugo bio razmatran
kao podrod u okviru roda Lysiphlebus, sa kojim deli mnoge morfološke karaktere. Pored
nerazjašnjenog statusa ovog roda, postoje velike poteškoće pri identifikaciji vrsta u
okviru samog roda Adialytus. U ovom radu su analizirane tri najčešće evropske vrste:
Adialytus ambiguus, A. salicaphis i A. thelaxis. Ukupno je analizirano 228 jedinki, pri
čemu analiza obuhvata isključivo ženke. Korišćene su tri metode za morfološku i
genetičku karakterizaciju vrsta roda Adialytus. Metodom „tradicionalne“ morfometrije su
analizirani meristički i kontinuirani karakteri, pri čemu su kontinuirani karakteri
predstavljeni kao dužinski odnosi. Metoda gemetrijske morfometrije je korišćena za
ispitivanje obrasca varijabilnosti oblika prednjih krila. Molekularne analize su
sprovedene korišćenjem mitohondrijalnog gena za citohrom oksidazu 1 (mtCOI) i gena
za veliku ribozomalnu subjedinicu 28S (28S rRNK). Upotrebom „tradicionalne“
morfometrije utvrđena je statistički značajna varijabilnost između tri analizirane vrste
roda Adialytus. Najveću varijabilnost su pokazali karakteri kao što je broj mirisnih brazdi
na prvom, drugom i petom flagelarnom članku antena. Takođe, značajne razlike su
uočene i u samim proporcijama analiziranih flagelarnih članaka. Geometrijskom
morfometrijom detektovane su izvesne razlike u obliku prednjih krila. Distalni deo krila
ima znatno veću varijabilnost u odnosu na proksimalni. Jedinke A. ambiguus imaju
relativno uska krila i dugačak metakarpus, za razliku od A. salicaphis i A. thelaxis.
Molekularnom analizom na osnovu mtCOI gena potvrđeno je da je A. thelaxis „dobra“
vrsta. Genetičke razlike između A. ambiguus i A. salicaphis su relativno male i na
graničnim vrednostima za specijsko razdvajanje. Vrste A. ambiguus i A. salicaphis su
veoma bliske, kako na morfološkom, tako i na genetičkom nivou. Međutim, one se ipak
dosta razlikuju u ekološkom pogledu. Analiza na generičkom nivou u okviru subtribusa
Lysiphlebina korišćenjem ribozomalnog gena za 28S rRNK je pokazala da se rod
Lysiphlebia izdvaja sa visokom podrškom od zajedničke klade koju čine rodovi Adialytus
i Lysiphlebus. Međutim poslednja dva roda su veoma bliska i razdvajanje nije jasno
definisano. | The genus Adialytus is a member of the subtribe Lysiphlebina. Beside this genus
the subtribe encompasses two more genera: Lysiphlebus and Lysiphlebia. So far, seven
valid Adialytus species have been recognized which can be found throughout the
Palaearctic. The genus Adialytus was previously considered as a subgenus within the
genus Lysiphlebus, which it resembles to. Beside the unclear status of this genus, there
are many difficulties concerning identification of Adialytus species. In this work, three
most common European species were analyzed: Adialytus ambiguus, A. salicaphis and
A. thelaxis. In total, 228 specimens were included into analysis, all being females. Three
methods were involved into morphological and genetic characterization of the Adialytus
species. Traditional morphometrics is used for the analysis of meristic and continuous
characters. However, continuous characters were represented as a relation of lengths
and widths. Geometric morphometrics is used in forewing shape analysis. For the
molecular analysis on the species level, the mitochondrial gene for cytochrome oxidase
1 is used (mtCOI) , while for the relation among the genera of the subtribe Lysiphlebina,
the ribosomal gene 28S (28SrRNK) is used. According to the traditional morphometrics
a statistically significant variability is detected among the all three Adialytus species.
The greatest variability is detected in the number of olfactory grooves (rhinaria) on the
first, second and fifth flagellar segment of antenna. Furthermore, significant differences
are detected in the proportions of the very segments. The differences in the forewing
shape are detected by the geometric morphometrics method. The distal part of the
forewing is much more variable than proximal. The species A. ambiguus has relatively
narrow forewings and long metacarpus in contrast to A. salicaphis and A. thelaxis.
According to the molecular analysis of the mtCOI gene, it was confirmed that A. thelaxis
is “bona species”. The genetic differences between A. ambiguus and A. salicaphis are
relatively small and on the margin for species delimitation. Although very close, both
genetically and morphologically, A. ambiguus and A. salicaphis the two species are
nevertheless ecologically different, i.e. they occupy different ecological niches. The
analysis on the generic level of the subtribe Lysiphlebina by using the 28S rRNK gene
showed that the genus Lysiphlebia is clearly delimited from the other two genus with
great support. However, relations between the genera Adialytus and Lysiphlebus are
not so clear and the two genera are very closely related. | true |
Ispitivanje genetičke varijabilnosti lokalnih populacija kukuruza (Zea mays L.) molekularnim markerima | Genetic variability of maize landraces (zea mays l.) assessed by molecular markers | Agronomski biodiverzitet je širok pojam koji uključuje sve komponente
biološkog diverziteta od značaja za hranu i poljoprivredu. On predstavlja rezultat
interakcije između genetičkih resursa, životne sredine i upravljanja sistemima i prakse
koji čine poljoprivrednu proizvodnju. Biljni genetički resursi, smatraju se izuzetno
značajnim u obezbeđivanju dovoljne količine hrane neophodne za ljudsku ishranu.
Procenjuje se da danas ukupno 30 useva obezbeđuju 95% čovekovih potreba za hranom.
Kukuruz je jedna od najznačajnijih useva koja se gaji širom sveta. Iako poseduje
izuzetno veliku genetičku varijabilnost, u komercijalnoj upotrebi se nalazi svega oko
5% ukupne germplazme kukuruza, koja obezbeđuje visoke prinose.
Banka gena Instituta za kukruz „Zemun Polje“ održava kolekciju od 2217
lokalnih populacija kukuruza klasifikovanih u 18 agroekoloških grupa, sakupljenih na
prostoru bivše Jugoslavije. Ispitivanje diverziteta lokalnih populacija predstavlja
osnovni preduslov za njihovu efikasnu klasifikaciju, čuvanje i korišćenje, i ima za cilj
procenu genetičke raznovrsnosti i strukture populacija
Ispitana je genetička varijabilnost 54 lokalne (po tri populacije svake
agroekološke grupe) i 6 introdukovanih populacija kukuruza (po dve iz Francuske,
Gruzije i Kine). Za ispitivanje genetičke varijabilnosti populacija korišćeno je 18
morfoloških osobina i 10 RAPD (Random Amlified Polymorphic DNA) i 10 SSR
(Simple Sequence Repeat) markera. Na osnovu morfoloških osobina urađena je analiza
varijanse i uočene su značajne razlike kod svih osobina za različite izvore variranja što
govori o visokom stepenu fenotipskog diverziteta između populacija. Takođe, dobijene
su visoke vredosti heritabilnosti u širem smislu (preko 0,6) za skoro sve osobine, osim
dužine granatog dela metlice. Rezultati PCA analize su ukazali da lokalne populacije
kukuruza mogu biti okarakterisane pomoću osobina kao što su rast biljke, osobine
metlice i karakteristike zrna, a zapaženo je i veće grupisanje tvrdunaca/polutvrdunaca,
odnosno poluzubana/zubana. Na osnovu morfoloških osobina i molekularnih markera,
pomoću UPMGA metode dobijeni su klasteri, koristeći NTSYSpc statistički program.
Morfološka, SSR i RAPD analiza nisu dovele do jasnog grupisanja lokalnih populacija
prema poreklu, ali je uočeno delimično poklapanje između grupa populacija povezanih
u kastere/subklaste i putevima introdukcije, odnosno njihovog nastanka od originalnih
populacija. Na osnovu vrednosti dobijenih genetičkih sličnosti i grupisanja populacija u
skladu sa agroekološkim grupama, zaključeno je da su SSR markeri bolji izbor od
RAPD markera za analizu varijabilnosti. | Agricultural biodiversity is a broad term which includes all components of
biological diversity of relevance to food and agriculture. It represents the result of
interaction between genetic resources, environmental protection and both management
systems and practices that make agricultural production. Plant genetic resources are
considered to be very important in providing sufficient amounts of food for human
consumption. It is estimated that today a total of 30 crops provide 95% of human needs
for food. Corn is one of the most important crops that are grown around the world.
Although it has a very high genetic variability, only about 5% of the germplasm is in the
commercial use, which provides high yields.
Maize Research Institute „Zemun Polje“ genebank maintains a collection of
2217 maize landraces classified into 18 agro-ecological groups, collected in the former
Yugoslavia. Evaluation of genetic diversity of the local population represents basic
precondition for their effective classification, storage and use. It aims to assess the
genetic diversity and population structure.
Assessment of genetic variability was done on 54 maize landraces (three
landraces from each agro-ecological group) and six introduced maize landraces (two of
each from France, Georgia and China). In order to analyze genetic variability of maize
landraces, 18 morphological traits, 10 RAPD (Random Amplified Polymorphic DNA)
and 10 SSR (Simple Sequence Repeat) markers were used. Analysis of variance was
performed for evaluate morphological traits. Significant differences were observed for
all traits for different sources of variation which indicates a high degree of phenotypic
diversity between populations. Also, high broad-sense heritability (over 0.6) were
obtained for almost all the traits except branched tassel length. The results of PCA
analysis indicated that local maize populations can be characterized by traits such as
plant growth, tassel traits and kernel characteristics. It was also observed the larger
grouping of flint/semi-flint respectively to semident/dent. Unweighted Pair Group
Method with Arithmetic Mean (UPGMA) method was applied for cluster analysis. All
marker data analyses were performed using NTSYSpc statistical program.
Morphological, SSR and RAPD analysis did not lead to a clear grouping of the
landraces by origin, but was observed partial correlation between groups of landraces
connected in cluster/subcluster and ways of introducing from their orgin. Based on the
obtained values of genetic similarity and grouping landraces according to agro-
ecological groups, it was concluded that SSR markers are a better choice than RAPD
markers for estimation of genetic variability of maize landraces. | true |
Efekat mimetika superoksid dismutaze na mitohondrijalni i funkcioni status humanih spermatozoida | The effect of superoxide dismutase mimic on mitochondrial and functional state of human spermatozoa | Neplodnost je globalni problem u svetu. Uzroci i frekvenca različitih oblika
neplodnosti variraju, a novije studije pokazuju da reaktivne vrste kiseonika (ROS) i
azota (RNS), superoksid (O2•─) i azot oksid (NO) pre svega, igraju važnu ulogu, posebno
kod neplodnosti muškaraca. Pokazano je da su mitohondrije ne samo mesto visoke
produkcije reaktivnih vrsta, već organele od čijeg funkcionog statusa zavisi, odnosno sa
njim pozitivno korelira, fertilizacioni potencijal i kvalitet humanih spermatozoida.
Međutim, molekulska osnova delovanja ROS/RNS na funkcionost spermatozoida,
posebno na funkcioni status mitohondrija, još uvek je nepoznata.
Cilj doktorske disertacije je da se ispitaju efekti modulacije redoks statusa
spermatozoida na ključne parametre koji determinišu funkcioni status, odnosno
aktivnost mitohondrija, kao i kvalitet spermatozoida, podjednako na molekulskom i
strukturnom nivou.
Iz tih razloga, redoks status spermatozoida je specifično moduliran
pentaazamakrocikličnim Mn(II) mimetikom SOD (engl. superoxide dismutase),
M40403, koji selektivno uklanja O2•─ i indirektno modulira bioraspoloživost NO. U cilju
rasvetljavanja signalnih puteva regulacije fertilizacionih procesa, naročito eventualnog
uticaja NO na poboljšanje funkcionog statusa mitohondrija i fertilizacionih svojstava
spermatozoida, ispitivali su se sledeći parametri: pokretljivost spermatozoida,
koncentracija NO elektrohemijski, korišćenjem NO specifične elektrode, kao i
korišćenjem fluorescentne probe specifične za NO (Daf2-DA, diamino-fluorescein-2-
acetat); lokalizacija produkcije NO u spermatozoidima; endogena produkcija NO
praćenjem ekspresije izoformi NOS (engl. NO synthase) – endotelske (eNOS),
neuronalne i inducibilne; enzimi ključni za produkciju O2•─ (NADPH oksidaza) i
njegovo uklanjanje (CuZnSOD, engl. cooper zink SOD i MnSOD, engl. manganese
SOD); ekspresija enzima odgovornih za uklanjanje H2O2, nastalog aktivnošću SOD i
SOD mimetika, katalaze i GSH-Px (engl. glutathione peroxidase); mitohondrijalna
dinamika i metabolički status, praćenjem mitohondrijalnog membranskog potencijala
(katjonska fluorescentna proba Mitotracker Green FM) i ekspresija kompleksa
oksidativne fosforilacije (kompleks I, kompleks III, kompleks IV, citohrom c, citohroma
b i ATP sintaza). Pored toga ispitivani su osnovni parametri semene plazme i
spermatozoida, kao i korelacija sa nivoom enzima antioksidativne odbrane u semenoj
plazmi.
U disertaciji su korišćeni spermatozoidi normospermičnih pacijenata. Prečišćeni
spermatozoidi su podeljeni u tri eksperimentalne grupe. Prva grupa je resuspendovana u
Tirodovom medijumu i ispitivana odmah (kontrola), dok su druga (resuspendovana u
Tirodovom medijumu) i treća (resuspendovana u Tirodovom medijumu koji sadrži 50
µM M40403) ispitivane nakon inkubacije od tri časa. Vreme i uslovi inkubacije su
identični onima koji se koriste tokom obrade spermatozoida za fertilizaciju in vitro
(IVF). Pre ispitivanja spermatozoida, čistoća uzorka je ispitivana i potvrĎena
svetlosnom mikroskopijom i odsustvom amplifikacije iRNK za specifične markere
germinativnih ćelija, epitelskih ćelija i leukocita.
Rezultati disertacije pokazuju da primena SOD mimetika poboljšava funkcioni
kapacitet spermatozoida, posebno energetski, vezan za mitohondrijalnu aktivnost.
Naime,
inkubacija
spermatozoida
u
Tirodovom
medijumu
(tri
sata
pod
nekapacitirajućim uslovima) praćena je smanjenjem pokretljivosti spermatozoida. Pri
istim uslovima, tretman SOD mimetikom restaurira pokretljivost spermatozoida, kao
jedan od ključnih parametara kvaliteta spermatozoida za IVF. Paralelno tome, posle
inkubacije spermatozoida u Tirodovom medijumu dolazi do smanjenja količine NO u
medijumu, dok mimetik ne samo da rastaurira nivo NO već je njegova koncentracija
iznad nivoa u poreĎenju sa kontrolnim spermatozoidima (pre inkubacije). U skladu sa
tim, uočeno je da se populacija Daf2-DA pozitivnih spermatozoida povećava pri
tretmanu sa M40403. Uz to, SOD mimetik indukuje povećanje ekspresije iRNK za
eNOS. Stoga se može zaključiti da SOD mimetik, M40403, modulira redoks status u
spermatozoidima, posebno NO-zavisni, delujući ne samo na povećanje NO
biodostupnosti usled smanjenja koncentracije O2
•─, već i na indukciju njegove endogene
sinteze. SOD mimetik direktno afektuje funkcioni status mitohondrija spermatozoida
održavanjem populacije Mitotracker Green FM pozitivnih ćelija na kontrolnom nivou.
Takvi spermatozoidi tokom asistirane reprodukcije imaju veći fertilizacioni potencijal.
Na molekulskom nivou to se ogleda u tome što SOD mimetik dovodi do restauracije
ekspresije nukleusno kodiranih subjedinica kompleksa I (Ndufa6) i IV (COX IV, engl.
cytochrome c oxidase) elektron transportnog lanca mitohondrija. SOD mimetik takoĎe
indukuje povećanje genske ekspresije MnSOD i katalaze. Sinhronizovanje puta
uklanjanja O2
•─ i H2O2 SOD mimetikom važno je za održavanje ukupnog redoks stanja u
spermatozoidima.
Ispitivanje parametara antioksidativne odbrane u semenoj plazmi pokazuje da
postoji korelacija sa parametarima kvaliteta spermatozoida i da se oni mogu koristiti kao
potencijalni biomarkeri u analizi kvaliteta spermatozoida.
SOD mimetik predstavlja relevantno farmakološko sredstvo u terapiji
neplodnosti kod muškaraca preko poboljšanja funkcionih parametara spermatozoida i
njihove mitohondrijalne aktivnosti. Njegova primena može biti dobra strategija u
asistiranoj fertilizaciji, kao i tretiranja subfertilnih (infertilnih) stanja koja se karakterišu
oštećenjima mitohondrija i/ili izmenjenim redoks statusom spermatozoida. | Infertility is a global problem worldwide. The frequency and origin of different
forms of infertility vary, but recent studies showed that reactive oxygen species (ROS)
and reactive nitrogen species (RNS), first of all, superoxide (O2
•─) and nitric oxide
(NO), play an important role, especially in male infertility. It has been shown that
mitochondria play essential role in regulation of reactive species production. Moreover,
mitochondrial functional status in spermatozoa positively correlates with fertilizing
potential and quality of human sperm. However, the molecular basis of ROS/RNS
action on sperm functionality, especially on the functional status of sperm mitochondria,
is still unknown.
The aim of the dissertation was to examine the effects of redox status
modulation in human spermatozoa on the key parameters determining functional status
(activity) of mitochondria and sperm quality, at the molecular and structural levels.
The
redox
status
of
the
sperm
is
specifically
modulated
using
pentaazamacrocyclic Mn(II) mimic SOD (eng. superoxide dismutase), M40403, that
selectively removes O2
•─ and indirectly modulates the bioavailability of NO. In order to
shed more light on the signalling pathways underlying fertilizing processes, particularly
possible beneficial effects of NO on mitochondria functional status and sperm fertilizing
properties, the following parameters were examined: sperm motility; concentration of
NO, electrochemically, using NO specific electrodes, as well as using fluorescent
probes specific for NO (Daf2-DA, fluorescein-diamino-2-acetate); localization of NO
production in the spermatozoa; endogenous NO production by determining the
expression of NOS isoforms (NO synthase) – endothelial (eNOS), neuronal and
inducible; enzymes essential for production of O2
•─ (NADPH oxidase) and its removing
(CuZnSOD, cooper zinc SOD and MnSOD, manganese SOD); expression of enzymes
removing H2O2, generated by activity of SOD and SOD mimic, catalase and glutathione
peroxidase (GSH-Px); mitochondrial dynamic and metabolic status, using cationic
fluorescent probe Mitotracker Green FM for monitoring mitochondrial membrane
potential and finally the expression of complexes of the oxidative phosphorylation
(Complex I, Complex III, Complex IV, cytochrome c, cytochrome b and ATP
synthase). Besides, basic parameters of the seminal plasma and sperm, as well as their
correlation with the level of antioxidant enzymes, were determined.
To do this, sperm of normozoospermic patient were used in this dissertation.
Purified spermatozoa were divided into three experimental groups. The first group was
resuspended in Tyrode’s medium and tested immediately (control), while the second
(resuspended in Tyrode’s medium) and the third (resuspended in Tyrode’s medium
containing 50 μM M40403) groups were examined after incubation of three hours. Time
and incubation conditions are identical to those for the processing of sperm for in vitro
fertilization (IVF). Before sperm examinations, the purity of the sample was tested and
confirmed by light microscopy and the absence of amplification of mRNA for specific
markers of germ cells, epithelial cells and leukocytes.
The results of the thesis show that treatment with SOD mimic improves the
functional capacity of spermatozoa, especially related to mitochondrial energy state.
The incubation of spermatozoa in Tyrode’s medium (three hours under non-capacitating
conditions) leads to decrease in sperm motility. In the same conditions, the treatment of
SOD mimic restores sperm motility, as one of the key parameters of sperm quality for
IVF. In parallel, incubation of spermatozoa in Tyrode’s medium induces decrease of
NO level in the medium, while SOD mimic restores and evenmore increases it
compared to control sperm (before incubation). Accordingly, the population of Daf2-
DA positive spermatozoa is increased after M40403 treatment. In addition, the SOD
mimic induces the increase of eNOS iRNK expression. Thus, it seems likely that SOD
mimic, M40403, modulates the redox environment in spermatozoa, especially NO-
dependent, not only by increasing NO bioavailability, due to removing O2
•─, but also
upregulating its endogenous synthesis. Moreover, SOD mimic directly leads to
maintaining of mitochondrial functional state in spermatozoa, since it restores
population of Mitotracker Green FM positive cells at the control level. Such
spermatozoa have a higher fertilizing potential during assisted reproduction. In line with
this are M40403-stimulated changes at mitochondria molecular level. Namely, SOD
mimic induces restoration of the iRNK expression of nucleus encoded subunits of
complex I (Ndufa6) and IV (COX IV, eng. cytochrome c oxidase) of mitochondrial
electron transport chain. SOD mimic increases the gene expression of MnSOD and
catalase, as well. Such coordinated upregulation of O2
•─ and H2O2 removing enzymes is
important to maintain the overall redox state of spermatozoa.
Our results also show that there is correlation between components of
antioxidant defence in seminal plasma with sperm quality parameters and thus they can
be used as potential biomarkers in the analysis of sperm quality.
These results suggest that M40403 can be promising pharmacological tool in the
treatment of male infertility acting beneficially on mitochondrial activity and functional
parameters of spermatozoa. Using of SOD mimic can be a good strategy in assisted
fertilization, as well as treatment of infertile states accompanied by mitochondrial
impairments and/or disturbed sperm redox state. | true |
Efekti estradiola u srcu pacova sa insulinskom rezistencijom izazvanom ishranom bogatom fruktozom | Effects of estradiol in the heart of rats with fructose-rich diet-induced insulin resistance | Fruktoza je prirodni šećer, ĉiji se unos poslednjih decenija dramatiĉno povećao, najvećim delom zbog povećane upotrebe kukuruznog sirupa sa visokim sadrţajem fruktoze. Povećan unos fruktoze vodi ka razvoju metaboliĉkog sindroma kod ljudi i eksperimentalnih ţivotinja. Pored promena u jetri, adipoznom tkivu i skeletnim mišićima, ishrana bogata fruktozom je praćena i razvojem insulinske rezistencije u srcu. Poznato je da insulin ostvaruje svoje efekte na ćelije srca i da u stanju insulinske rezistencije u srcu dolazi do poremećaja u signalnom putu Akt/eNOS i balansu korišćenja energetskih supstrata. Posledice povećanog unosa fruktoze su polno zavisne, pri ĉemu je protektivni efekat pripisan estrogenim hormonima. Fiziološke koncentracije estrogena poboljšavaju osetljivost na insulin i deluju kardioprotektivno. TakoĊe, estradiol utiĉe na specifiĉno delovanje insulina u srcu. Cilj ove studije bio je analiza efekta estradiola na procese u srcu regulisane insulinom u stanju insulinske rezistencije izazvane ishranom bogatom fruktozom. Ţenke pacova su podvrgnute standardnoj ishrani ili ishrani obogaćenoj 10% rastvorom fruktoze tokom 9 nedelja i ovarijektomisane 2 nedelje pre ţrtvovanja, pri ĉemu je polovina ţivotinja na fruktoznoj ishrani dan nakon ovarijektomije podvrgnuta tretmanu estradiolom. U cilju izuĉavanja efekata ishrane bogate fruktozom i estradiola na insulinom regulisanu fosforilaciju i unutarćelijsku lokalizaciju ispitivanih molekula, polovina ţivotinja je tretirana insulinom 40 minuta pre ţrtvovanja. Analizirani su molekuli iz insulinskog signalnog puta (IRS-1, Akt i ERK1/2), kao i efektorni molekuli ĉiju funkciju reguliše insulin, kao što su eNOS, Na+/K+-ATP-aza i molekuli ukljuĉeni u transport i metabolizam energetskih supstrata u srcu (GLUT1, GLUT4, CD36, lipin 1 i CPTI) i lipidni profil srca. Ispitivana je ekspresija ovih molekula na nivou proteina, kao i njihova fosforilacija ili unutarćelijska lokalizacija. Ishrana bogata fruktozom je povećala unos teĉnosti i smanjila unos hrane, dok je povećala ukupan kalorijski unos i koncentraciju leptina u plazmi. Estradiol je većim delom poništio štetne efekte ishrane na regulaciju apetita, verovatno preko povećanja centralne osetljivosti na leptin. Ţivotinje na fruktoznoj ishrani su imale povišene koncentracije triglicerida i insulina u plazmi i povećan HOMA indeks, smanjenu koncentraciju slobodnih masnih kiselina, dok je koncentracija glukoze bila nepromenjena... | Fructose is natural sugar whose intake has increased dramatically over the past decades, mostly due to increased consumption of high-fructose corn syrup. Increased intake of fructose initiates development of metabolic syndrome phenotype in humans and experimental animals. In addition to changes in liver, adippose tissue and skeletal muscle, fructose-rich diеt is accompanied by cardiac insulin resistance. Cardiac muscle is a target of insulin. Insulin resistance is accompanied by disturbances in Akt/eNOS signalling and altered cardiac usage of energetic substrates. Consequences of enhanced fructose intake are shown to be sex-dependent and the protective effect is attributed to estrogens. Physiological concentrations of estrogens improve insulin sensitivity and they are cardioprotective. Also, estrogens affect specific cardiac insulin action. The aim of the present study was to analyze effects of estradiol on insulin-regulated processes in the heart in insulin resistance state. Female rats were subjected to standard diet or diet containing 10% fructose in drinking water during 9 weeks. Two weeks before sacrifice, all animals were bilaterally ovariectomized and half of the fructose-fed animals were subjected to estradiol replacement treatment, day after ovariectomy. In order to study fructose-rich diet and estradiol effects on the insulin regulated phosphorylations and subcellular localization of analyzed molecules, half of the animals were treated with insulin, 40 min before killing. Insulin signaling molecules (IRS-1, Akt i ERK1/2) were analyzed, as well as insulin regulated effector molecules, such as eNOS, Na+/K+-ATPase and molecules involved in the transport and metabolism of energetic substrates in the heart (GLUT1, GLUT4, CD36, lipin 1 i CPTI), as well as profile of cardiac lipids. We examined the protein exppression of the molecules, as well as their phosphorylation or subcellular localization. Fructose-rich diet increased liquid intake and decreased food intake, while it increased total caloric intake and plasma leptin concentration. Estradiol abolished most of the detrimental effects of diet on appetite regulation, probably through increase in central leptin sensitivity. The fructose-fed rats had increased plasma triglycerides and insulin levels and increased HOMA index, decreased plasma free fatty acid level, while glucose concentration was unaltered... | false |
Mehanizmi preživljavanja astrocita pacova u medijumu bez glukoze i u uslovima hipoksije in vitro | The mechanisms of rat astrocytes survival in the medium without glucose and in hypoxic conditions in vitro | Uvod. Poznato je da astrociti preživljavaju duže izlaganja deprivaciji kiseonika i
glukoze (OGD) i da preživljavaju danima bez nutrijenata u poređenju sa izrazito
osetljivim neuronima. Razlozi ovako smanjene osetljivosti astrocita i dalje nisu
dovoljno razjašnjeni. Šta više, promene membranskog potencijala mitohondrija
(ψm) astrocita, kao indikatora ćelijskog energetskog metabolizma i vijabilnosti,
nisu ispitane tokom simulirane reperfuzije nakon dužeg izlaganja OGD (odnosno
1 % O2 bez glukoze u prisustvu ograničene količine alternativnih nutrijenata).
Smanjena osetljivost astrocita može biti posledica korišćenja alternativnih
vanćelijskih izvora energije, kao i unutarćelijskih zaliha energije u cilju održanja
membranskog potencijala mitohondrija. Strategija preživljavanja astrocita tokom
takvih metaboličkih izazova i dalje nije jasna.
Metode. U ovoj studiji, kultura astrocita izložena je deprivaciji glukoze (GD),
OGD i njihovoj sukcesivnoj kombinaciji različitih trajanja. Promene ψm, praćene
putem promena u fluorescenciji JC-1, ispitane su u toku jednog sata tokom
simulirane reperfuzije u cilju modelovanja uslova in vivo. Fluorescentni obeleživač
mitohondrija JC-1 ulazi u matriks mitohondrije u zavisnosti od potencijala
membrane mitohondrije, pomerajući pri tome maksimum fluorescencije iz zelenog u
crveni deo spektra. Takođe, efekti farmakološke
inhibicije dva bitna
metabolička puta: autofagije hlorokinom (CQ) i lipolize orlistatom, ispitani su
tokom deprivacije nutrijenata. Praćen je uticaj pomenutih inhibicija na promene
vijabilnosti astrocita bojenjima sa akridin narandžastim (AO) i propidijum
jodidom (PI), kao i promene membranskog potencijala mitohondrija (JC-1).
Rezultati. Pokazali smo da su astrociti otporni na duže periode OGD (u trajanju
od 6 i 8 časova) koja je imala slab uticaj na ψm tokom simulirane reperfuzije, dok je
GD dovela do hiperpolarizacije membranskog potencijala mitohondrija astrocita.
Naknadna hemijska hipoksija izazvana tretmanom sa natrijum azidom dovela je do
depolarizacije mitohondrija, ali je njihova reakcija na takav tretman nakon OGD
bila odložena u odnosu na normoksične uslove. Kada je GD u trajanju od 12 časova
prethodila OGD, porast ψm izazavan je i sa GD i sa naknadnom OGD, ali značajna
interakcija između njihovih efekata nije zabeležena. Međutim, kada je GD
produžena na 48 časova, imala je sinergističan efekat sa naknadnom OGD
izazivajući dodatno povećanje membranskog potencijala mitohondrija astrocita.
Inhibicija autofagije nije pokazala značajan efekat na preživljavanje astrocita sve
do 8-9 časa deprivacije nutrijenata. Pokazali smo i da kratkotrajna inhibicija
autofagije čini astrocite osetljivim na deprivaciju nutrijenata. Inhibicija
lipolize dovela je do smrti ćelija, ali je do nje došlo znatno kasnije u poređenju sa
inhibicijom autofagije. Takođe, oba inhibitora sprečila su porast membranskog
potencijala mitohondrija izazvan deprivacijom nutrijenata.
Zaključci. Jedan od najbitnijih zaključaka ove studije jeste da astrociti održavaju
membranski potencijal mitohondrija hiperpolarizovanim tokom deprivacije
nutrijenata i OGD. Takođe smo istakli značaj dva osnovna supstrata lanca
transporta elektrona (glukoze i kiseonika) u hiperpolarizaciji
membrane
mitohondrija i relevantnost dobijenih rezultata za otpornost astrocita na
ishemičnu povredu in vitro. Ovi rezultati takođe pokazuju da su autofagija i lipoliza
esencijalne za preživljavanje astrocita tokom deprivacije nutrijenata, što može
predstavljati adaptaciju koja je blisko povezana sa ulogom astrocita u podršci
neuronima u obavaljanju njihove funkcije. | Introduction. Astrocytes are known to tolerate long periods of oxygen-glucose deprivation
(OGD) and they survive nutrient deprivation (ND) for days as compared to rather vulnerable
neurons. The reasons for this reduced vulnerability of astrocytes are not well understood. In
fact, changes in mitochondrial membrane potential (ψm), as the indicator of the cellular energy
metabolism and viability, have not been investigated during simulated reperfusion after
extended OGD exposure (i.e. 1 % of О2 without glucose in the presence of limited
alternative nutrients). This reduced vulnerability could be due to utilization of the alternative
extracellular sources of energy, as well as utilization of the internal energy stores, in
maintenance of mitochondrial membrane potential. The pro-survival strategy of astrocytes
under such metabolic challenge is still not clear.
Methods. Here, we subjected astrocytes in culture to glucose deprivation (GD), OGD and
combinations of both conditions varying in duration and sequence. Changes in ψm, visualized
by the change in the fluorescence of JC-1, were investigated within one hour after
reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion.
Fluorescent probe JC-1 enters the mitochondrial matrix in a potential-dependent manner,
thus shifting its emission from green to red. Furthermore, the effects of inhibition of two
potential steps in energy acquisition during ND: autophagy using chloroqione (CQ) and
lipolysis using orlistat were investigated. Changes in astrocytes viability were followed with
acridine orange (AO) and propidium iodide (PI) staining, and ψm was followed with JC-1.
Results. We showed that astrocytes were resilient to extended periods of OGD (6 and 8 h),
which had little effect on ψm during reperfusion, whereas GD contributed to a more negative
ψm. Subsequent chemical oxygen deprivation induced by sodium azide caused depolarization,
which, however, was significantly delayed as compared to the normoxic group. When GD
preceded OGD for 12 h, mitochondrial membrane hyperpolarization was induced by both
GD and subsequent OGD, but significant interaction between these conditions was not
detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization
enhanced during reperfusion. This implicates synergistic effects of both conditions in that
sequence.
The inhibition of autophagy did not show significant effects on cell viability until 8-9 h of
ND. We observed that early inhibition of autophagy makes astrocytes vulnerable to later
ND. The inhibition of lipolysis also provoked cell death, but it emerged much later compared
to the effects of autophagy inhibition. Furthermore, the application of these inhibitors
prevented ND-related hyperpolarization of mitochondrial membrane.
Conclusions. One of the most important original findings of this work is that astrocytes
maintain ψm at hyperpolarization after extended GD, ND or OGD. We also point to the role
of the two main substrates of the electron transport chain (glucose and oxygen) in the
hyperpolarization of the mitochondrial membrane, and emphasize how this result may
further be relevant in terms of astrocyte resilience to prolonged ischemic injury in vitro. These
results also show that autophagy and lipolysis are essential for astrocytes survival under
nutrient deprivation, which may add to their role as neuron-supporting cells. | true |
The role of NO-synthetic pathway in structural remodeling of rat myocardium | Uloga NO-sintaznog puta u strukturnom remodeliranju miokarda pacova | Azot oksid (NO), endogeno sintetisan NO sintazama (NOS, engl. NO
synthases) ima važnu ulogu u optimizaciji adaptivnog odgovora srca. Poznato je da
NO pri fiziološkim koncentracijama ima pozitivno inotropno dejstvo, kao i da je
hronično redukovan fiziološki nivo NO spregnut sa izmenjenom funkcijom srca i da
stoga može doprineti razvoju srčane insuficijencije.
Polazeći od ovih osnova, cilj teze je bio da razjasni mehanizme remodeliranja
miokarda desne komore srca pacova u odgovoru na nisku temperaturu, kao i da se
ispita uloga NO, modulacijom NO-sintaznog puta. U tu svrhu pacovi Mill Hill soja,
starosti 2 meseca izlagani su niskoj temperaturi (4±1 ºC) u vremenu od 45 dana i
poređeni sa životinjama aklimiranim na sobnu temperaturu (22±1 ºC). Obe grupe
životinja, aklimirane na sobnu i nisku temperaturu dodatno su podeljene u tri
podgrupe: 1. netretirane; 2. životinje tretirane supstratom NO sintaza NOS – L-
arginin•HCl (2.25%) u pijaćoj vodi; 3. životinje pojene inhibitorom NOSs, L-
NAME•HCl (0.01%).
U poređenju sa kontrolnim, životinje aklimirane na hladnoću imaju značajno
nižu ekspresiju transkripcionog faktora proliferacije ćelija. Mada se beleži trend
hipertrofije kardiomiocita i smanjenje kapilarnosti, volumenska gustina komponenti
miokarda desne komore se ne menja kod pacova aklimiranih na hladnoću, u
poređenju sa životinjama aklimiranim na sobnu temperaturu. Zajedno, rezultati ne
pokazuju značajnu hipertrofiju desne komore prouzrokovane hladnoćom, barem ne na
histološkom nivou. Rezultati pokazuju da strukturne promene nakon L-arginin
tretmana odgovaraju fiziološkoj srčanoj hipertrofiji. Naime, L-arginin tretman pacova
aklimiranih na sobnu temperaturu, u poređenju sa kontrolama vodi hipertrofiji
kardiomiocita, praćeno istovremenim povećanjem kapilarnosti i vezivnog tkiva.
Međutim, relativni odnos komponenti miokarda, količina kolagena i strukturna
organizacija miofibrila se ne menja nakon L-arginin tretmana, u poređenju sa
kontrolnim životinjama.
Sa druge strane, inhibicija enzimske sinteze NO L-NAME tretmanom rezultira
pojavom fibroze, odnosno povećanjem volumenske gustine vezivnog tkiva uz
povećano deponovanje kolagena u intersticijumu. Pored toga, uprkos smanjenju
volumenske gustine, očigledna je hipertofija kardiomiocita. Opisane promene su
prisutne u L-NAME-tretiranim grupama, na sobnoj i niskoj temperaturi. U okviru L-
NAME-tretiranih grupa, fibrozna je izraženija u grupi pacova aklimiranih na
hladnoću. Smanjena veličina nukleusa kardiomiocita je detektovana u netretiranoj
grupi pacova aklimiranih na hladnoću, kao i nakon L-arginin- i L-NAME- tretmana.
Rezultati teze ukazuju da povećanje binuklearnosti, zajedno sa opisanim smanjenjem
veličine nukleusa imaju bitnu ulogu u hipertrofiji kardiomiocita, kao i samog
miokardijuma. Značajno je da su opisane nukleusne promene najizrazitije u L-NAME-
tretiranim grupama. Podaci koji se odnose na nukleusne promene i njihovu ulogu u
hipertrofiji kardiomiocita nakon hroničnog L-NAME tretmana po prvi put su istaknuti
u ovoj tezi, te su kao takvi jedinstveni u literaturi. Osim nukleusnih, značajne
ultrastrukturne promene uočene su na nivou mitohondrijalnih populacija u svim
ispitivanim grupama; u poređenju sa netretiranom grupom, smanjen površinski profil
svih mitohondrijalnih subpopulacija je zabeležen u L-arginin-tretiranoj grupi pacova
aklimiranih na sobnu temperaturu. Takođe, u poređenju sa kontrolom, smanjen
površinski profil perinukleusnih i intersarkomeralnih mitohondrijalnih subpopulacija
je zabeležen u grupi životinja aklimiranih na hladnoću. Međutim, L-arginin-tretirana
grupa pacova aklimirana na hladnoću je imala veći površinski profil mitohondrija u
poređenju sa netretiranom grupom sa hladnoće, kao i u poređenju sa L-arginin-
tretiranom grupom koja je boravila na sobnoj temperaturi. Slično, povećan površinski
profil mitohondrija u perinukleusnom i subsarkolemalnom kompartmentu, odnosno
smanjen u intersarkomeralnom regionu zabeležen je u L-NAME-tretiranoj grupi
životinja aklimiranih na hladnoću, u poređenju sa istim tretmanom na sobnoj
temperaturi. Pored toga, ispitivanje ekspresije koneksina 43, jednog od koneksina
električnih sinapsi interkalarnih diskova, je pokazalo da se ekpresija koneksina 43
značajno smanjuje nakon primenjenih tretmana u poređenju sa netretiranom grupom
na sobnoj temperaturi. Sa druge strane, u miokardu pacova aklimiranih na hladnoću
nema značajnih promena u ekspresiji koneksina 43 u poređenju sa kontrolnim
pacovima, kao ni nakon primenjenih tretmana na hladnoći, u poređenju sa
netretiranom grupom sa hladnoće. Na osnovu toga se može zaključiti da nema
izrazitih promena u kapacitetu za prenos akcionog potencijala u miokardu desne
komore, barem ne kod grupa pacova aklimiranih na hladnoću. U cilju pojašnjavanja
uloge endogene sinteze NO u pokazanom strukturnom remodeliranju miokarda desne
komore srca pacova ispitivane su ekspresija i lokalizacija NOS. Rezultati pokazuju da
L-arginin povećava ekspresiju endotelijalne NOS (eNOS), ukazujući da ova izoforma
posreduje u produkciji NO u desnoj komori srca pacova. Sa druge strane, L-NAME,
pored toga što deluje kao nespecifični inhibitor NOS, deluje i na translacioni nivo
konstitutivnih izoformi, smanjujući proteinsku ekspresiju nNOS i održavajući nivo
eNOS na kontrolnom nivou. Obzirom da aklimacija na hladnoću povećava eNOS, a
suprimira ekspresiju nNOS, može se zaključiti da eNOS ima protektivnu ulogu u
srčanom afterload-u i posledičnom razvoju hipertrofije desne komore srca.
Na osnovu dobijenih rezultata može se zaključiti da hronični L-arginin tretman
indukuje umerenu hipertofiju desne komore srca pacova na sobnoj temperaturi i
značajno je povećava u uslovima hronične stimulacije na hladnoći. L-argininom-
indukovana ventrikularna hipertofija se može smatrati fiziološkom, obzirom na
odsustvo fibroze. Sa druge strane, hronična inhibicija NOS, posredovana L-NAME je
asocirana sa patološkom hipertrofijom miokarda desne komore sa karakterističnim
znacima fibroze, ističući značaj deplecije NO u razvoju kardiovaskularne bolesti. | Nitric oxide (NO), endogenously synthesized by nitric oxide synthases (NOSs),
generally acts to fine tune and optimise cardiac pump function. Several experimental
studies have shown that low (submicromolar) doses of NO exert small positive
inotropic effects, while at higher, but still physiological levels, NO enhances
cardiomyocyte relaxation and diastolic function. It has been known that reduced NO
bioavailability plays an important role in the development of heart failure, but the
mechanisms are still uncovered. This thesis deals with structural remodeling of right
ventricle (RV) myocardium of rats, induced by chronic modulation of NO-producing
system and cold acclimation. In order to examine the possible effects of NO on
myocardial structure and molecular basis of its remodeling at room temperature
(22±1 ºC) or cold (4±1 ºC) acclimation, adult male rats (Mill Hill hybrid hooded, 2-
month-old), were treated with L-arginine or Nω-nitro-L-arginine methyl ester (L-
NAME) for 45 days, respectively.
Our results demonstrated that cold acclimation per se does not significantly
alter the volume densities of myocardial tissue components, but leads to a trend in
cardiomyocyte hypertrophy followed by a downward trend in capillarity. Also, cold-
acclimated control animals showed a significant decrease of proliferating cell nuclear
antigen expression in comparison with room temperature-kept control animals. In all,
these results do not indicate the cold-induced RV hypertrophy, at least at histological
level. Results demonstrated that the structural alterations observed after the L-arginine
treatment corresponded to physiologic cardiac hypertrophy, since no pathological
changes in the myocardial structure (increased collagen deposition and/or myofibril
distortion) after chronic L-arginine treatment were seen. Namely, L-arginine treatment
in RT-acclimated rats led to cardiomyocyte hypertrophy which was followed by a
simultaneous increase in capillarity and interstitial connective tissue in the
myocardium, maintaining the relative ratio of tissue components unaltered. The most
prominent effect of L-NAME on rat myocardia observed was fibrosis, demonstrated as
an increase in connective tissue volume density accompanied by increased collagen
abundance in the interstitium. This effect was followed by cardiomyocyte
hypertrophy, despite a decrease in their volume density. These alterations were
observed in both L-NAME-treated groups, with additional enhancement of interstitial
fibrosis after cold acclimation. The reduced size of cardiomyocytes’ nuclei was
detected in the cold-acclimated control group, and in all L-arginine- and L-NAME-
treated groups. This nuclear alteration, as well as the increased occurrence of
cardiomyocytes’ binucleation seems to play an important role in the cardiomyocytes
and myocardial hypertrophy. Decribed nuclear changes were the most evident after
the chronic treatment with L-NAME. According to our evidence, this is the first
demonstration of nuclear alterations in cardiomyocytes after chronic L-NAME
treatment which indicate their role in the cardiomyocyte hypertrophy. Besides nuclear
alterations, significant ultrastructural changes were observed in the cardiomyocytes
mitochondrial population in all examined groups. Namely, decreased profile surface
of all mitochondrial subpopulations was observed in L-arginine room temperature-
acclimated group, while L-NAME per se did not affect mitochondrial size, compared to
untreated group. Cold acclimation decreased profile size of cardiomyocytic
perinuclear and intersarcomeral mitochondrial subpopulations. Cold-acclimated L-
arginine-treated animals had increased mitochondrial profile size, compared to both,
untreated cold-acclimated and L-arginine-treated room temperature-acclimated group.
Also, L-NAME-treated animals acclimated to cold had increased profile surface of
mitochondria in perinuclear and subsarcolemal region, while in intersarcolemal region
mitochondrial profiles decreased, compared to L-NAME-treated room temperature-
acclimated group. Regarding the cardiomyocytes synapses, protein level of connexin
43 declined after L-arginine and, to a lesser extent, after L-NAME treatment at room
temperature. Neither the cold acclimation per se, nor drug treatments during cold
acclimation affected the connexin 43 expression level in the RV of rats, indicating that
there were no profound changes in cardiac electric coupling during the structural
remodeling, at least at the level of gap junctions. To reveal the involvement of
endogeneous NO-synthesis in the observed structural RV remodeling the expression
levels and the localisation of NOSs were examined. NO precursor, L-arginine,
upregulates eNOS expression in rats myocardium, thus indicating the primary effects
of eNOS in the NO production, at least in the RV of rats. On the other hand, L-NAME,
a potent non-selective inhibitor of NOSs acts also at the level of translation on
constitutive NOS isoforms in RV, by decreasing the expression of nNOS and by
maintaining the expression of eNOS at the control level. Since herein demonstarted
cold-induced upregulation of eNOS expression and downregulation of nNOS
expression are not shown to induce any structural signs of RV hypertrophy in rats, we
can assume a protective role of eNOS during increased cardiac afterload.
In conclusion, chronic L-arginine supplementation induces low level RV
hypertrophy
in
RT-acclimated
rats
and
significantly
augments
ventricular
hypertrophy in cold-acclimated animals. L-arginine-induced ventricular hypertrophy
could be considered physiological, since no signs of myocardial fibrosis were
observed. In contrast, chronic treatment with L-NAME, an inhibitor of NOSs, caused
pathological right ventricular hypertrophy with signs of myocardial fibrosis,
demonstrating the importance of NO depletion in the development of cardiovascular
disease. | true |
Varijabilnost genotipa i fenotipske ekspresije spinalne mišićne atrofije kod pacijenata iz Srbije | Variability of genotype phenotypic expression of spinal muscular atrophy in patients from Serbia | Spinalna mišićna atrofija (SMA) je drugo najčešće autozomno recesivno oboljenje
kod ljudi. Prema uzrastu kada se pojavljuju prvi simptomi i težini kliničke slike SMA je
klasifikovana u tri tipa. Mutacije u telomernoj kopiji gena SMN1 (eng. survival of mo-
tor neuron), koji se nalazi u 5q13 regionu, dovode do nastanka bolesti, dok ostali geni u
ovom regionu predstavljaju potencijalne modifikatore SMA fenotipa.
Detekcija homozigotne delecije egzona 7 i 8 gena SMN1, primenom PCR ampli-
fikacije i polimorfizma dužine restrikcionih fragmenata (PCR/RFLP, eng. Polymerase
Chain Reaction/Restriction Fragment Length Polymorphism) urađena je na uzorcima
107 SMA pacijenata iz Srbije i 100 kontrolnih uzoraka DNK poreklom od zdravih oso-
ba. Kod pacijenata bez homozigotne delecije kod kojih je klinička reevaluacija potvrdila
dijagnozu SMA je primenom multipleks ligaciono-zavisne amplifikacije proba (MLPA,
eng. Multiplex Ligation-dependent Probe Amplification) određivan broj kopija prisu-
tnog gena SMN1 u cilju detekcije složenih heterozigotnih nosilaca delecije na jednom i
tačkaste mutacije na drugom hromozomu. Kod potvrđenih heterozigota sekvenciran je
egzon 6 gena SMN u cilju detekcije intragenskih mutacija. Za ispitivanje varijabilnosti
genotipa 5q13 regiona i njegove povezanosti sa fenotipskom ekspresijom bolesti prvo
je na istom uzorku analizirana homozigotna delecija gena NAIP (eng. neuronal apop-
tosis inhibitory protein). Nakon toga, primenom MLPA metode je kod 30 pacijenata
sa prethodno dijagnostikovanom homozigotnom delecijom gena SMN1 određivan broj
kopija gena iz 5q13 regiona: NAIP, SMN2 (centromerna kopija gena SMN), GTF2H2
(eng. general transcription factor IIH subunit 2) i gen koji kodira protein nepoznate
funkcije - H4F5. Studija je obuhvatila i ispitivanje 39 zdravih osoba sa porodičnom
SMA anamnezom kod kojih je analizom broja kopija gena SMN1 određivan status he-
terozigotnih nosilaca delecije ovog gena. Kod 66 fetusa iz 44 porodice sa visokim rizi-
kom za dobijanje obolelog potomstva sprovedena je prenatalna dijagnostika za direktnu
detekciju homozigotne SMN1 delecije.
Homozigotna delecija gena SMN1 je uočena kod 81% (87/107) ipitivanih SMA
pacijenata: delecija egzona 7 i 8 kod 76,6% (82/107) i delecija samo egzona 7 kod 4,7%
(5/107). Učestalost homozigotne delecije bila je najveća u grupi pacijenata sa SMA tip
I - 93,1% (54/58), u odnosu na SMA tip II - 71,4% (25/35) i SMA tip III - 57,1% (8/14).
Kod 23,1% (3/13) pacijenta bez homozigotne delecije gena SMN1 kod kojih je klinička
reevaluacija potvrdila dijagnozu SMA detektovana je jedna SMN1 kopija i potvrđena
je heterozigotnost. Kod dva ovakva pacijenta, klasifikovana kao SMA tip II, dokazano
je prisustvo mutacije koja dovodi do aminokiselinske zamene (eng. missense mutation)
c.821C>T. Homozigotna delecija gena NAIP je detektovana kod 21,5% (23/107) SMA
pacijenata i 1% (1/100) kontrola. Uočena je statistički značajna razlika u učestalosti
ove delecije i tipa SMA: najučestalija je bila kod SMA tip I pacijenata - 36,2% (21/58)
dok je učestalost u SMA tip II iznosila - 2,9% (1/35), a u SMA tip III - 7,1% (1/14).
Komparativna analiza homozigotne delecije gena SMN1 i NAIP je pokazala da su veće
homozigotne delecije učestalije u grupi SMA tip I pacijenata - 36,2% (21/58), dok su
manje delecije - 42,9% (6/14) i 7,1% (1/14) ili njihovo odsustvo - 42,9% (6/14) naju-
čestaliji u grupi SMA tip III. Analizom 5q13 regiona utvrđeno je da broj individualnih
kopija gena NAIP i SMN2 statistički značajno korelira sa fenotipom SMA pacijenata –
kako se smanjuje broj kopija gena tako tako klinička slika postaje sve teža. Pored toga,
homozigotne i heterozigotne delecije detektovane kroz prisustvo 0 i 1 kopije gena NAIP
su prisutne kod 80% svih ispitivanih pacijenata. Komparativna analiza ova dva gena je
pokazala da smanjenje broja kopija gena NAIP prati smanjenje broja kopija gena SMN2
i rezultira u težem obliku bolesti. Nije pronađena statistički značajna korelacija između
tipa SMA i broja kopija gena GTF2H2 i H4F5 u ispitivanoj grupi. Kod 61,5% (24/39)
analiziranih zdravih osobe sa porodičnom anamnezom SMA je kroz detekciju jedne ko-
pije gena SMN1 potvrđen status heterozigotnih nosilaca. Prenatalnom dijagnostikom je
prisustvo homozigotne delecije gena SMN1 detektovano kod 19,7% (13/66) ispitivanih
fetalnih uzoraka.
Na osnovu dobijenih rezultata zaključeno je da je ovakav pristup u analizi gena
SMN1 neophodan u cilju pouzdane prenatalne i postnatalne dijagnostike i izračunavanja
preciznog rizika za dobijanje obolelog potomstva. Analizom ostalih gena iz 5q13 regiona
uočen je visok stepen njegove varijabilnosti i nestabilnoti što ukazuje na njegovu ulogu
u fenotipu SMA, kao i na složenost mehanizama koji se nalaze u osnovi ove bolesti. Re-
zultati ovog rada su pokazali da prilikom predviđanja SMA fenotipa kod analiziranog
pacijenta treba imati u vidu sledeće: broj kopija gena SMN2, veličinu homozigotne ili
heterozigotne delecije koja pored SMN1 uključuje i susedne gene kao i tip intragenske
SMN1 mutacije ukoliko je prisutna. | Spinal muscular atrophy (SMA) is the second most frequent autosomal recessive
disease in humans. According to the age of onset and severity of clinical manifestations,
SMA is classified into three types. Mutations in the telomeric copy of the survival of
motor neuron gene (SMN1) cause the disease while other genes in 5q13 region has been
considered as modifying factors of the SMA severity.
Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/
RFLP) method was used for direct detection of homozygous deletions of SMN1 exons
7 and 8 in 107 SMA patients and 100 healthy controls from Serbia. Patients without
homozygous SMN1 deletion in which clinical re-evaluation confirmed SMA diagnosis
were further analysed using Multiplex Ligation-dependent Probe Amplification (MLPA)
for determination of SMN1 copy number to identify compound heterozygotes of deletion
and point mutation. Sequencing of SMN exon 6 for detection of subtle mutations was
performed in patients with confirmed heterozygosity. For examination of genotype vari-
ability in 5q13 region and its correlation with phenotype homozygous deletion of neu-
ronal apoptosis inhibitory protein (NAIP) gene was first analysed using PCR in the same
sample of patients and controls. After that, 30 patients with homozygous SMN1 deletion
were further examined using MLPA to determine copy number of genes in 5q13 region:
NAIP, centromeric copy of SMN gene (SMN2), general transcription factor IIH subunit 2
(GTF2H2) and putative RNA-binding
protein (H4F5). The study also included the analy-
sis of SMN1 copy number for carrier status determination in 39 healthy individuals with
SMA history. Prenatal diagnosis for direct detection of homozygous SMN1 gene deletion
was performed on 66 fetal samples from 44 families at high SMA risk.
Homozygous SMN1 gene deletion was detected in 81.3% (87/107) of SMA pa-
tients: deletion of exons 7 and 8 in 76.6% (82/107), deletion of exon 7 in 4.7% (5/107).
The highest deletion frequency was among SMA type I patients – 93.1% (54/58) com-
pared to SMA type II – 71.4% (25/35) and SMA type III – 57.1% (8/14). One SMN1 gene
copy was detected in 23.1% (3/13) patients without homozygous deletion of this gene
and with clinically reassessed confirmation of SMA diagnosis and heterozygosity was
confirmed. Among them two patients, classified as SMA type II, had missense mutation
c.821C>T. Homozygous deletion of NAIP gene was present in 21.5% (23/107) of SMA
patients and 1% (1/100) of controls. Statistical analyses showed significant difference
between frequency of this deletion and SMA type - deletion was the most frequent among
SMA type I patients compared to SMA type II and SMA type III: 36.2% (21/58) vs. 2.9%
(1/35) and 7.1% (1/14). Comparative analyses of homozygous deletions of SMN1 and
NAIP genes showed that extended gene deletions were more frequent among SMA type
I patients – 36.2% (21/58), while shorter deletions – 42.9% (6/14) and 7.1% (1/14) or
absence of deletion – 42.9% (6/14) were the most frequent among SMA type III patients.
The analysis of 5q13 region revealed negative correlation between copy number of SMN2
and NAIP genes separately and SMA phenotype and this negative correlation was statisti-
cally significant. Beside, homozygous and heterozygous deletions of NAIP gene, detected
as 0 and 1 gene copy, were present in 80% of all analysed patients. Comparative analysis
of these two genes showed that lower copy number of NAIP gene reflects lower copy
number of SMN2 gene and is related to severe phenotype. No statistically significant cor-
relation between the type of SMA and copy number of GTF2H2 and H4F5 genes, among
analysed patients was found.
Carrier status with one SMN1 gene copy was confirmed in 61.5% (24/39) of an-
alysed healthy individuals with SMA history. Among analysed fetuses, 19.7% (13/66)
showed homozygous deletion in SMN1 gene.
The results from this study revealed that the strategy used in SMN1 gene analyses,
is essential for reliable SMA prenatal and postnatal diagnosis as well for accurate risk as-
sessment. Copy number analysis of genes in 5q13 region showed the presence of a high
degree of variability and therefore instability of this region indicating its role in SMA
phenotype and revealed complex mechanisms underlying the disease. Consequently, gen-
otype-phenotype analysis of SMA should take into account the following: SMN2 copy
number, extent of homozygous or heterozygous deletion which may include other genes
from 5q13 region in addition to SMN1 and if present, SMN1 subtle mutations. | true |
Molekularna osnova auto-inkompatibilnog sistema heljde (Fagopyrum esculentum Moench) | Molecular basis of self-incompatibility system in buckwheat (Fagopyrum esculentum Moench) | Heljda (fam. Polygonaceae) je heteromorfna biljna vrsta koja sadrži cvetove dve
morfologije: "pin" (dugačak stubić tučka, kratke antere, manja polenova zrna) i "tram"
(kratak stubić tučka, dugačke antere, veća polenova zrna). Oplođenje je dozvoljeno samo
između cvetova različite morfologije, dok je sprečeno između cvetova iste morfologije
delovanjem sistema auto-inkompatibilnosti (AI), koji ne dozvoljava ukrštanje u srodstvu.
Heljda se zbog svojih dobrih nutritivnih osobina gaji širom sveta, a osnovni problem u
njenom uzgajanju predstavljaju nizak i nepouzdan prinos i visoka otpornost na introgresiju
novih osobina iz srodnih vrsta, što su direktne posledice njene AI. Ovakvo delovanje AI
sistema može predstavljati prepreku u uzgajanju i oplemenjivanju poljoprivredno značajnih
vrsta (npr. badem, heljda, itd.), odatle i veliki interes za proučavanje ovih sistema. Kroz
njihovo upoznavanje može se omogućiti proizvodnja samooplodnih i hibridnih linija
željenih svojstava, kao i razvijanje novih markera, pomoću kojih se ubrzava proces
selekcije, što bi dovelo do povećanja prinosa uz snižavanje troškova gajenja. Ono što je
poznato o heljdinom AI odgovoru jeste da predstavlja genetički determinisanu kaskadu
biohemijskih reakcija, koja dovodi do zaustavljanja rasta polenove cevi ako je polen
prepoznat kao sopstveni. Rast sopstvene polenove cevi se zaustavlja na spoju žiga i stubića
"tram" tučka, odnosno na 2/3 dužine stubića "pin" tučka. Ova reakcija je pod kontrolom S-
supergena, koji pored AI gena sadrži i gene koji određuju dužinu stubića tučka, visinu
antera i veličinu polenovog zrna, koji se zajedno vezano nasleđuju. Između alela AI gena
postoji dominantno-recesivan odnos, u kom su "pin" biljke recesivni homozigoti za AI gen,
ss, dok su "tram" biljke dominantni heterozigoti, Ss. Pošto se pri oplođenju "tram" polen
ponaša u skladu sa diploidnim genotipom roditeljske biljke, a ne sopstvenim haploidnim
genotipom, zaključeno je da kod heljde postoji sporofitna determinacija AI. Poslednjih
godina razvijeni su molekularni markeri za fino mapiranje S-lokusa heljde, a sekvenciran je
i transkriptom cveta heljde, što omogućuje detekciju novih S-sekvenci. Identifikovan je i
prvi kandidat gena S-lokusa heljde, gen S-ELF3, koji najverovatnije određuje "tram"
fenotip tučka. Cilj ovog istraživanja bio je ispitivanje molekularne osnove AI odgovora
heljde na dva nezavisna načina: metodom polimeraznog lančanog umnožavanja uz
upotrebu prajmera karakterističnih za sekvencu blisku S-lokusu, odnosno prajmera
specifičnih za AI gene Prunus i Brassica (čija fiziologija AI odgovora podseća na AI
odgovor dva morfa heljde) i metodom 2D-PAGE za razdvajanja ukupnih proteina polena,
neoprašenih i inkompatibilno/kompatibilno oprašenih tučkova oba morfa. Na ovaj način
dobijeno je 14 novih genomskih sekvenci bliskih S-lokusu heljde, a analiza njihovih
otvorenih okvira čitanja ukazala je na postojanje homologije sa proteinima uključenim u
signalnu transdukciju i regulaciju transkripcije. Pokazano je da u genomu heljde nema gena
homolognih S-RNaznim i SFB genima (AI geni roda Prunus), kao ni SRK, SLG i SP11
genima (AI geni roda Brassica), ali da postoji homolog MLPKf2 genu Brassica. Ovaj gen
kodira M-lokus vezanu protein kinazu izoforme 2, koja je pozitivni medijator signala u AI
ogovoru kupusa. Izvedena aminokiselinska sekvenca MLPKFe heljde pokazala je 80%
homologije sa MLPKf2 Brassica. Grupa miristilovanih protein kinaza C (npr. za membranu
vezne Ca2+/kalmodulin-zavisne protein kinaze) nema ulogu u AI odgovoru heljde, što je
utvrđeno primenom inhibitora ovih kinaza. 2D-PAGE analizom detektovano je preko 200
reproducibilnih proteina po uzorku i uočena je visoka sličnost sastava proteoma među
uzorcima, što ukazuje da najveći relativni udeo u proteomima svih uzoraka imaju proteini
opšte uloge u polen-tučak interakciji. Detektovano je preko 100 proteina različitih među
uzorcima, od kojih je 21 morf-specifično ili specifično za AI odgovor. Istraživanje bi
trebalo nastaviti kroz identifikaciju/sekvenciranje detektovanih specifičnih proteina i kroz
dalje sekvenciranje regiona bliskog S-lokusu, pomoću prajmera dizajniranih iz novih 14 S-
sekvenci, kao i prajmera dizajniranih na osnovu dobijenih proteinskih sekvenci. | Buckwheat (fam. Polygonaceae) is heteromorphic species with two flower morphs: "pin"
(long style, short anthers, and smaller pollen grains) and "thrum" (short style, long anthers,
and larger pollen grains). Fertilization is allowed only between flowers of different
morphology, while it is prevented between flowers of the same morphology, trough self-
incompatibility (SI) system, which prevents inbreeding. Buckwheat is grown worldwide
due to its good nutritive characteristics, but the main issue in its breeding is low and
uncertain yield and high resistance of buckwheat to introgression of new characteristics
from related wild species, all of which are direct cosequences of buckwheat's SI. These
effects of SI systems may present obstacles in breeding of agriculturally important species
(i.e. almond, buckwheat, etc.), hence the interest in SI system studies. The aim of SI
systems studies is to provide self-fertile and hybrid lines of desired characteristics, as well
as to provide new markers for marker-assisted selections, all of which would improve the
yield, while lowering the costs of breeding. Current understanding of buckwheat SI system
considers SI response as genetically determined cascade of biochemical reactions that lead
to pollen tube arrest when pollen has been recognized as self. The site of self-pollen tube
arrest is different in two morphs: in "thrum" morph it occurs at the stigma-style junction,
while in "pin" it takes place at 2/3 of style’s length. This reaction is under the control of the
S-supergene which comprises SI genes and closely linked genes that determine style’s
length, anthers’ height and pollen grain size, inherited as a single unit. Between SI alleles
there is a dominant-recessive relationship, in which “pin” plants act as recessive
homozygotes, ss, while “thrum” plants act as dominant heterozygotes, Ss. Since “thrum”
pollen behaves in accordance with a diploid genotype of its parental plant and not its own
haploid genotype, it was concluded that SI in buckwheat is sporophytically determined.
Recently, molecular markers for fine mapping of S-locus were developed, and flower
transcriptome was sequenced, which enables detection of new S-sequences. First
buckwheat S-gene was identified as S-ELF3 gene, which is the most probable candidate for
the "thrum" pistil phenotype determinant. The aim of this research was to analyze
molecular basis of buckwheat’s SI response in two independent ways: trough PCR by using
primers specific for S-linked region and primers specific for SI genes of Prunus and
Brassica (species in which SI response resembles SI response observed in two buckwheat
morphs) as well as trough 2D-PAGE separation of total protein extracts isolated from
pollen, unpollinated, self- and cross-pollinated pistils of both morphs. In this way 14 new
genomic sequences, closely linked to S-locus, were obtained and open reading frame
analysis suggested their homology to proteins involved in signal transduction and
transcription regulation. No genes homologous to S-RNase and SFB genes (SI genes in
Prunus) or to SRK, SLG and SP11 genes (SI genes in Brassica) were found in buckwheat
genome, but a gene homologous to Brassica’s MLPKf2 was found. It codes for M-locus
protein kinase isoform 2 involved in Brassica’s SI response as a positive mediator of SI
signal. MLPKFe showed 80% homology with Brassica’s MLPKf2 at deduced amino acid
sequence level. Test involving inhibitor of myristoylated protein kinases C showed these
kinases (i.e. Ca2+/calmoduline-dependant protein kinase) have no role in buckwheat’s SI
response. 2D-PAGE proteome analysis revealed over 200 reproducible proteins in each
sample and high overall similarity of proteome composition between samples, suggesting
most proteins are shared in all samples and have general roles in pollen-pistil interactions.
Over 100 proteins were found to be different between samples. Among them 21 were found
to be specific to the particular morph or its SI response. This research should be continued
trough identification/sequencing of these specific proteins and also trough further
sequencing of S-locus linked region by using primers designed form 14 new S-sequences as
well as primers designed according to obtained specific protein sequences. | true |
Uticaj obrade hrane i interakcija komponenti matriksa hrane na strukturu i funkciju alergena hrane | Impact of food processing and interactions with other food components on the structure and function of food allergens | β-laktoglobulin (BLG) je glavni protein surutke mleka preživara, ali je prisutan i u
mleku mnogih drugih vrsta sisara. Poznato je da BLG poseduje niz funkcionalnih osobina
(npr. geliranje, stvaranje pene, emulzifikacija) važnih za industriju hrane kao i veliku
nutritivnu vrednost zbog visokog sadržaja esencijalnih aminokiselina. Iz tog razloga se
često koristi kao aditiv u prehrambenoj industriji. Međutim, BLG je jedan od glavnih
alergena mleka; 45% pacijenata alergičnih na mleko reaguje na BLG. Stoga se sve više
nameće potreba za razvijanje metoda za modifikaciju BLG-a u cilju smanjenja alergenosti i
poboljšanja funkcionalnih osobina. Takođe, sva istraživanja u oblasti hrane ne smeju
isključiti prisustvo drugih komponenti u matriksu hrane.
Predmet rada ove teze je bio ispitivanje uticaja interakcija komponenti matriksa
hrane (protein-polifenolna jedinjenja) i odabranih tehnika za obradu hrane (ultrazvuk i
enzimsko umrežavanje) na strukturu i funkciju BLG-a.
U okviru rada su okarakterisane nekovalentne interakcije između polifenolnih
jedinjenja iz ekstrakata kafe, kakaoa, zelenog i crnog čaja i BLG-a. Pokazano je da se usled
formiranja nekovalentnih interakcija između polifenolnih jedinjenja i BLG-a smanjuje
digestibilnost proteina i da dolazi do maskiranja ukupnog antioksidativnog kapaciteta.
Tretiranjem BLG-a ultrazvukom visokog intenziteta generisani su stabilni
modifikati sa različitim promenama u sekundarnoj i tercijarnoj strukturi. Kontrolisanje
temperature u toku tretmana je imalo odlučujući efekat na vrstu modifikacije. Izrazite
promene u tercijarnoj strukturi BLG-a su dovele do formiranja polimera, gubitka afiniteta
za vezivanje retinola i brže degradacije pepsinom. Promene u sekundarnoj strukturi nisu
uslovile promene u digestibilnosti i vezivanju retinola. Afinitet sonifikovanih formi za
vezivanje IgE antitela i indukciju IgE posredovane aktivacije bazofila i mastocita nije bio
narušen.
BLG je kompaktan protein koji je slabo podložan enzimski katalizovanoj reakciji
umrežavanja. Pospešenje reakcije umrežavanja BLG-a lakazom iz Trametes versicolor
(fenol oksidaza) je postignuto uz prisustvo kafeinske kiseline kao pomoćne supstance i
tretmana BLG-a ultrazvukom visokog intenziteta pre enzimske reakcije. Umreženi BLG
(CL-BLG) je bio molekulske mase približno 50-60 MDa i fibrilarne strukture. CL-BLG je
izazvao alergijski imunski odgovor kod miševa na efikasniji način od nativnog proteina.
Smatramo da je to posledica preuzimanja umreženog BLG-a preko Pejerovih ploča crevnog
trakta za razliku od nativnog proteina koji se transportuje kroz crevni epitel. Takođe, CL-
BLG je bio preuzet i obrađen od strane dendritičnih ćelija na način koji je rezultovao
većom ekspanzijom BLG specifičnih T-ćelijskih klonova i produkcijom IL-13, Th2
citokina.
Rezultati proistekli iz ove teze ukazuju da je potrebno dobro definisati uslove pri
obradi hrane i ispitati uticaj svakog novog tretmana na alergenost proteina iz hrane. U
našem slučaju, prisustvo polifenolnih jedinjenja i tretman ultrazvukom mogu imati uticaja
na alergenost BLG-a usled promena u digestibilnosti. Međutim, najizraženije efekte na
alergeni potencijal BLG-a je pokazalo enzimsko umrežavanje proteina kojim se drastično
menjaju fizičko-hemijska svojstava proteina. | β-lactoglobulin (BLG) is the major whey protein of ruminant species, but is also
present in the milk of many other species. It is well known that BLG has many benefitial
functional properties such as gelling, foaming and emulsifying properties. Although, BLG
is considered to be a valuable protein in view of nutritional science, and is frequently used
as an additive in food industry, BLG is known to be a potent allergen responsible for milk
allergy; around 45% of patients allergic to milk are sensitive to BLG. Hence, it is strongly
desirable to develop new methods that would decrease the allergenicity and enhance the
functional properties of BLG. Also, all the research in the field of food science can not
exclude the presence of other components in the food matrix.
The subject of this thesis was to examine the impact of the interactions formed
between food matrix components (protein-polyphenol compounds) and selected food
processing techniques (ultrasound and enzyme cross-linking) on the structure and function
of BLG.
In the paper, non-covalent interactions between polyphenolic compounds from
extracts of coffee, cocoa, green and black tea and BLG are characterized. It is shown that
due to the formation of non-covalent interactions between polyphenolic compounds and
BLG, protein digestibility is reduced and total antioxidant capacity is masked.
Treatment of BLG by high intensity ultrasound generated stable modified protein
forms with marked changes in the secondary and tertiary structure. Temperature control
during treatment had a determining effect on the type of modification. Striking changes in
the tertiary structure of BLG led to the formation of polymers, the loss of affinity for retinol
binding and degradation by pepsin. Alterations in the secondary structure did not cause
changes in digestability and retinol binding. Affinity of sonicated forms for IgE binding and
induction of IgE-mediated activation of basophils and mast cells was not compromised.
BLG is a compact protein that is poorly susceptible to enzyme-catalyzed
crosslinking reactions. Efficient BLG cross-linking by laccase from Trametes versicolor
(phenol oxidase) was achieved by using caffeic acid as an auxiliary compound and treating
BLG with high intensity ultrasound prior to the enzymatic reaction. Cross-linked BLG (CL-
BLG) had molecular weight of approximately 50-60 MDs and fibrillar structure. CL-BLG
induced allergic immune response in mice in a more efficient way than BLG. We assume
that this is due to the transport of CL-BLG to Peyer’s patches in contrast to the native
protein which is transported through the intestinal epithelium. Also, CL-BLG was taken up
and processed by dendritic cells in a manner that resulted in higher expansion of BLG-
specific T-cell clones and production of IL-13, a Th2 cytokine.
These findings suggest that it is necessary to define well food processing conditions
and to examine the impact of each new treatment on the allergenicity of food proteins. In
our case, the presence of phenolic compounds and ultrasound treatment can influence the
allergenicity of BLG due to changes in digestability. However, the most pronounced effect
was shown by enzymatic cross-linking that dramatically alters the physical and chemical
properties of BLG. | true |
Primena nelinearnih metoda u analizi promena EEG teta ritma pri formiranju kratkoročne auditivne memorije kod ljudi | Aplication of nonlinear methods in analysis of EEG theta rhythm changes during auditory short-term memory formation in human. | Kratkoročna auditivna memorija (KAM) predstavlja kratkotrajno zadržavanje auditivno
primljenih informacija u memoriji. Kratkoročna memorija, iako zaseban sistem, nije u potpunosti
nezavisna od dugoročne memorija. Memorija se ne nalazi na jednom području u mozgu, već
obuhvata široko - distribuiranu mrežu koja se sastoji, uglavnom, od mreža koje formiraju
prefrontalni, temporalni i parijetalni regioni i njihove interakcije sa subkortikalnim regijama
limbičkog sistema. Prethodna istraživanja promena EEG-a tokom formiranja KAM naglašavaju
značaj aktivnosti teta ritma. Postoje brojna istraživanja EEG pokazatelja (korelata) formiranja
KAM kod ljudi. Međutim, skoro da nema studija koje su primenjivale nelinearne metode za
kvantifikaciju promena kompleksnosti EEG signala u procesu formiranja KAM.
U ovoj studiji primenjene su nelinearne metode – fraktalna dimenzija (FD), fraktalna
dimenzija u okviru pokretnog prozora (prozorska fraktalna dimenzija – pFD) i entropija uzorka
(SampEn) – za kvantifikaciju promena EEG signala tokom procesa formiranja KAM. Analiziran
je period zadržavanja stimulusa u kratkoročnoj memoriji (eng. „retention period“– retencija) u
klasičnoj Sternbergovoj paradigmi za tri nivoa memorijskog opterećenja (MO) – tri, pet i sedam
stimulusa. Rezultati primene nelinearnih metoda su upoređeni sa linearnim metodama –
spektralna snaga, koherenca i analiza izvora EEG signala (analiza dipola).
Primenom nelinearnih metoda utvrđeno je da pri formiranju KAM za stimuluse sa
značenjem (reči) dolazi do promene kompleksnosti EEG signala u posteriornim regionima
korteksa a anteriornim tokom formiranja KAM za stimuluse bez značenja (nereči). Primena FD i
SampEn omogućila je detekciju promena stepena MO. Po prvi put je pokazano da sa povećanjem
MO dolazi do linearnog povećanja ili smanjenja vrednosti FD i SampEn EEG signala. pFD
analizom utvrđeno je da tokom formiranja KAM za reči dolazi do maksimuma kompleksnosti
EEG signala u intervalu od 600-800 ms perioda retencije. Pri formiranju KAM za nereči
maksimum kompleksnosti se javlja ranije u odnosu na reči, u intervalu od 200-600ms sa
cikličnim ponavljanjem porasta i smanjenja kompleksnosti do kraja perioda retencije.
Primenom spektralnih mera pokazana je aktivacija središnje frontalne regije (Fz) u teta
ritmu. Sa povećanjem MO vrednost spektralne snage teta ritma je rasla. Sličan trend povećanja
spektralne snage teta ritma u Fz regiji dobijen je i pri formiranju KAM za reči i za nereči. U
drugim ritmovima (alfa i beta) dobijene su regionalne razlike pri formiranju KAM za reči i nereči
ali bez mogućnosti gradacije stepena MO. Analiza generatora EEG teta ritma (analiza dipola)
pokazala je aktivaciju središnje frontalne regije Fz. Gradacija stepena MO nije dobijena za sve
nivoe.
Dobijeni podaci dali su dodatna objašnjenja o promenama elektrofizioloških (EEG)
korelata formiranja kratkoročne auditivne memorije. Poseban doprinos ove studije je u detekciji
stepena memorijskog opterećenja primenom nelinearnih metoda. Nova saznanja dobijena ovom
studijom omogućiće dalju primenu nelinearnih metoda u istraživanjima kratkoročne memorije
kod neurodegenerativnih oboljenja i razvojnih poremećaja. | Short-term auditory memory (STAM) is a short-term retention of auditory received
information in memory. Short-term memory, although a separate system, is not entirely
independent of long-term memory. Memory is not located in one area of the brain, but also
includes a wide - distributed network consisting mainly of networks that form the frontal,
temporal and parietal regions and their interaction with subcortical regions of the limbic system.
Previous studies of changes in the EEG during the formation of STAM emphasize the
importance of theta rhytm activities. There are numerous studies of EEG correlates of STAM
forming in humans. However, there are almost no studies that have applied non-linear method
for quantification of changes in the complexity of the EEG signal during formation of STAM.
In this study, we applied the non-linear methods – fractal dimension (FD), fractal
dimension with moving window (wFD), and sample entropy (SampEn) – to quantify the changes
in the EEG signal complexity during formation of STAM. We analyzed the period of retention in
the classic Sternberg’s paradigm for three levels of memory load (ML) - three, five, and seven
stimuli. Results of nonlinear methods were compared with linear methods - spectral power,
coherence and analysis of the sources of EEG signals (analysis of the dipoles).
During formation of the STAM for stimuli with meaning (words) there is a change in the
complexity of EEG signal in the posterior regions of the cortex, and the anterior during the
formation of the STAM for the stimuli without meaning (nonwords). Application of FD and
SampEn has enabled the detection of changes in the level of ML. For the first time it is shown
that with increasing ML comes a linear increase (decrease), respectively, in the value of the FD
and SampEn. Window fractal analysis showed that during the formation of STAM for words,
there is a maximum of complexity in the range of 600-800 ms during retention period. In
forming STAM for nonwords, maximum of complexity occurs earlier than for words, in the
range of 200-600ms with cyclic repetition of the complexity increase and decrease towards the
end of the retention period.
Spectral measures detected activation of the central frontal region (Fz) in theta rhythm.
With the increasing ML theta spectral power increased for both words and nonwords. Alpha and
beta rhythm's spectral power had regional differences during formation of STAM for words and
nonwords but without the possibility of detecting ML influence. Analysis of EEG theta rhythm
generator (dipole analysis) showed activation of the central frontal region Fz. Detection of ML
was not obtained for all levels.
The obtained results provided additional understanding of the changes exhibited in
electrophysiological (EEG) correlates of the short-term auditory memory formation, specificaly
in detecting the memory load degree by using non-linear methods. The knowledge geneated
through this study will enable further application of nonlinear methods in studies of short-term
memory in neurodegenerative diseases and developmental disorders. | true |
Molekularno-citogenetička karakterizacija hromozomskih prekida u limfocitima periferne krvi pacijenata obolelih od Fankonijeve anemije u različitim fazama bolesti | Molecular-cytogenetic characterization of chromosomal breakages in peripheral blood lymphocytes of Fanconi anemia patients in different stages of the disease | Česta fragilna mesta (CFS) su hromozomski regioni skloni lezijama, suženjima i
prekidima u uslovima replikativnog stresa in vitro. Najčešće se mogu naći kod
sindroma hromozomskih nestabilnosti kao što je Fankonijeva anemija (FA), retka
nasledna bolest koju karakteriše sklonost ka razvoju maligniteta i progresivna
insuficijencija koštane srži, povećan broj hromozomskih prekida i radijala i
telomerna
fragilnost. Cilj
ovog istraživanja je molekularno-citogenetička
karakterizacija hromozomskih prekida u limfocitima periferne krvi, ispitivanje
zajedničkog mesta javljanja - kolokalizacije prekida sa CFS i analiza mutacija u
FANCD2 genu kod FA-D2 pacijenata iz Srbije kako bi se utvrdilo da li CFS zavise
od tipa mutacija u FANCD2 genu. Navedeni parametri su analizirani kod pacijenata
u različitim fazama bolesti kako bi se identifikovali prognostički parametri bolesti.
Rezultati su pokazali da FA-D2 hromozomski prekidi kolokalizuju sa CFS,
karakteristični su za komplementacionu grupu i distribucija im se menja sa
progresijom bolesti. Učestalost radijala i telomernih fuzija značajno je viša kod
pacijenata sa teškom insuficijencijom koštane srži i može predstavljati prognostički
parametar bolesti. Radijali se formiraju između nehomologih hromozoma,
uključujući i polne i autozome. Otkriveno je novo fragilno mesto u regionu 1q42.2.
Sangerovim sekvenciranjem je otkriveno 10 varijanti FANCD2 gena, uglavnom
dubokih intronskih, od kojih su tri novootkrivene (c.2396 C>A, c.206-246delG i
c.2715+573 C>T). In-silico analizom identifikovane su tri patogene varijante kod
više pacijenata koji nisu u srodstvu, koje mogu biti specifične za populaciju u Srbiji.
Poređenje rezultata molekularno-citogenetičke i analize mutacija pokazalo je da ne
postoji povezanost CFS i varijanti FANCD2 gena. | Common fragile sites (CFSs) are chromosomal regions prone to gaps,
constrictions and breaks under conditions of replication stress in vitro. They are
mostly found in chromosomal instability syndromes such as Fanconi anemia (FA).
FA is rare inherited disease characterized by cancer predisposition, progressive bone
marrow failure, increased level of chromosomal breakages, radial figures and
marked telomere fragility. The aim of this study was to perform molecular-
cytogenetic characterization of chromosomal breakages and co-localization with
CFSs in peripheral blood lymphocytes, as well as to analyze FANCD2 gene
mutations in FA-D2 patients from Serbia. These parameters were monitored in
patients at different stages of the disease in order to identify the prognostic
parameters of the disease.
The results of this study showed that FA-D2 chromosomal breakages co-localize
with CFS, they are specific for the complementation group and their distribution
pattern changes with the disease progression. The frequency of radials and telomere
fusions is significantly higher in patients with severe bone marrow failure and could
be of predictive value. Radials are composed of non-homologous chromosomes,
including sex chromosomes as well as autosomes. One novel fragile site is found in
region 1q42.2. Ten variants of FANCD2 gene are detected by Sanger sequencing,
mostly in deep intronic regions, among which three are novel (c.2396 C>A, c.206-
246delG i c.2715+573 C>T). In-silico analysis revealed three pathogenic variants in
several unrelated patients, indicating their possible association to Serbian
population. Matching the results of molecular-cytogenetic characterization and
mutation analysis showed that there is no relationship between CFS and FANCD2
variants. | true |
Diverzitet mekušaca Dunava (1260-863,5 rkm) i taksonomska analiza rodova Planorbarius, Radix, Physella, i Ferrissia (Pulmonata:Basommatophora) | Diversity of Molluscs in the Danube river (1269-863.5 rkm) and taxonomic analysis of the genera Planorbarius, Radix, Physella and Ferrissia (Pulmonata: Basommatophora) | Analiza slatkovodnih mekušaca razmatrana je kroz tri zone glavnog toka
Dunava (1260 - 863,5 rkm) kao i plavnoj zoni, na levoj obali reke, od 1082 do 1085
rkm. Uzorci su prikupljeni sezonski (april, jun, septembar i novembar), u tri
ponavljanja, uključujući uzorke za analizu fizičkih i hemijskih parametara kvaliteta
vode i sedimenta. Ovim istraţivanjima dat je detaljan pregled sastava i distribucije
malakofaune istraţivanog dela rečnog toka pri različitim uticajima grupe antropogenih
pritisaka. Primenom molekularnih metoda razmatrani su filogenetski odnosi autohtonih
i alohtonih vrsta iz reda Basommatophora.
Analizom zajednica akvatičnih makroinvertebrata, uočeno je da plavnu zonu
reke karakteriše najveća procentualna zastupljenost insekatskih grupa (61,69%) kao i
fitofilnih vrsta slatkovodnih mekušaca (15%). Lokalitete rečne, prelazne i jezerske zone
karakteriše velika procentualna zastupljenost pre svega Oligochaeta (37,89%, 63,83%,
83,99%), zatim Bivalvia (38,21%, 24,35%, 16,72%) i Crustacea (15,82%, 31,11%,
13,73%), što predstavlja karakteristične odnose u zajednicama makroinvertebrata reka
potamon tipa.
Analizom zajednica slatkovodnih mekušaca zabeleţeno je 32 taksona iz 28
rodova i 13 familija u okviru dve klase - Bivalvia i Gastropoda.
Klasa Bivalvia zastupljena je sa 12 taksona iz četiri familije u okviru redova
Veneroida i Unionida. Najveći procenat, 92% od ukupnog broja zabeleţenih taksona iz
klase Bivalvia, zabeleţen je na lokalitetima prelazne zone; nešto manji procenat od 75%
zabeleţen je u jezerskoj zoni; dok je najmanja zastupljenost zabeleţenih taskona uočena
na lokalitetima rečne zone (41%).
Klasa Gastropoda zastupljena je sa 20 taksona, iz dve potklase, Prosobranchia
(Orthogastropoda) i Pulmonata. Posmatrajući raznovrsnost taksona iz klase Gastropoda
na istraţivanim lokalitetima, najmanji broj taksona uočen je u gornjem delu toka
Dunava (20%), dok je najveća raznovrsnost zabeleţena u plavnoj zoni gde je naĎeno
55% od ukupnog broja zabeleţenih taksona. U srednjem delu toka Dunava, prelaznoj
iv
zoni, naĎeno je 40% taksona, dok je u jezerskoj zoni zabeleţeno prisustvo 45% od
ukupnog broja zabeleţenih taksona.
U zabeleţenoj malakofauni, šest vrsta su introdukovane u vode Dunava. Ponto-
kaspijski relikti su Dreissena polymorpha i Dreissena rostriformis bugensis, zatim
istočno azijske školjke Corbicula fluminea i Sinanodonta woodiana i Severno Američki
puţevi Physella acuta i Ferrissia sp. Nova vrsta za faunu mekušaca Srbije zabeleţena u
ovim istraţivanjima je Dreissena rostriformis bugensis u klasi Bivalvia i rod Ferrissia
sp. u klasi Gastropoda.
Rezultati CCA ukazuju na izdvajanje taksona iz familije Dreissenidae na
lokalitetima rečne zone, čije je prisustvo uslovljeno uslovima staništa i to ukupnom
tvrdoćom vode, koncentracijom Mg i pH vrednošću. Druga grupa izdvojenih taksona
pokazuju pozitivnu korelisanost sa prisustvom nitrita u vodi (Unio crassus, Bithynia
tentaculata i Viviparus viviparus).
Dobijeni rezultati CCA zavisnosti varijabilnosti malakofaune i prisustva
organskih mikropolutanata, teških i toksičnih metala u sedimentu, ukazuju na izdvajanje
taksona iz familije Dreissenidae, usled pozitivne korelacije sa prisustvom Cd, Hg i PAH
u sedimentu. Vrsta Teodoxus fluviatilis je u korelaciji sa prisustvom PAH, dok je
Lithoglyphus naticoides bio korelisan sa prisustvom Ni u sedimentu.
Analiza filogenetskih odnosa odabranih vrsta iz reda Basommatophora sa
prostora Evrope, Azije i SAD, uraĎena je na osnovu 54 sekvenci (16S rDNK i COI).
Ispitivane sekvence za 16S rDNK bile su duţine 359 bp odnosno 315 bp za COI. Kao
spoljna grupa korišćena je – Siphonaria sp., marinska vrsta iz reda Basommatophora.
Dobijeni filogrami Bayesian analizom i ML metodom bili su slične topologije, i
jasno su se izdvojile očekivane haplogrupe, prema taksonomskoj podeli u tri familije:
Planorbidae, Lymnaeidae i Physidae. Genetičke p-distance za 16S rDNK gen, izmeĎu
haplogrupa Physidae i Planorbidae kretale su se od 24% do 25%, izmeĎu haplogrupa
Physidae i Lymnaeidae 30%, dok je izmeĎu haplogrupa Planorbidae i Lymnaeidae
genetička p-distanca iznosila 23%.
Rezultati analize povezanosti haplotipova alohtone vrste Physella acuta,
prikupljene tokom naših istraţivanja sa haplotipovima iz SAD (preuzeto iz banke gena -
kao nativna populacija) pokazuju razlike od 4 do 25 baznih parova. Prikupljeni i
analizirani uzorci ove vrste sa područja Srbije pokazuju značajnu genetičku varijabilnost
v
od 1% do 6,2% razlika za analizirani 16S rDNK gen. Znatno manja intraspecijska
varijabilnost za isti gen utvrĎena je poreĎenjem populacija iz Srbije (rezultati analize
naših uzoraka) sa podacima iz Danske (preuzeto iz baze gena) za autohtonu vrstu
Planorbarius corneus (0,2% do 0,6%). Ovi nalazi ukazuju da velika intraspecijska
genetička varijabilnost povećava ekološku plastičnost kod invazivnih vrsta. | Fresh water molluscs analysis was performed through three zones of The
Danube main flow (1260-863.5 rkm) as well as flooding zone, on the left river bank
from 1080 to 1085 rkm. Samples were collected seasonally (April, June, September and
November), in three repetitions, along with samples for physical and chemical analysis
of water and sediment quality. This research presents detailed summary of composition
and distribution of malacofauna in analysed part of the river flow under different
influence of anthropogenic pressure. Molecular methods were used for establishing
phylogenetic relationships among native and alien species from Basommatophora order.
Analysis of aquatic macroinvertebrate communities showed that river flooding
zone is characterized by highest percentage of occurrence for insect groups (61.69%) as
well as phytophilic species of freshwater molluscs (15%). Localities from riverine,
transitional and lacustrine zone are characterized by large percentage of occurrence of
Oligochaeta (37.89%, 63.83%, 83.99%), then Bivalvia (38.21%, 24.35%, 16.72%) and
Crustacea (15.82%, 31.11%, 13.73%), which is characteristic for macroinvertebrate
communities of potamon river type.
Analysis of aquatic molluscs communities showed that 32 taxa from 28 genera
and 13 families within two classes Bivalvia and Gastropoda were recorded.
Class Bivalvia is represented with 12 taxa from four families within orders
Veneroida and Unionida. Highest percentage, 92% from total number of all recorded
taxa within class Bivalvia, was found in localities in transitional zone, about 75% in
lacustrine zone; lowest percentage of identified taxa was recorded at riverine zone
localities (41%).
Classis Gastropoda is represented with 20 taxa from two subclasses,
Prosobranchia (Orthogastropoda) and Pulmonata. Taxonomic diversity of class
Gastropoda at investigated localities was lowest in riverine zone of The Danube (20%
of all recorded taxa) and highest diversity was recorded in flooding zone where 55% of
vii
all recorded taxa were present. In the middle part of The Danube flow (transitional
zone) 40% of taxa were found and in lacustrine zone 45% of total recorded taxa were
present.
Six species of recorded malacofauna were introduced in the Danube water.
Ponto Caspian relicts are Dreissena polymorpha and Dreissena rostriformis bugensis,
as well eastern Asian shells Corbicula fluminea and Sinanodonta woodiana, and North
American snails Physella acuta and Ferrissia sp. New species for Serbian mollusc
fauna recorded during this research are: Dreissena rostriformis bugensis in class
Bivalvia and genus Ferrissia sp., in class Gastropoda.
Results of CCA analysis showed differentiation of taxa from the family
Dreissenidae at riverine zone localities, whose presence is conditioned with habitat
characteristics such as total water hardness, Mg concentration and pH value. Second
segregated group of taxa showed positive correlation with nitrite presence in water
(Unio crassus, Bithynia tentaculata and Viviparus viviparus).
The CCA results of correlation between malacofauna variability and the
presence of organic micropollutants, heavy and toxic metals in sediments, also showed
the differentiation of taxa of the family Dreissenidae that are positively correlated with
Cd, Hg and PAH presence in sediment. Species Teodoxus fluviatilis was correlated with
the presence of PAH, while Lithoglyphus naticoides showed a positive correlation with
the presence of Ni in sediments.
Analyses of phylogenetic relationships of selected species from order
Basommatophora from Europe region, Asia an USA were performed using 54
sequences (16s rDNA and COI). Analysed sequences of 16s rDNA were 359 bp long
and COI sequences were 315 bp long. Siphonaria sp, marine species from order
Basommatophora was used as outgroup. Phylogenetic trees obtained both with Bayesian
analysis and ML method had similar topology, with three clearly differentiated
haplogroups, according to taxonomic division in three families: Planorbidae,
Lymnaeidae and Physidae. Genetic p-distances for 16S rDNA between haplogroups
Physidae and Planorbidae ranged from 24% to 25%, while p-distance between
haplogroups Physidae and Lymnaeidae was 30% and between haplogroups Planorbidae
and Lymnaeidae was 23%.
viii
Haplotype analysis of alien species Physella acuta collected during this research
showed 4 to 25 base pairs difference compared with haplotypes from USA (sequences
of native population from USA were taken from a gene bank). Collected and analysed
samples of this species from Serbian area showed significant genetic variability 1% to
6.2% difference for analysed 16S rDNA gene. Native species Planorbarius corneus
showed significantly lower intraspecies diversity (0.2% to 0.6%) for the same gene
when Serbian and Danish population were compared (sequences from Denmark were
taken from a gene bank). These results suggest that high intraspecies genetic variability
increases ecologic plasticity in invasive species. | true |
Struktura globalnih finansijskih mreža i transfer rizika na iskustvu bankarskih sistema zemalja zapadnog Balkana | The structure of global financial networks and risk transfer in the experience of the banking system of Western Balkan countries | U ovoj disertaciji je analizirana struktura globalne finansijske
mreže u periodu poslednje finansijske krize, od 2007-2013. godine. Osnovni
izvor podataka koji je u istraživanju korišćen jeste Konsolidovana bankarska
statistika Banke za međunarodna poravnanja (BIS). Posebno je analizirana
struktura jednog užeg segmenta globalne finansijske mreže, kojem pripadaju
bankarski sistemi zemalja zapadnog Balkana. U tu svrhu su korišteni osnovni
pokazatelji strukture finansijske mreže. To su deskriptivne statistike kao
rang čvorišta, prosečan najkraći put, koeficijent gomilanja čvorišta,
veličina mreže, red mreže, indeks povezanosti, Herfindal-Hiršman indeks
kao pokazatelj koncentracije na strani izvora sredstava, kao i grafičko
predstavljanje finansijske mreže. Osnovna karakteristika ove finansijske
mreže je visok nivo centralizacije i koncentracija na mali broj čvorišta.
Navedena čvorišta reprezentuju bankarske sisteme, tzv. regionalne zajedničke
poverioce (bankarski sistemi Austrije, Italije i Grčke). Regionalni
zajednički poverioci su svoje inostrane plasmane dominanto usmerili u region
centralne, istočne i jugoistočne Evrope. Zaključak koji proizlazi iz strukture
analizirane finansijske mreže je da postoji visok potencijal transfera
rizika ka regionu zapadnog Balkana. U slučaju nastanka finansijskog poremećaja
u nekoj od zemalja centra ili u nekoj od zemalja centralne, istočne i
jugoistočne
Evrope
struktura
finansijske
mreže
bi
imala
efekat
transmitera rizika. Dalje, u disertaciji je analizirana uloga i mesto velikih
bankarskih grupacija aktivnih u regionu zapadnog Balkana u finansijskoj
mreži. U posmatranom regionu su uglavnom prisutne bankarske grupacije koje su
svoje poslovanje usmerile isključivo na region centralne, istočne i jugoistočne
Evrope. Posledica je da postoji visok potencijal transfera rizika kroz
navedene bankarske grupacije ka zemljama zapadnog Balkana i da zbog niske
geografske diversifikacije svog poslovanja navedene bankarske grupacije ne mogu
ostvariti ulogu apsorbera rizika. U disertaciji je urađena i dinamička
analiza razvoja događaja u slučaju difolta neke od 42 evropske zemlje koje su
uzete u uzorak zemalja. Simulacija je sprovedena koristeći metodološki okvir
kao u Espinosa-Vega i Sole (2010), ali na primeru druge finansijske mreže.
Simulacija je pokazala da postoji visok rizik za zemlje zapadnog Balkana u
slučaju difolta nekog od bankarskih sistema regionalnih poverilaca (Austrija,
Italija i Grčka) ili u slučaju difolta nekog od bankarskih sistema globalnih
poverilaca. Takođe, postoji visok potencijal za transfer finansijske zaraza ka
regionu zapadnog Balkana iz nekih evropskih zemalja u razvoju. | The structure of the global financial network in the period of the last financial
crisis, from 2007 to 2013, was analyzed in this dissertation. The main data source used
in the research is the Consolidated banking statistics from the Bank for International
Settlements (BIS). The structure of the smaller part of the global financial network,
which the banking systems of the Western Balkan countries belong to, was especially
analyzed. The main indicators of financial network structure were used for this purpose.
These are descriptive statistics like node’s degree, average shortest path length, node’s
clustering coefficient, network’s size, network’s order, connectivity index, Herfindahl-
Hirschman index as the concentration indicator on the side of fund sources, as far as
graphical presentation of financial network. The main feature of this financial network
is the high centralization level and concentration on a small number of nodes. The
mentioned nodes represent the banking systems, so called regional common lenders (the
banking systems of Austria, Italy and Greece). The foreign claims of the regional
common lenders are dominantly directed to the region of Central, Eastern and
Southeastern Europe. According to the structure of the analyzed financial network it
can be concluded that there is a high potential of risk transfer to the region of the
Western Balkans. In the case that a financial disturbance appears in any central
country or any country in Central, Eastern and Southeastern Europe, the structure of
the financial network would have an effect as a risk transmitter. Further, the role and
place of the large banking groups operating in the region of the Western Balkans area
in the financial network was analyzed in the dissertation. The banking groups, which
operate in the considered region, have mostly directed their businesses towards the
region of Central, Eastern and Southeastern Europe. The consequence is that there is a
high potential of risk transfer through considered banking groups on the Western
Balkan countries and that they can’t realize the role of a risk absorber because of the
low level of geographical diversification of their businesses. The dynamical simulation
analysis was done in the dissertation for the case of default for some of the 42 European
countries chosen in the sample of countries. The simulation is conducted using the
methodological framework as used by Espinosa-Vega and Sole (2010), but on the
example of a different financial network. The simulation has shown there is a high risk
for the Western Balkan countries in the case of default of some of the banking systems
regional common lenders (Austria, Italy and Greece) or in the case of default of some of
the banking systems of global common lenders. In addition to this, there is a high
potential for financial contagion transfer to the region of the Western Balkans from any
other European developing countries. | true |
Uticaj varijanti u kodirajućim i nekodirajućim regionima gena uzročnika i gena modifikatora na fenotip pacijenata sa hiperfenilalaninemijom | Impact of variants in coding and noncoding regions of disease-causing and modifier genes on phenotype of patients with hyperphenylalaninemia | Hiperfenilalaninemija
(HPA)
predstavlja
najčešći
nasledni
poremećaj
metabolizma aminokiselina (učestalost 1:10 000) koji se karakteriše povišenim nivoom
fenilalanina u krvi. Višak fenilalanina ima toksičan efekat na razvoj mozga, i ukoliko se
pravovremeno ne uvede odgovarajuća terapija, dolazi do teške i ireverzibilne mentalne
retardacije pacijenta. U oko 98% slučajeva, HPA je uzrokovana mutacijama u genu za
fenilalanin hidroksilazu (PAH) i tada je poremećaj poznat kao fenilketonurija (PKU). U
1-2% slučajeva HPA je posledica deficijencije esencijalnog kofaktora enzima PAH,
tetrahidrobiopterina (BH4), koja nastaje usled mutacija u genima odgovornim za
biosintezu (gen za piruvoil tetrahidropterin sintazu – PTS i gen za GTP ciklohidrolazu –
GCH1) ili metabolizam BH4 (gen za dihidropteridin reduktazu – QDPR i gen za pterin-
4a-karbinolamin dehidratazu, PCBD). S obzirom na ozbiljne i ireverzibilne posledice
koje HPA izaziva, od presudne važnosti je precizno postavljanje dijagnoze i što ranija
primena odgovarajuće terapije. U Srbiji je molekularno genetička karakterizacija gena
PAH uvedena 2004. godine, međutim, neophodno je uspostaviti dodatne metode radi
sveobuhvatne identifikacije i funkcionalne karakterizacije varijanti identifikovanih u
različitim genima uzročnicima HPA. Ovakav pristup imao bi značajnu primenu za
diferencijalnu dijagnostiku pacijenata sa HPA u Srbiji i pravovremeno uvođenje
odgovarajuće terapije specifične za genotip pacijenta. U slučaju fenilketonurije, iako
mutirani genotip PAH predstavlja glavnu odrednicu PKU fenotipa, primećeno je da nije
uvek moguće predvideti fenotip samo na osnovu genotipa. Smatra se da genetički
faktori koji doprinose razvoju finalnog PKU fenotipa (posebno kognitivnog PKU
fenotipa) još uvek nisu dovoljno istraženi. Stoga bi identifikacija novih intra-genskih
(npr., novih elementa uključenih u regulaciju transktipcije) modulatora PKU fenotipa,
kao i novih gena modifikatora doprinela njegovom boljem razumevanju.
U prvom delu ovog istraživanja je izvršena sveobuhvatna molekularna
karakterizacija varijanti u genima uzročnicima kod 76 pacijenata sa HPA u Srbiji.
Primenom DNK sekvenciranja kompletnog kodirajućeg regiona gena PAH, egzon-
intron granica, promotorskog regiona, kao i primenom MLPA (multiplex ligation-
dependent probe amplification) metode, detektovano je 29 poznatih i 1 nova mutacija.
Po prvi put je kod pacijenta iz Srbije detektovana velika delecija (c.510-?_706+?del), i
to sa učestalošću od 1,33%. Osim toga, optimizovana je metoda za detekciju mutacija u
genu PTS (DNK sekvenciranje kompletnog kodirajućeg regiona), zaduženom za
biosintezu BH4, i tom prilikom je kod jednog pacijenta detektovana mutacija
p.Asp136Val, u homozigotnom stanju. Ovo je prvi slučaj BH4 deficijencije u Srbiji,
koji je identifikovan zahvaljujući primeni molekularne genetike i uvođenju metoda za
detekciju mutacija u genu PTS. Učestalost deficijencije PTS u analiziranoj grupi HPA
pacijenata iznosi 1,31%. Zahvaljujući kombinovanju različitih metoda, postignut je nivo
detekcije mutacija od čak 99,34% za HPA u Srbiji, a na osnovu spektra detektovanih
mutacija je konstruisan dijagnostički algoritam za Srbiju, koji omogućava bržu, lakšu i
pristupačniju detekciju mutacija u genima uzročnicima HPA. Dalje je, na osnovu
genotipa analiziranih PKU pacijenata, izvršena i predikcija korisnosti nove
suplementacione terapije sa BH4. Uočeno je da bi 32% pacijenata sa PKU iz Srbije
najverovatnije imalo koristi od primene terapije sa BH4, dok maksimalan procenat
pacijenata koji bi mogli imati koristi od ovog novog leka iznosi 79%.
U drugom delu ovog istraživanja je izvršena funkcionalna analiza novootkrivene
aminokiselinske zamene p.Gln226Lys PAH (c.676C>A). U in vitro hepatoma sistemu je
pokazano da količina mutiranog p.Gln226Lys PAH-FLAG detektovanih Western blot
metodom, iznosi samo 1,2% u odnosu na nemutirani PAH-FLAG. Dodavanje
sepiapterina, intraćelijskog prekursora BH4 koji ima protektivnu šaperonsku ulogu, nije
značajno povećalo količinu mutiranog proteina. Stoga je zaključeno da je p.Gln226Lys
patološka mutacija, koja pritom ne odgovara na suplementaciju sa BH4.
U trećem delu ovog istraživanja je izvršena identifikacija i funkcionalna
karakterizacija novih transkripcionih regulatora u nekodirajućim regionima gena PAH.
U funkcionalnoj CAT esej analizi je pokazano da deo promotora gena PAH koji sadrži
citozin na poziciji -170 (koji odgovara nemutiranoj sekvenci) funkcioniše kao novi cis-
regulatorni element koji dovodi do smanjenja nivoa transkripcije, i to za 50% manje u
odnosu na kontrolni plazmid sa bazalnom promotorskom aktivnošću. Dalje je u EMSA
(electrophoretic mobility shift assay) supershift esejima pokazano da ovaj regulatorni
element vezuje KLF1 transkripcioni faktor. Osim toga, izvršena je i funkcionalna
analiza VNTR regiona u 3' kraju gena PAH, pri čemu je pokazano da VNTR region,
nezavisno od tipa ponovaka, predstavlja novi regulatorni element, koji dovodi do
smanjenja intenziteta transkripcije, i dovodi do oko 60% smanjenja reporterske
aktivnosti u odnosu na kontrolni plazmid sa bazalnom promotorskom aktivnošću. U
EMSA supershift esejima je pokazano da tip ponovka koji je najčešći kod PKU
pacijenata u Srbiji, VNTR3, vezuje C/EBP alpha transkripcioni faktor. Dva nova
transkripciona regulatorna elementa, identifikovana u nekodirajućim regionima,
konkretno promotorskom i 3' regionu gena PAH, doprinose razumevanju fine regulacije
PAH ekspresije i potpunijem shvatanju kompleksnog fenotipa PKU.
U četvrtom delu ovog istraživanja je izvršena analiza varijanti u potencijalnim
genima modifikatorima kognitivnog PKU fenotipa. Ova analiza je bila sprovedena
korišćenjem dva različita metodološka pristupa, gde je prvi pristup podrazumevao
studiju odabranog gena-kandidata, dok je drugi pristup za cilj imao identifikaciju novog
gena modifikatora na nivou celog genoma. Prvo je urađena studija asocijacije u
literaturi opisane varijante m.10398G>A iz mitohondrijskog gena MT-ND3 sa
kognitivnim fenotipom 64 pacijenata sa PKU (evaluiranom u obliku vrednosti IQ), pri
čemu je pokazano da između nosilaca m.10398A i m.10398G nije pronađena statistički
značajna razlika u srednjoj vrednosti IQ. Zatim je u istraživanju koje je za cilj imalo
identifikaciju novih gena modifikatora kognitivnog PKU fenotipa, kompletna genomska
sekvenca 10 nelečenih PKU pacijenata (iz 6 porodica) sa normalnim kognitivnim
razvojem analizirana metodom sekvenciranja nove generacije. Tom prilikom su se,
nakon ekstenzivne bioinformatičke obrade, kao potencijalni geni modifikatori PKU
kognitivnog fenotipa izdvojili SHANK geni, s obzirom da su njihovi proteinski produkti
uključeni u sinaptičku transmisiju i da su kod čak 4 od 6 analiziranih porodica u nekom
od 3 SHANK gena (SHANK1, SHANK2 ili SHANK3) identifikovane benigne varijante
(ukupno 6 varijanti). Za SHANK proteine koji nose identifikovane varijante je in silico
analizom pokazano da mogu zadobiti nove karakteristike, te mogu imati protektivni
modifikatorski efekat na razvoj kognitivnih poremećaja pacijenata sa PKU. Njihovo
otkriće može imati ogroman značaj za razvoj nove strategije lečenja pacijenata sa PKU. | Hyperphenylalaninemia (HPA) is the most frequent inborn error of amino acid
metabolism (incidence 1:10 000) and it is characterized by an increased level of blood
phenylalanine. Excessive phenylalanine has a toxic effect on brain development, and if
left untreated, leads to a severe and irreversible mental retardation of a patient. In
approximately 98% of all cases, HPA is caused by mutations in phenylalanine
hydroxylase (PAH) gene and in such case the disorder is known as phenylketonuria
(PKU). In other 1-2% of the cases, HPA results from deficiency of an essential cofactor
of the PAH enzyme, tetrahydrobiopterin (BH4). BH4 deficiency can therefore result
from mutations in two genes responsible for BH4 biosynthesis, 6-pyruvoyl-
tetrahydropterin synthase (PTS) gene and GTP cyclohydrolase I (GCH1) gene, or it can
be due to mutations in genes responsible for BH4 metabolism, dihydropteridine
reductase (DHPR) gene and pterin-4a-carbinolamine dehydratase (PCBD) gene. Due to
serious and irreversible consequences of HPA, it is essential to establish an accurate
diagnosis of a patient, and start appropriate treatment as soon as possible. In Serbia,
molecular genetics characterization of the PAH gene was introduced in 2004. However,
it is necessary to establish additional methods for comprehensive identification and
functional characterization of variants detected in various genes that cause HPA. This
approach can have an important application for differential diagnosis of HPA patients in
Serbia and it enables a prompt introduction of adequate genotype-specific treatment. In
case of PKU, although mutant PAH genotype represents the main determinant of PKU
phenotype, it is not always possible to predict the phenotype based only on genotype.
Genetic factors which attribute to development of final PKU phenotype (especially
cognitive PKU phenotype) are still poorly understood. Therefore, the identification of
the new intragenic modulators (eg. new elements involved in transcription regulation) of
PKU phenotype, as well as the new modifier genes, would lead to its better
understanding.
The first part of this research featured a comprehensive molecular
characterization of variants in disease-causing genes in 76 patients with HPA in Serbia.
DNA sequencing of the whole coding region of the PAH gene, exon-intron boundaries,
promoter region and MLPA (multiplex ligation-dependent probe amplification) analysis
led to a detection of 29 known and 1 new mutation. For the first time in Serbia, a large
deletion (c.510-?_706+?del) was detected in one patient in homozygous state, therefore
having a frequency of 1,33%. Also, mutation detection in the PTS, gene responsible for
BH4 biosynthesis, was optimized, and it involved DNA sequencing of the whole coding
region. At the time, mutation p.Asp136Val was detected in one patient, in a
homozygous state. This is the first case of BH4 deficiency in Serbia, which was
identified owing to the application of molecular genetics and optimization of the PTS
gene mutation detection. Frequency of PTS deficiency in the analyzed group of HPA
patients is 1,31%. By combining various methods, the level of mutation detection
reached 99,34% for HPA in Serbia. Based on the spectrum of detected mutations, a
diagnostic algorithm was established for Serbia, which enables faster, easier and more
affordable detection of mutations in HPA-causing genes. Furthermore, a prediction of
BH4 responsiveness was conducted based on the analyzed PKU patients’ genotypes,
and it was shown that 32% of PKU patients from Serbia would most probably benefit
from BH4 therapy, while the maximum of the patients that could benefit from the BH4
treatment is up to 79%.
The second part of the research consisted of a functional analysis of detected
nonsynonymous substitution p.Gln226Lys PAH (c.676C>A). Using an in vitro
expression hepatoma system, it was demonstrated that the amount of mutant
p.Gln226Lys PAH-FLAG detected by Western blot was only 1.2% compared to wt
PAH-FLAG. Furthermore, the addition of sepiapterin, intracellular precursor of BH4
with a protective chaperone-like function, did not significantly increase the amount of
mutant protein. Therefore, it was concluded that p.Gln226Lys is a disease causing
mutation, and it does not respond to BH4.
The third part of the research comprised the identification and functional
characterization of new transcription regulators in noncoding regions of the PAH gene.
Functional CAT assay analysis demonstrated that the PAH promoter region with
cytosine at -170 position (analogous to wt sequence) functions as new cis-regulating
element. This element decreases the level of transcription up to 50% compared to the
control plasmid with basal promoter activity. In addition, EMSA (electrophoretic
mobility shift assay) supershift assay showed that this regulatory element binds KLF1
transcription factor. Furthermore, functional analysis of VNTR region at 3’ part of PAH
gene showed that this VNTR region, regardless of the tandem repeat type, acts as a new
regulatory element. This element decreases level of transcription approximately up to
60% in comparison with control plasmid with basal promoter activity. EMSA supershift
assays demonstrated that the most frequent type of repeats in PKU patients in Serbia,
VNTR3, binds C/EBP alpha transcription factor. These two new transcription regulatory
elements, identified in noncoding regions (promoter and 3’ region of the PAH gene)
contribute to better understanding of PAH expression regulation and can lead to a more
comprehensive knowledge of complex PKU phenotype.
The fourth part of the research involved analysis of variants in putative PKU
cognitive phenotype modifier genes. This analysis was conducted using two different
approaches; the first proposed study of a selected gene-candidate, whereas the second
approach aimed at identifying new modifier genes at the level of the whole genome.
Firstly, a study was conducted to assess the possible association of m.10398G>A
variant in mitochondrial MT-ND3 gene and cognitive phenotype of 64 patients with
PKU (evaluated as IQ scores), which exhibited no statistically relevant difference in
average IQ between carriers of m.10398A and m.10398G. Then, in research which
aimed to identify new modifier genes of cognitive PKU phenotype, complete genome
sequence of 10 untreated PKU patients (from 6 families) with normal cognitive
development was analyzed using new generation sequencing method. After extensive
bioinformatics analysis, SHANK genes were selected as potential modifier genes of
PKU cognitive phenotype, given that their protein products are involved in synaptic
transmission. Furthermore, for 4 of 6 analyzed families, benign variants were identified
in one of the 3 SHANK genes (SHANK1, SHANK2 or SHANK3). In silico analysis
showed that SHANK proteins which carry identified variants could gain new
characteristics, and thus, could have a protective modifier effect on development of
cognitive dysfunction in PKU patients. Their discovery could hold great value in
development of new strategies for treatment of patients with PKU. | true |
Ekofiziološke adaptacije odabranih vrsta zeljastih biljaka na deponiji pepela termoelektrane 'Nikola Tesla - A' u Obrenovcu | Ecophysiological adaptations of selected herbaceous plants growing on the fly ash deposits of thermoelectric plant 'Nikola Tesla - A' in Obrenovac | Zasnivanje biljnog pokrivača na odlagalištima pepela obezbeđuje fizičku i
hemijsku stabilizaciju pepela, sprečava eroziju vetrom i smanjuje disperziju toksičnih
hemijskih elemenata u životnu sredinu. Istraživanja u okviru ove disertacije su
obavljena na području deponije pepela termoelektrane 'Nikola Tesla – A' u Obrenovcu i
to na pasivnim kasetama (K3 – stara tri godine i K2 – stara jedanaest godina), kao i na
kontrolnim staništima (KKo - nasip reke Kolubare; KBb – Botanička bašta „Jevremovac“
u Beogradu i KBk – naselje Bežanijska kosa u Beogradu), a obuhvatala su analize
biljaka, kao i supstrata na kojima one rastu. Za ekofiziološka istraživanja bile su
odabrane zeljaste biljke koje se obično koriste za biorekultivaciju pepelišta: Festuca
rubra L. (K3, K2 i KBb) i Dactylis glomerata L. (K3 i KKo), kao i vrste koje spontano
kolonizuju prostor na deponiji pepela: Calamagrostis epigejos (L.) Roth. (K3, K2 i KKo)
i Oenothera biennis L. (K3, K2 i KBk). Osnovni cilj doktorske disertacije je utvrđivanje
limitirajućih faktora staništa za nesmetani razvoj biljnog pokrivača, kao i
ustanovljavanje ekofizioloških adaptivnih strategija ispitivanih biljaka koje opstaju u
uslovima višestrukog stresa na deponiji pepela.
Fitocenološka analiza deponije pepela TENT – A u Obrenovcu je pokazala da su
na K3 najveću brojnost i pokrovnost imale sejane vrste F. rubra i D. glomerata, dok su
na K2 bile dominantne spontano kolonizovane vrste C. epigejos i O. biennis. Tokom
vremena na K2 se stvaraju povoljni fizičko - hemijski uslovi za rast i opstanak biljaka
na pepelu, što se ogleda u smanjenju procenta ukupnog peska, a povećanju frakcije
gline i glina+prah, smanjenju koncentracije rastvorljivih soli, slabo bazne reakcije
pepela i povećanju kiselih katjona u adsorptivnom kompleksu, što je u velikoj meri
uslovljeno aktivnošću biljaka koje su kolonizovale ovu kasetu. Količina dostupnih
oblika K2O je bila najveća na K2, a P2O5 na K3, dok je količina N bila generalno mala.
Na deponiji pepela, koncentracije As, B i Cu su bile toksične, (izuzev B na K2),
dok su koncentracije Mn i Zn (K2) bile deficitarne. Na deponiji pepela, DTPA – As i
DTPA – B, kao i udeo DTPA – dostupnih frakcija u odnosu na ukupan sadržaj ovih
elemenata je bio velik, što ukazuje na potencijalna strukturna i funkcionalna oštećenja
biljaka vezana za njihovu toksičnost. Količina DTPA – Cu, DTPA – Mn i DTPA – Zn,
kao i udeo DTPA – dostupnih frakcija u odnosu na ukupan sadržaj istih je bio mali, te
rastvorna količina koja je dostupna biljkama može biti odgovorna za metabolička
oštećenja vezana za njihov deficit.
Na deponiji pepela u listovima ispitivanih biljaka bile su izmerene toksične
koncentracije As i B, izuzev B kod C. epigejos. Koncentracije Mn i Zn u listovima svih
biljaka su bile u deficitu, dok je Cu bio na granici deficita, osim kod O. biennis. Sejane
vrste F. rubra i D. glomerata se odlikuju smanjenim kapacitetom transporta As, Cu i
Mn iz korenova u listove (BCF<1, TF<1), osim Mn kod F. rubra (K3). Međutim,
spontano kolonizovane vrste C. epigejos i O. biennis pokazuju veću sposobnost
translokacije As, Cu, Mn iz korena u listove (BCF<1, TF >1). Se se kod F. rubra i O.
biennis (K2) akumulira u korenu (BCF>1, TF<1), dok se kod ostalih vrsta efikasno
transportuje u listove (BCF>1, TF>1). Zn se kod svih ispitivanih biljaka na deponiji
pepela nalazi u većoj količini u korenu u odnosu na listove (izuzev kod O. biennis, K2),
dok se B i Mo efikasno transportuju u listove.
Efikasnost fotosinteze kod svih biljaka na deponiji pepela je bila ispod optimalnog
opsega, što ukazuje na fotoinhibiciju fotosistema II (PSII) i smanjenu vitalnost. Količina
hlorofila i karotenoida je u listovima svih ispitivanih biljaka bila manja u odnosu na
kontrolna staništa, dok je količina antocijana u listovima F. rubra bila najveća na K3.
Količina malondialdehida (MDA) je bila veća u listovima svih ispitivanih biljaka na
deponiji pepela, što ukazuje da su one izložene oksidativnom stresu. Količina fenola,
askorbinske kiseline (AsA) i ukupna DPPH antioksidativna aktivnost bile su velike kod
svih biljaka koje rastu na pepelištu, što ukazuje na sposobnost aktivacije adaptivnih
mehanizama zaštite. Na deponiji pepela, vrsta O. biennis se najviše razlikuje od ostalih
biljaka po većoj količini vezanih fenola (VF) i ukupnoj DPPH antioksidativnoj
aktivnosti. Morfološke promene listova biljaka bile su izražene u vidu hloroza i nekroza
izazvanih toksičnim sadržajem As i B, kao i deficitarnim koncentracijama Cu, Mn i Zn.
Sejane vrste F. rubra i D. glomerata su značajne tokom prve faze biološke
rekultivacije, jer stabilizuju pepeo dobro razvijenim korenovim sistemom i u uslovima
stresa poseduju potencijal aktivacije antioksidativne zaštite. Međutim, tokom vremena
spontano kolonizovane vrste C. epigejos i O. biennis osvajaju prostor deponije pepela,
zahvaljujući dobro razvijenom multirizomskom sistemu, velikoj količini viabilnih
semena i ekofiziološkim adaptacijama koje im omogućavaju rast i opstanak u
nepovoljnim uslovima sredine, što ih čini pogodnim za revitalizaciju pepelišta. | Establishment of plant cover on fly ash deposits provides the physical and
chemical stabilization of ash, prevents wind erosion and reduces dispersion of toxic
chemical elements in the environment. Research within this thesis was carried out in the
area of the fly ash deposits thermoelectric plant 'Nikola Tesla - A' in Obrenovac: passive
cassette (K3 - three years old and K2 - eleven years old), as well as the control sites
(KKo - embankment of the river Kolubara; KBb - Botanical Gardens "Jevremovac" in
Belgrade and KBK - settlement Bežanijska kosa in Belgrade), and included analysis of
plants, as well as the substrate on which they grow. For ecophysiological studies were
selected herbaceous plants which are commonly used for the biorecultivation on the fly
ash: Festuca rubra L. (K3, K2 and KBb) and Dactylis glomerata L. (K3 and KKo), as
well as species that spontaneously colonized space in fly ash deposits: Calamagrostis
epigejos (L.) Roth. (K3, K2 and KKo) and Oenothera biennis L. (K3, K2 and KBk). The
main objective of the doctoral dissertation is to determine the limiting factors of site for
the smooth development of the vegetation cover, as well as the establishment of
ecophysiological adaptive strategies of examined plants that survive in conditions of
multiple stress on the fly ash.
Phytocoenological analysis of the fly ash deposit TENT - A in Obrenovac
showed that on the K3, the largest number and coverage had sown species F. rubra and
D. glomerata, while on the K2 were spontaneously colonized dominant species C.
epigejos and O. biennis. Over time, on the K2 to create good physico - chemical
conditions for the growth and survival of plants in the fly ashes, which is reflected in the
reduction of the percentage of total sand, and increasing fractions of clay and clay +
powder, reducing the concentration of soluble salts, weak base reactions of fly ash and
increasing acidic cation of the adsorptive complex, which is largely conditioned by the
activity of the plants that have colonized this disposal site. The content of available
forms of K2O was the highest on the K2, and P2O5 on the K3, while the content of N
was generally small. On the fly ash deposit sites, the concentrations of As, B and Cu
were toxic (except B on the K2), while concentrations of Mn and Zn (K2) were in
deficiency. On the fly ash deposit sites, DTPA - As and DTPA - B, and the percentage
of DTPA - available fraction of the total content of these elements was large, indicating
the potentialy structural and functional damage to plants related to their toxicity. The
content of DTPA - Cu, DTPA - Mn, DTPA - Zn as well as the percentage of DTPA -
the available fraction of Cu, Mn and Zn of the total content was small, and the soluble
content which is accessible to the plants may be responsible for the metabolic disorders
related to their deficiency.
Thus, on the fly ash deposit sites, toxic concentrations As and B in leaves of
examined plants were measured, with the exception of B in C. epigejos. The
concentrations of Mn and Zn in the leaves of all plants were deficient, while Cu was on
the deficiency limit, except for O. biennis. Sown species F. rubra and D. glomerata are
characterized by a reduced transport capacity of As, Cu and Mn from roots to leaves
(BCF<1, TF<1), except for Mn in F. rubra (K3). However, spontaneously colonized
species C. epigejos and O. biennis showed greater ability of translocation of As, Cu and
Mn from the roots to the leaves (BCF<1, TF>1). In F. rubra and O. biennis (K2), Se is
accumulated in the roots (BCF>1, TF>1), while in other species, Se is efficiently
transported to the leaves (BCF>1, TF>1). In all the species examined at the disposal
site, greater amount of Zn is retained in the roots (except in O. biennis, K2), while B
and Mo are efficiently transported to the leaves.
Photosynthetic efficiency of all plants which grow on fly ash deposits was below
the optimum range, which indicates on photoinhibition of photosytem II (PSII) and
decreased vitality. The chlorophyll and carotenoids content in leaves of all plants was
lower compared to the control site, while the anthocyanins content in leaves of F. rubra
was the highest on the K3. The malondialdehyde (MDA) content was higher in leaves
of all plants at fly ash deposits, indicating that they were exposed to oxidative stress.
The phenolics content, ascorbic acid (AsA) and total DPPH antioxidant activity was
high in all plants, indicating the ability to activate the protective adaptive mechanisms.
Species O. biennis is the most different from the other species by the higher content of
bound phenolics (BPh) and the total DPPH antioxidant activity. Morphological changes
in plant leaves were expressed in the form of chlorosis and necrosis caused by toxic
levels of As and B, as well as deficient concentrations of Cu, Mn and Zn.
Sown species F. rubra and D. glomerata are important in the first stage of
biological cultivation, because their well-developed root system stabilizes the substrate.
Furthermore, under conditions of stress they have the potential to activate antioxidant
protection. However, over time, spontaneously colonized species C. epigejos and O.
biennis have conquered the area of fly ash. This was made possible by their well-
developed multirhizome system, large amount of viable seeds and ecophysiological
adaptations that enable them to grow and survive in adverse environmental conditions.
These traits make them suitable for the revitalization of fly ash . | true |
Biomarkeri oksidacionog stresa i koncentracija metala u odabranim tkivima belouške (Natrix natrix) i ribarice (Natrix tessellata) sa područja Obedske bare i Pančevačkog rita | Oxidative stress biomarkers and metal concentrations in selected tissues of grass snake (Natrix natrix) and dice snake (Natrix tessellata) from Obedska bara and Pančevački rit | Ekotoksikološka istraživanja su značajna zbog činjenice da mogu imati
praktičnu primenu u zaštiti životne sredine. Semiakvatične vrste zmija konstantno su
izložene dejstvu kompleksa različitih ekoloških faktora kako u vodenoj tako i u
terestričnoj sredini. Zmije su dugoživeći organizmi, nalaze se na vrhu lanca ishrane,
imaju manji radijus kretanja u odnosu na većinu ptica i sisara i to ih čini idealnim model
organizmima za praćenje efekta izloženosti različitim vrstama ksenobiotika u prirodnim
uslovima.
Metali predstavljaju najrasprostranjeniju grupu zagađivača. Zbog svoje
toksičnosti, biomagnifikacije duž lanca ishrane, kao i bioloških efekata koje izazivaju
predstavljaju neizostavnu kariku u ekotoksikološkim istraživanjima.
Cilj ove doktorske disertacije bio je da se ispita sezonska dinamika biomarkera
oksidacionog stresa, uticaj metala prisutnih u životnoj sredini kao i njihova
bioakumulacija u nekim tkivima Natrix natrix (belouška) i Natrix tessellata (ribarica) sa
područja Specijalnog rezervata prirode Obedska bara i industrijske zone Pančevački rit,
u periodu pre i posle hibernacije. U krvi, jetri, bubregu, mišiću i masnom tkivu su
analizirane enzimske komponente sistema zaštite od oksidacionih oštećenja: aktivnost
ukupne, mangan i bakar cink sadržavajuće superoksid-dismutaze (Uk SOD, Mn SOD,
CuZn SOD), katalaze (CAT), glutation-peroksidaze (GSH-Px) i glutation-reduktaze
(GR). Ispitivane su i neenzimske komponente sistema zaštite od oksidacionih oštećenja:
koncentracija glutationa (GSH) i slobodnih sulfhidrilnih grupa (SH). Takođe su
analizirani: biohemijski pokazatelj oksidacionih oštećenja (TBARS), enzim faze I
biotransformacije: citohrom P4501A (CYP1A), enzim faze II biotransformacije:
glutation-S-transferaza (GST), biomarkeri izloženosti metalima (metalotioneini) i
biomarker neurotoksičnosti (ChE). U vodi i tkivima (jetri i mišiću) analizirani su alkalni
(Li, K) zemnoalkalni (Ba, Ca, Mg), prelazni (Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Zn), slabi
(Al, Pb) metali kao i metaloid As.
U jetri N. natrix aktivnost Uk SOD i CuZn SOD bila je značajno veća u
periodu posle hibernacije, dok je kod N. tessellata uočen suprotan trend. Aktivnosti Uk
SOD i CuZn SOD u jetri N. natrix i N. tessellata nisu se značajno razlikovale između
lokaliteta u oba ispitivana perioda. Aktivnost CAT u jetri, bubregu i mišiću N. tessellata
iz Pančevačkog rita, jetri N. natrix sa Obedske bare kao i bubregu N. natrix sa oba
ispitivana lokaliteta bila je veća u periodu posle hibernacije. Aktivnost GSH-Px bila je
veća u periodu posle hibernacije u jetri ispitivanih vrsta sa oba lokaliteta. Smanjena
aktivnost GSH-Px dobijena je u krvi obe vrste u Pančevačkom ritu, u periodu pre
hibernacije, kao i u jetri i masnom tkivu N. natrix posle hibernacije, mišiću N. natrix pre
hibernacije i bubregu N. tessellata posle hibernacije. Indukcija aktivnosti GR u jetri,
bubregu i masnom tkivu N. natrix pre hibernacije i bubregu N. tessellata u oba
ispitivana perioda u Pančevačkom ritu rezultat je povećane potrošnje GSH. Dobijena je
značajno manja koncentracija SH grupa u jetri, bubregu, mišiću i masnom tkivu
belouške iz Pančevačkog rita pre hibernacije i ribarice posle hibernacije. Povećan nivo
TBARS zabeležen je u krvi N. tessellata iz Pančevačkog rita u periodu posle
hibernacije, bubregu pre hibernacije i masnom tkivu u obe ispitivane sezone. Kod N.
natrix iz Pančevačkog rita u krvi, jetri i masnom tkivu postoji značajno smanjenje nivoa
TBARS. U periodu pre hibernacije nivo ekspresije enzima faze I biotransformacije
CYP1A u jetri N. tessellata iz Obedske bare bio je značajno veći u poređenju sa nivoom
ekspresije ovog enzima jetri N. tessellata iz Pančevačkog rita. U jetri i bubregu obe
ispitivane vrste sa Obedske bare aktivnost GST značajno je niža u posthibernacijskom
periodu u odnosu na period pre hibernacije. U krvi N. tessellata u obe sezone, krvi N.
natrix u periodu pre hibernacije i jetri obe ispitivane vrste u periodu posle hibernacije
aktivnost ChE bila je inhibirana kod jedinki iz Pančevačkog rita. Inhibicija ChE uočena
je i kod životinja iz Obedske bare, u krvi N. natrix posle hibernacije, bubregu i mišiću
pre hibernacije kao i u bubregu N. tessellata posle hibernacije. U jetri jedinki N. natrix
iz Obedske bare u oba ispitivana perioda, kao i kod N. tessellata u periodu posle
hibernacije zabeležen je povećan nivo ekpresije MT, što može biti direktan odgovor na
intraćelijsko povećanje koncentracije metala. U periodu pre hibernacije u jetri N. natrix
koncentracija mangana je bila značajno veća u jedinkama sa Obedske bare, dok je
koncentracija aluminijuma bila veća u jetri jedinki iz Pančevačkog rita. U jetri jedinki
vrste N. tessellata iz Obedske bare u periodu pre hibernacije, koncentracije gvožđa,
molibdena i cinka bile su takođe značajno veće u odnosu na jedinke iz Pančevačkog
rita. Izmerene koncentracije bakra, mangana, molibdena i cinka u jetri N. tessellata
posle hibernacije bile su takođe značajno veće u jedinkama sa Obedske bare.
Istraživanja sprovedena u ovoj doktorskoj disertaciji pokazala su da biomarkeri
oksidacionog stresa, biotransformacije i neurotoksičnosti u ispitivanim vrstama zmija
ispoljavaju varijacije u zavisnosti od ispitivane vrste, sezone, ispitivanog tkiva,
lokaliteta, a samim tim i kompozicije metala u životnoj sredini. Utvrđen je različit
stepen akumulacije metala u ispitivanim tkivima, koji ne korelira u potpunosti sa
koncentracijom metala izmerenom u životnoj sredini što je rezultat individualne
varijabilnosti i uticaja složenih ekoloških faktora. | Ecotoxicological studies are important due to the fact they can have a practical
application in environmental protection. Semiaquatic snakes are continuously exposed
to various complex environmental factors both in aquatic and terrestrial environment.
They are long-living organisms, on the top of the food chain, with smaller movement
radius compared to most birds and mammals, which makes them ideal model organisms
for monitoring effects of exposure to different types of xenobiotics in natural
conditions.
Metals are the most widespread group of pollutants. Due to their toxicity,
biomagnification along the food chain, as well as biological effects they cause, they
represent indispensable link in ecotoxicological research.
The aim of this doctoral thesis was to examine seasonal dynamics of oxidative
stress biomarkers, the impact of metals present in the environment, as well as their
bioaccumulation in some tissues of Natrix natrix (Grass snake) and Natrix tessellata
(Dice snake) from Special Nature Reserve sites, Obedska bara and Pančevački rit,
industrial zone, for periods before and after hibernation. In blood, liver, kidney, muscle
and adipose tissue, enzyme components of system protection against oxidative damage
were analyzed: activities of total, manganese and copper zinc superoxide dismutase (Tot
SOD, Mn SOD, CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and
glutathione reductase (GR), non-enzymatic components of system protection against
oxidative damage: concentration of glutathione (GSH) and free sulfhydryl groups (SH),
biochemical indicator of oxidative damage (TBARS), phase I biotransformation
enzyme: cytochrome P4501A (CYP1A), phase II biotransformation enzyme: glutathione-
S-transferase (GST), biomarkers of exposure to metals, metallothionein and activity of
cholinesterase (ChE), neurotoxicity biomarker.
In water, liver and muscle tissues, alkaline (Li, K), alkaline earth metal (Ba, Ca,
Mg), transient (Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Zn), weak (Al, Pb ) metals and
metalloid As were analyzed.
In liver of N. natrix Uk SOD and CuZn SOD activities were significantly higher
during the period after hibernation, while in N. tessellata opposite trend was obtained.
Tot SOD and CuZn SOD activities in liver of N. natrix and N. tessellata did not
significantly differ between locations during both studied periods. CAT activity in liver,
kidney and muscle of N. tessellata from Pančevački rit, liver of N. natrix from Obedska
bara, as well as kidney of N. natrix within both locations was higher after hibernation.
GSH-Px activity was higher in the period after hibernation in liver of both studied
species from both locations. Reduced GSH-Px activity in blood of both species before
hibernation in Pančevački rit was obtained, as well as in liver and adipose tissue of N.
natrix after hibernation, muscle of N. natrix before hibernation, and kidney of N.
tessellata after hibernation. Induction of GR activity in liver, kidney and adipose tissue
of N. natrix before hibernation and kidney of N. tessellata in both studied periods in
Pančevački rit, is result of increased consumption of GSH. Significantly lower
concentration of SH groups in liver, kidney, muscle and adipose tissue of N. natrix
before hibernation and N. tessellata after hibernation in Pančevački rit were obtained.
Increased level of TBARS was recorded in N. tessellata in blood after hibernation,
kidney before hibernation and adipose tissue in both studied periods in Pančevački rit.
In N. natrix in blood, liver and adipose tissue there was a significant reduction in the
level of TBARS in Pančevački rit. Prior to hibernation expression level of phase I
biotransformation enzymes of CYP1A in liver of N. tessellata was significantly higher
in Obedska bara. In liver and kidney of both species in Obedska bara, GST activity was
significantly lower in post hibernation period compared to period before hibernation. In
blood of N. tessellata in both seasons, blood of N. natrix before hibernation and liver of
both species after hibernation ChE activity was inhibited in individuals from Pančevački
rit. Inhibition of ChE was obtained in Obedska bara, in blood of N. natrix after
hibernation, kidney and muscle before hibernation, as well as in kidney of N. tessellata
after hibernation. In liver of specimen of N. natrix in both studied periods, as with N.
tessellata after hibernation, significantly higher level of MT expression in Obedska bara
was recorded, which can be a direct response to an increase in intracellular
concentrations of metals. Prior to hibernation in liver of N. natrix manganese
concentration was significantly higher in individuals in Obedska bara, while the
concentration of aluminum was higher in liver in individuals from Pančevački rit. In
liver samples of N. tessellata before hibernation concentrations of iron, molybdenum
and zinc were also significantly higher in Obedska Bara in relation to Pančevački Rit.
Measured concentrations of copper, manganese, molybdenum and zinc in liver of N.
tessellata after hibernation were also significantly higher in individuals from Obedska
bara.
Research carried out in this doctoral thesis showed that oxidative stress
biomarkers, biotransformation and neurotoxicity in examined snakes species exhibit
variations that depend on studied species, season, tested tissue, locations, and thus the
composition of metals in the environment. Different degree of metal accumulation in
studied tissues was determined, which does not correlate exactly with the measured
metal concentration in the environment, which is result of individual variability and
impact of complex environmental factors. | true |
Obavezna odbrana u krivičnom postupku | Mandatory legal aid in criminal proceedings | Obavezna odbrana je stručna odbrana, u zakonski određenim slučajevima obavezna u krivičnom postupku za okrivljene izloženim povećanim procesnim rizicima. Ti rizici uzrokovani su nekim ličnim hendikepima koji dovode do smanjene procesne kompetencije, težinom krivičnih dela, kao i nekim drugim procesnim okolnostima koje im otežavaju ili čak potpuno onemogućavaju ličnu odbranu. Analiziraju se ciljevi postojanja i funkcionisanja obavezne odbrane unutar krivičnog postupka kao i njen uticaj na tok postuka. U tezi se, pored domaće i inostrane teorije, koriste i rezultati četiri opsežna istrživanja: „Osuđenici o advokatima“, „Advokati o obaveznoj odbrani“, „100 okrivljenih Okružnog suda u Beogradu“ i „Stručna odbrana okrivljenih za krivična dela privrednog kriminaliteta“. Teza je sistematizovana u četiri poglavlja od kojih Prvo obuhvata definisanje obavezne odbrane, kao i odnos sa drugim srodnim vidovima odbrane, posebno odbranom siromašnih. U Drugom poglavlju se kroz istorijski i uporedno pravni prikaz uočava geneza i univerzalnost ovog procesnog instituta u prošlim i savremenim krivičnim postupcima. Treće poglavlje sadrži analizu pozicije i uloge obavezne odbrane u krivičnom postupku i njenu osnovanost na pocesnim načelima savesnosti, nezavisnosti, monofukcionalnosti i zamenljivosti. Analiziraju se posebni slučajevi obavezne odbrane kao i sličnosti i razlike sa srodnim institutima građanskog i upravnog postupka, kroz zajednički ratio legis upoređivanih instituta. Četvrto poglavlje sadrži izlaganje o položaju i ulozi obaveznog branioca u pojedinim fazama krivičnog postupka, od prethodnog postupka sve do postupka po vanrednim pravnim lekovima. Zaključna razmatranja daju moguće smernice daljeg razvoja tog instituta na osnovu svih sakupljenih, sistematizovanih i izloženih saznanja o obaveznoj odbrani. Predlaže se da obaveznost počne sa prvim ispitivanjem okrivljenog i da se mora popraviti kvalitet odbrane po službenoj dužnosti, pošto se najveći broj obaveznih odbrana realizuje upravo na taj oficijelni način. Predlaže se niz mera kako da obavezna odbrana bude pre svega put profesionalne afirmacije advokata a ne samo neudobna dužnost. Uočava se da je poverenje između branioca i okrivljenog presudno za kvalitet odbrane, više nego materijalni momenat pa se predlažu mere za jačanje tog elementa obavezne odbrane. | Mandatory legal aid is defence by attorney, implemented in criminal proceedings in cases when defendants are exposed to high procedural risks, e.g. if charged for serious crimes, or when there are reasons of a personal nature which made them incompetent for personal defence or even to realise the need of having an attorney to defend him. Mandatory legal aid reflects concern of society for defendants in these cases. There is analysed the existence and functioning of mandatory defense within the criminal proceedings as well as its influence on the proceedings. Iin addition to domestic and foreign theories, in the thesis are used the results of four extensive researches: "Prisoners of lawyers", "Lawyers on mandatory legal aid", "100 defendants in cases of the District Court in Belgrade" and "Expert defense of the defendants chareged for offenses of fraud". The thesis is systematized in four chapters of which the First one involves defining the mandatory legal aid and the relationship with other related aspects and other kinds of defense, especially defense of the poor. In the Second chapter, through comparative and historical legal display, it was shown the genesis, universality and versatility of this institute in modern criminal proceedings. The Third Chapter comprises the analyse of the position and role of mandatory legal aid in criminal proceedings, special cases of mandatory legal aid, as well as the similarities and differences with related institutes of civil and administrative proceedings. There are also analysis of the legal aid ground on procedural principles of conscientiousness, independence, mono- funcionality and substitutability. Thus is provided the complete image displayed through the establishment of relationships between these elements, and the consequences on other procedural institutions and relationships that are considered. The Fourth Chapter contains a presentation on the status and role of mandatory legal aid counsel in all stages of criminal proceedings, from the previous procedure to proceedings of the extraordinary legal remedies. Concluding observations provide possible directions of further development of the institute on the basis of all collected, systematized and presented findings on mandatory legal aid. It is proposed that the mandatoru legal aid starts with the first examination of the defendant and that it must be improved the quality of ex officio legal aid since the largest number of mandatory legal aid is realized officially. Series of measures are also proposed as to reform mandatory legal aid as prominent attorneys’duty leading to professional affirmation, rather than only uncomfortable, undesired duty. It is emphasizes that the trust between attorney and the defendant is crucial for the quality of defense, more than its financial aspects. Therefore several measures are proposed to strengthen the relation of the defence attorney and the defendants. | false |
Bioakumulacija i biomagnifikacija toksičnih metala i elemenata u tragovima kod slatkovodnih riba različitog trofičkog nivoa iz Dunava kod Beograda | Bioaccumulation and biomagnifications of toxic metals and trace elements in freshwater fishes from different trophic levels in the Danube near Belgrade | Iako je i dalje u upotrebi, termin „teški metal“ različiti autori
definišu na različite načine brojnim parametrima. Izvori zagađenja teškim
metalima mogu biti prirodni, ali su najčešće antropogeni. Ova klasa
zagađivača predstavlja značajnu pretnju za životnu sredinu i vrste koje žive u
njoj, kao i potencijalnu pretnju za ljudsko zdravlje. Najbitnija je opasnost koju
predstavljaju toksični elementi – arsen, živa, kadmijum i olovo. Osim
toksičnih
elemenata,
i
elementi
u
tragovima,
kao
i
esencijalni
mikronutrijenti, mogu pokazivati toksična svojstva ako su prisutni u velikim
koncentracijama.
U ovom radu analizirane su koncentracije 18 elemenata (Al, As, B, Ba, Cd,
Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Se, Sr, Zn) u tkivima 11 vrsta riba – Esox
lucius (štuka), Alburnus alburnus (ukljeva), Cyprinus carpio (šaran), Pelecus
cultratus (sabljar), Silurus glanis (som), Lota lota (manić), Gymnocephalus cernua
(balavac), Perca fluviatilis (grgeč), Sander lucioperca (smuđ), Babka gymnotrachelus
(glavoč trkač) i Neogobius melanostomus (glavoč okruglak). Osnovni cilj bio je
da se ustanovi da li postoje sličnosti i/ili razlike u bioakumulaciji
elemenata u određenim tkivima kod različitih vrsta. Analizirane vrste su
predstavnici različitih trofičkih nivoa, što ima značajan uticaj na
biokoncentraciju u tkivima, a može ukazati i na biomagnifikaciju pojedinih
elemenata duž trofičkog lanca.
Vrste su hvatane u periodu između 2010. i 2012. godine na dva lokaliteta,
između 1168. i 1170. rečnog kilometra Dunava u Beogradu. Ovi lokaliteti su
birani zbog blizine ušća Save (koja donosi dodatno zagađenje) u Dunav, blizine
urbanog dela Beograda, kao i blizine poljoprivrednih površina. Analiza 18
elemenata u tkivima riba urađena je pomoću induktivno spregnute plazma-
optičke emisione spektrometrije (ICP-OES). Za određivanje starosti jedinki
korišćene su krljušti, žbice peraja (som) i otoliti (manić).
IV
Uzorci mišićnog tkiva odlikuju se najmanjim brojem statistički
značajnih razlika između ispitivanih vrsta riba u odnosu na koncentracije
elemenata. Ovo tkivo se takođe ne odlikuje najvećom koncentracijom bilo kog
elementa, u poređenju sa ostalim analiziranim tkivima. Nasuprot tome, u jetri
je pronađen veći broj statistički značajnih razlika. Sličnost između mišićnog
tkiva i jetre jeste u činjenici da ni u jednom od ovih tkiva ni kod jedne vrste
nije pronađena statistički značajna razlika u odnosu na koncentraciju
aluminijuma.
Koncentracije arsena i žive su, u svim analiziranim tkivima, najčešće
više u odnosu na rezultate istraživanja na drugim lokalitetima. Ovo može biti
posledica zagađenosti ispitivanih lokaliteta u Dunavu, a u slučaju arsena
objašnjenje može biti i u prirodnoj koncentraciji ovog elementa u podzemnim
vodama i aluvijalnim formacijama u slivu Dunava.
Linearna regresija i korelaciona analiza ukazuju na pozitivnu vezu između
koncentracije nekih elemenata kod riba-plena (glavoči) i predatorskih vrsta
(smuđ, som), a potvrđen je i trend biomagnifikacije žive. Na osnovu
koncentracije elemenata u jetri, moguće je funkcionalno odvojiti predatorske
vrste riba od nepredatorskih, a na osnovu koncentracije elemenata u škrgama,
moguće je razdvojiti vrste riba prema preferiranom tipu staništa. Većina
uočenih statistički značajnih korelacija između koncentracije elemenata i
težine riba jesu negativne korelacije.
Koncentracije As, Cd, Cu, Fe i Zn u mišićnom tkivu ne prelaze propisane
vrednosti MDK ni kod jedne analizirane vrste. U mišićnom tkivu manića,
smuđa, soma, ukljeve i šarana, živa ne prelazi MDK graničnu vrednost, što nije
slučaj sa mišićnim tkivom balavca, grgeča, sabljara i štuke. Korišćenje vrsta
riba kod kojih koncentracija žive prelazi MDK granice u ishrani može
predstavljati potencijalnu opasnost po ljudsko zdravlje. | Although still in use, the term “heavy metal” is defined in different ways, based
on a number of parameters, by different authors. Sources of heavy metal contamination
can be natural, yet they are mostly anthropogenic. This class of pollutants poses a
significant threat to environment and species living in it, as well as a potential threat to
human health. The most significant threat are the toxic elements – arsenic, mercury,
cadmium, and lead. Apart from these toxic elements, both trace elements and essential
micronutrients can also show toxic properties if present in large concentrations.
The concentrations of 18 elements (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn,
Mo, Ni, Pb, Se, Sr, Zn) in tissues of eleven fish species – Esox lucius (pike), Alburnus
alburnus (common bleak), Cyprinus carpio (common carp), Pelecus cultratus (sichel),
Silurus glanis (catfish), Lota lota (burbot), Gymnocephalus cernua (ruffe), Perca
fluviatilis (perch), Sander lucioperca (pikeperch), Babka gymnotrachelus (racer goby),
and Neogobius melanostomus (round goby), were analyzed in this study. The principal
aim was to find out if there were any similarities and/or differences in element
bioaccumulation in assorted tissues of different species. The analyzed species represent
different trophic levels, which has a significant impact on bioconcentration in tissues,
and it can as well as point to biomagnification of certain elements along the trophic
chain.
Species were caught between 2010 and 2012 on two locations, between 1168th
and 1170th river kilometer of the Danube River in Belgrade. These locations were
selected for their proximity to the confluence of the Sava River (who carries additional
pollution) into the Danube, their proximity to the urban area of Belgrade, as well as
their proximity to agricultural areas. The analysis of 18 elements in fish tissues was
done using an inductively coupled plasma optical emission spectrometry (ICP-OES).
For the determination of fish age, scales, ray fins (catfish), and otoliths (burbot) were
used.
VI
Muscle samples are characterized by the fewest number of statistical differences
between analyzed fish species and element concentrations. Moreover, this tissue does
not have the highest concentration of any analyzed element, in comparison to other
analyzed tissues. Contrary to this, in liver we have found a higher number of statistical
differences between analyzed fish species and element concentrations. Similarity
between muscle and liver tissues is in the fact that in these tissues there are no statistical
differences in aluminum concentration between the fish species.
Concentrations of arsenic and mercury, in all analyzed tissues, were usually
higher in comparison to the results from different locations. This can be the result of
pollution at the sampling locations in the Danube, and, in the case of arsenic, the
explanation could also be the natural concentration of this element in the groundwater
and alluvial formations in the Danube basin.
Linear regression and correlation analysis indicate a positive relation between the
concentration of some elements in prey fishes (gobies) and predatory fishes (pikeperch,
catfish), and the trend of mercury biomagnification is confirmed. Based on the
concentration of elements in the liver, it is possible to functionally separate predatory
species from non-predatory. Based on the concentration of elements in gills, it is
possible to separate fish species based on their preferred habitat type. Most of the
detected statistically significant correlations between element concentrations and fish
weight were negative correlations.
Concentrations of As, Cd, Cu, Fe, and Zn in muscle tissue do not exceed the
prescribed MAC values in any of the analyzed species. In the muscle tissue of burbot,
pikeperch, catfish, common bleak, and carp, mercury does not exceed the MAC level,
which is not the case with the muscle tissue of ruffe, perch, sichel, and pike. The
consumption of the fish species in which the mercury concentration is above the MAC
threshold may present a potential risk to human health. | true |
Detekcija elokventnih govornih moždanih zona funkcionalnom magnetnom rezonancom | FMRI identification of eloquent language brain areas | Funkcionalna magnetna rezonanca (fMR), u poslednjoj deceniji dala je
značajan doprinos u ispitivanju različitih aspekata moždanih funkcija
kako kod zdravog mozga tako i u različitim patološkim stanjima. Svrha ove
disertacije bila je da ispita mogućnosti fMR u detekciji elokventnih
govornih moždanih zona kako kod zdravih volontera tako i kod pacijenata sa
tumorima mozga.
U grupi zdravih volontera bilo je 12 ispitanika, 6 muškaraca i 6
žena. Grupu pacijenata činilo je 17 ispitanika (9 muškaraca i 8 žena) sa
tumorima
mozga
Kod
svih ispitanika
načinjeni
su
fMR
snimci
endokranijuma za dva različita stimulusa brojanje u napred i brojanje u
nazad, a potom su načinjene fMR mape aktiviranih moždanih zona. Pored
toga načinjene su i grupne analize za zdrave ispitanike i ispitanike sa
tumorima mozga kao i grupne analize prema polu u oviru obe grupe
ispitanika. Za sve ispitanike i oba stimulusa izračunat je indeks
lateralizacije govora.
Kod
zdravih
ispitanika
na
zadate
stimuluse
aktivirana
je
distribuirana mreža moždanih zona koja je osim kanonskih govornih zona u
inferiornom frontalnom girusu (IFG) i superiornom temporalnom girusu
(STG)
podrazumevala
i
aktivnost
u
primarnim
motornim
zonama,
suplementnoj motornoj zoni, prefrontalnim regijama, cerebelumu i
medijalnim aspektima temporalnog režnja a uočene su određene razlike u
odgovoru na zadate stimuluse između muškaraca i žena. Indeks
lateralizacije bio je u skladu sa procenom lateralizacije govora za sve
posmatrane zdrave ispitanike. Kod ispitanika sa tumorima mozga osim
primarnih govornih zona u IFG i STG identifikovana je aktivnost u
primarnim motornim zonama, prefrontalnim regijama, cerebelumu i u
vi
medijalnim aspektima temporalnog režnja. Osim toga uočena je i aktivnost
u senzori-motornim zonama, okcipitalnim i parijetalnim regijama. Uočene
su i određene razlike u moždanom odgovoru na zadate stimuluse između žena
i muškaraca. Indeks lateralizacije kod ispitanika sa tumorima mozga samo
delimično je bio u skladu sa procenom lateralizacije govora, za ovu grupu
ispitanika.
Predloženi
stimulusi
omogućili
su
detekciju
nižih
nivoa
distribuirne govorne mreže i dali dobru osnovu za interpretaciju statusa
funkcije govora kako kod zdravih ispitanika tako i kod pacijenata sa
tumorima mozga. | Functional magnetic resonance imaging (fMRI), in the last decade, has
made significant contributions in the study of various brain functions in the
healthy brain and in different pathological brain conditions as well. The purpose
of this thesis was to explore the possibilities of fMRI in detection of eloquent
language cortex in healthy volunteers and patients with brain tumors.
In the group of healthy volunteers there were 12 subjects, 6 males and 6
females. In the patient group there were 17 patients, (9 males and 8 females) with
brain tumors. For all participants fMR exam was made for two different stimuli,
counting forward and counting backward. After that fMR maps of activated brain
areas were made. In addition, group analysis for healthy individuals and patient
with brain tumors were made, as well as group analysis according to gender for
both groups. For all subjects and both stimulus laterality index was calculated.
In healthy subject for both stimulus distributed network of cortical areas
were detected. Besides of canonical language areas in inferior frontal gyrus (IFG)
and superior temporal girus (STG), activation in primary motor areas,
supplementary motor areas, prefrontal region, cerebellum and medial temporal
region were detected. There were some differences in response to given stimuli
between females and males. Laterality index was consistent with the assessment
of speech lateralization in all observed healthy subjects.
In patients with brain tumors besides the brain areas identified in healthy
volunteers, additional activity was observed in sensory motor areas, the occipital
and parietal region. There were certain differences in brain responses for given
stimuli between females and males in this group as well. Laterality index in
patients with brain tumors were only partially in line with the evaluation of
language lateralization in this group.
viii
The proposed stimuli allowed detection of lower levels of distributed
cortical language network and provide a good basis for interpreting the functional
status of language in healthy individuals and in patients with brain tumors as
well. | true |
Uticaj LPS i IL-33 na adultne matične tkiva periodoncijuma: funkcijske promene i aktivirani molekulski mehanizmi | The influence of LPS and IL-33 on adult stem cells of periodontal ligament:functional changes and activated molecular mechanisms | Savremeni načini lečenja različitih oboljenja usne duplje, nastalih
usled urođenih deformiteta, mehaničkih oštećenja ili poremećaja
uzrokovanim
inflamatornim
ili
tumorskim
oboljenjima
mogu
predstavljati kompleksan klinički zadatak s obzirom da se nameće zahtev
obnove funkcije, ali i estetike orofacijalnog regiona. Značajnu ulogu u
održanju strukture i funkcije orofacijalnog sistema ima potporno tkivo
zuba u okviru koga tkivo periodoncijuma predstavlja vezu između zuba i
alveolarne kosti. Tkivo periodoncijuma je bogato mezenhimskim matičnim
ćelijama (MMĆ), te može biti pogodan izvor populacije MMĆ za
potencijalnu terapijsku primenu. Usled oštećenja tkiva periodoncijuma
uspostavljanje strukture i funkcije zuba, a time i orofacijalnog sistema je
veoma otežano čak i uz primenu najsavremenijih protetskih tretmana. Ipak,
funkcijske osobine MMĆ periodoncijuma (PD-MMĆ) nisu u potpunosti
ispitane, što predstavlja neophodan uslov za njihovu primenu. Posebno
važan aspekt predstavlja poznavanje ponašanja PD-MMĆ u inflamatornoj
mikrosredini, budući da do razaranja tkiva periodoncijuma najčešće
dolazi
usled
parodontopatije,
hroničnog
inflamatornog
oboljenja
potpornog tkiva zuba koje može dovesti do gubitka zuba. Pored toga,
inflamacija potornog tkiva može biti prateća komplikacija nakon
postavljanja dentalnih implantata što takođe ukazuje na potrebu za
poboljšavanjem postojećih protetskih tretmana.
Istraživanja obuhvaćena ovom disertacijom imala su za cilj
dobijanje podataka o uticaju faktora koji se mogu naći u niši tkiva
periodoncijuma na funkcijska svojstva PD-MMĆ. Ispitivan je uticaj LPS,
kao snažnog inicijatora inflamatornog procesa, na regenerativna i
imunomodulatorna svojstva PD-MMĆ. Na molekulskom nivou utvrđivani su
mehanizmi koji posreduju u efektima LPS, pri čemu je analizirana
aktivnost MARK (od engl. Mitogen-Activated Protein Kinases) signalnih puteva.
Pored toga, utvrđivan je efekat IL-33, kao endogenog alarmina, na
regenerativna svojstva PD-MMĆ. U sklopu utvrđivanja molekulskih
mehanizama koji se nalaze u osnovi delovanja IL-33 ispitivana je
uključenost NF-κB (od engl. Nuclear Factor kappa-light-chain-enhancer of
activated B cells) i β-katenin signalnih puteva.
Dobijeni rezultati pokazuju da ispitivani faktori imaju značajan
uticaj na funkcijska svojstva PD-MMĆ, pri čemu ni LPS ni IL-33 ne utiču na
vijabilnost, morfološka kao ni fenotipska svojstva ovih ćelija. Efekat
LPS ogleda se u značajnoj inhibiciji osteogeneze, što je praćeno
stimulacijom hondrogene i adipogene diferencijacije posredstvom ERK1,2
(od engl. Extracellular signal-Regulated Kinases 1 and 2) signalizacije.
Istovremeno, LPS ne utiče na migratornu sposobnost PD-MMĆ, ali podstiče
njihovu kontraktilnost, odnosno sticanje miofibroblastnog fenotipa
kroz
stimulaciju
ekspresije
molekula
karakterističnih
za
miofibroblaste što je posredovano ERK1,2 signalnim putem. Rezultati
analize imunomodulatornih svojstava PD-MMĆ u prisustvu LPS pokazuju da,
aktivirajući
ERK1,2
signalni
put,
kod
PD-MMĆ
LPS
inhibira
transendotelsku migraciju (TEM) mononuklearnih ćelija periferne krvi
(MNĆ), dok blago utiče na smanjenje ekspresije površinskog markera CD29
kod MNĆ. Sa druge strane, LPS ne modifikuje sposobnost PD-MMĆ da
inhibiraju proliferaciju mitogenom stimulisane CD4+ subpopulacije
MNĆ, kao ni odnos zastupljenosti CD4+CD25visoko/CD4+CD25nisko limfocita.
U kontekstu analize modulacije svojstava ćelija mijeloidnog porekla,
istraživanja obuhvaćena ovom disertacijom pokazuju da LPS ne utiče na
sposobnost PD-MMĆ da inhibiraju TEM polimorfonuklearnih ćelija
periferne krvi (PMĆ), na sposobnost PD-MMĆ da modifikuju respiratorni
prasak PMĆ, kao ni na zastupljenost CD33+, CD45+ i CD34+ ćelija u
populaciji MNĆ. Sa druge strane, pretretman PD-MMĆ sa LPS značajno
inhibira zastupljenost CD14+ MNĆ uz učešće ERK1,2 signalnog puta. Pored
toga, LPS, aktivacijom ERK1,2 signalnog puta, blokira stimulatorni efekat
PD-MMĆ
na
formiranje
ukupnog
broja
kolonija
opredeljenih
hematopoetskih progenitora prisutnih u populaciji MNĆ periferene
krvi, među kojima su najzastupljenije CFU-GM kolonije. Analiza uticaja LPS
na ekspresiju imunomodulatornih molekula kod PD-MMĆ pokazuje da LPS ne
utiče na konstitutivnu ekspresiju HLA-A i HLA-DR gena, dok stimuliše
gensku i proteinsku ekspresiju COX-2 i IL-6 kod PD-MMĆ. Stimulacija
ekspresije COX-2 i IL-6 pod uticajem LPS kod PD-MMĆ posredovana je ERK1,2
signalnim putem. Pored toga, PD-MMĆ konstitutivno eksprimiraju ST2 i
IL-33 gen, pri čemu LPS povećava ekspresiju ST2 gena, ali smanjuje ekspresiju
gena za IL-33.
Rezultati ispitivanja uticaja IL-33 na regenerativna svojstva PD-
MMĆ
pokazuju
da
ovaj
citokin
stimuliše
ekspresiju
markera
pluripotentnosti kod PD-MMĆ (OCT-4, SOX-2 i NANOG) gde je uočena
uključenost NF-kB i β-katenin signalnih puteva. Sa druge strane, IL-33
inhibira osteogenu diferencijaciju PD-MMĆ posredstvom NF-kB i β-
katenin signalnix puteva, dok pretretman PD-MMĆ sa IL-33 stimuliše
osteogenu diferencijaciju na nivou aktivnosti enzima alkalne fosfataze
(ALP, od engl. Alkaline phosphatase). Nasuprot uticaju na osteogenezu, IL-33 ne
utiče na hondrogenu i adipogenu diferencijaciju PD-MMĆ. Međutim, tokom
osteogene diferencijacije IL-33 stimuliše proliferativnu aktivnost PD-
MMĆ posredstvom β-katenina. Istovremeno IL-33 podstiče sposobnost
formiranja CFU-F (od engl. Colony Forming Unit – Fibroblast) kolonija PD-MMĆ
u standardnom medijumu, ali ne modifikuje stimulatorni efekat samog
osteogenog medijuma. Uz to, ispitivanje ćelijskog starenja, pokazuje da IL-33
inhibira aktivnost enzima β-galaktozidaze kod PD-MMĆ kultivisanih u
standardnom i osteogenom medijumu, pri čemu sam osteogeni medijum
značajno redukuje procenat ćelija koje eksprimiraju β-galaktozidazu.
Na osnovu izloženih rezultata može se zaključiti da LPS, kroz
modulaciju diferencijacionog potencijala, predstavlja značajan faktor u
regulaciji regenerativnog potencijala PD-MMĆ. Istovremeno, LPS je i
regulator
imunomodulatornih
aktivnosti
ovih
ćelija
kako
kroz
modulaciju interakcije PD-MMĆ i imunskih ćelija, tako i kroz modulaciju
produkcije imunomodulatornih faktora. Za razliku od LPS, na osnovu
predstavljenih rezultata može se zaključiti da je IL-33 kao endogeni
alarmin, pretežno uključen u regulaciju osteogene diferencijacije
ispoljavajući protektivne efekte na populaciju progenitorskih ćelija
periodoncijuma,
kroz
sprečavanje
starenja
PD-MMĆ,
podsticanje
proliferacije i klonogenosti, odnosno matičnosti. Ovi nalazi doprinose
boljem sagledavanju ispoljavanja funkcijskih svojstava PD-MMĆ u uslovima
inflamatorne mikrosredine, što je važno za razumevanje fiziologije ovih
ćelija u normalnim i patološkim stanjima, kao i za razumevanje procesa
koji se nalaze u osnovi funkcionisanja PD-MMĆ. Istovremeno, ovi nalazi
značajni su i za unapređivanje postojećih metoda lečenja potpornog aparata
zuba, ali ujedno i osnova za razvoj novih pristupa lečenja u okviru
regenerativne medicine zasnovanih na primeni PD-MMĆ. | Contemporary methods for treating various diseases of the oral cavity, caused by
congenital deformities, mechanical damage or damage caused by inflammatory or
tumor diseases can be a complex clinical task, given that the requirement for restoration
of function and aesthetics of the orofacial region is imposed. The supporting tissue of
the tooth in which the tissue of the periodontium represents the connection between
the teeth and the alveolar bone, has an important role in maintaining the structure and
function of the orofacial system. The tissue of the periodontium is very rich with
mesenchymal stem cells (MSC) and can be a suitable source of MSC population for
potential therapeutic use. Due to periodontium damage, the establishment of teeth
structure and function, and consequently the orofacial system, is very difficult, even
with the application of the most modern prosthetic treatments. However, functional
features оf MSC from periodontium (PD-MSC) have not been fully revealed, which is a
necessary requirement for their application. A particularly important aspect is
understanding of PD-MSC’s behavior in the inflammatory microenvironment, since the
periodontium destruction is most often caused by periodontal disease, chronic
inflammatory disease of the supporting tissue of the teeth that can lead to tooth loss. In
addition, inflammation of the teeth supportive tissue may be an accompanying
complication after the dental implant, which also indicates the need to improve existing
prosthetic treatments.
The aim of the research covered by this dissertation was to obtain data about
effects of factors frequently associated with the periodontium niche on the functional
properties of PD-MSC. The influence of LPS, as a strong initiator of the inflammatory
process, on the regenerative and immunomodulatory PD-MSC was examined. On the
molecular level mechanisms mediating the effects of LPS were determined, wherein the
activity of MAPK (Mitogen-Activated Protein Kinases) signaling pathways was
determined. In addition, the effect of IL-33, as an endogenous alarm signal, on the
regenerative properties of PD-MSC was examined. As a part of the determination of the
molecular mechanisms underlying IL-33 effects, involvement of NF-kB (Nuclear Factor
cappa-light-chain-enhancer of activated B cells) and β-catenin signaling pathways was
examined.
Obtained results show that the investigated factors have significant effects on the
PD-MSC functional properties, while neither LPS nor IL-33 affect viability,
morphological or phenotypic properties of these cells. The LPS effect is manifested in a
significant inhibition of osteogenesis, followed by stimulation of chondrogenic and
adipogenic differentiation through ERK1,2 (Extracellular signal-Regulated Kinases 1 and
2) signaling. At the same time, LPS does not affect migratory ability of PD-MSC, but
stimulates acquisition of myofibroblast phenotype, by upregulating contractility, and
expression of myofibroblast’s molecules, via ERK1,2 a signaling pathway. Results of the
PD-MSC immunomodulatory properties in the presence of LPS show that, by activating
the ERK1,2 signaling pathway in PD-MSC, LPS inhibits transendothelial migration (TEM)
of mononuclear peripheral blood cells (MNC), while slightly affecting the expression of
surface marker CD29 in MNC. On the other hand, LPS does not modify the ability of PD-
MSC to inhibit the proliferation of mitogen-stimulated CD4+ subpopulation of MNC, nor
the ratio of the CD4+CD25high/ CD4+CD25low lymphocytes. In the context of modulation
of myeloid cells, studies covered by this dissertation, show that LPS does not affect the
ability of PD-MSC to inhibit TEM of polymorphonuclear peripheral blood cells (PMC),
the PD-MSC capability to modify PMC respiratory burst, as well as the presence of
CD33+, CD45+ and CD34+ cells in the MNC population. On the other hand, PD-MMC
pretreatment with LPS significantly decreased the presence of CD14+ MNC via ERK1,2
signaling pathway. In addition, LPS blocks the stimulatory effect of PD-MSC on the
formation of the total number of colonies of the hematopoietic progenitors present in
the peripheral blood MNC population, predominantly of CFU-GM, by activating the
ERK1,2 signaling pathway. Analysis of the LPS effect on the immunomodulatory
molecules expression in PD-MSC shows that LPS does not affect the constitutive
expression of HLA-A and HLA-DR gene, while stimulates both gene and protein
expression of COX-2 and IL-6 in PD-MSC. This stimulation of COX-2 and IL-6 expression
is mediated by ERK1,2 signaling pathway. Additionally, PD-MSC constitutively express
ST2 and IL-33 gene, whereby LPS increases the expression of ST2 gene, but reduces the
expression of the IL-33 gene.
The results of the IL-33 influence on the regenerative properties of PD-MSC show
that this cytokine stimulates the expression of pluripotencty markers in PD-MSC (OCT-
4, SOX-2 and NANOG) through the involvement of NF-kB and β-catenin signaling
pathways. Moreover, IL-33 inhibits osteogenic differentiation of PD-MSC by NF-kB and
β-catenin signaling pathways, although pretreatment of PD-MSC with IL-33 induced
stimulation of osteogenic differentiation at alkaline phosphatase activity (ALP, Alkaline
phosphatase) level. Contrary to the effect of IL-33 on osteogenesis, this cytokine does
not affect the chondrogenic and adipogenic differentiation of PD-MSC. However, during
osteogenic differentiation, IL-33 stimulates the proliferative activity of PD-MSC via β-
catenin. At the same time, IL-33 stimulates the ability to form CFU-F (Colony Forming
Unit - Fibroblast) colonies of PD-MSC in a standard medium, but does not modify the
stimulatory effect of osteogenic media alone. Additionally, cellular aging assays indicate
that IL-33 inhibits the activity of β-galactosidase enzyme in PD-MSC cultured in
standard and osteogenic medium, wherein the osteogenic medium alone substantially
reduces the percentage of cells expressing β-galactosidase.
Considering the presented results, it can be concluded that LPS, through the
modulation of the differentiation potential, represents a significant factor involved in
the regulation of the regenerative potential of PD-MSC. At the same time, LPS is the
regulator of immunomodulatory activity of these cells through modulation of PD-MSC
interaction with immune cells, as well as through the modulation of the production of
immunomodulatory factors. Unlike LPS, IL-33, as an endogenous alarmin, is mainly
involved in the regulation of osteogenic differentiation, showing protective effects, by
prevention of PD-MSC aging, promotion of their proliferation and clonogenicity, i.e
stemness.
Taken together, these findings contribute to better understanding of PD-MSC’s
functional properties under inflammatory conditions, which is important for
understanding the physiology of these cells in normal and pathological conditions, as
well as understanding of the processes underlying PD-MSC functioning. At the same
time, these findings can be significant for the improvement of existing methods of
treatment of the tooth support tissues, and also as a basis for the development of new
treatment approaches within regenerative medicine based on the application of PD-
MSC. | true |
Solvotermalna sinteza cink-oksida sa kontrolisanom veličinom čestica na nano i mikro skali | Solvothermal synthesis of zinc oxide with controlled nano-and microsized particles | Cink-oksid je poluprovodni materijal koji je zbog svojih specifičnih fizičkih i
hemijskih svojstava (širok energijski procep, hemijska i fotohemijska stabilnost, optička
propustljivost, piezoelektricitet, netoksičnost, antibakterijska svojstva) našao primenu u
izradi optoelektronskih uređaja, uređaja na bazi površinskih akustičnih talasa, emitera
polja, piezoelektričnih uređaja, propustljivih provodnih materijala, solarnih ćelija,
kozmetičkih preparata i slično.
U okviru ove doktorske disertacije detaljno je proučen uticaj uslova sol-gel
sinteze modifikovanim Španhelovim postupkom i sinteze solvotermalnim postupkom na
strukturna i mikrostrukturna svojstva dobijenih prahova ZnO. Ispitivan je uticaj pH-
vrednosti u modifikovanom Španhelovom postupku, kao i temperature, vremena
trajanja i pH-vrednosti koloidnih prekursora primenjenih u solvotermalnoj sintezi na
veličinu i morfologiju čestica praha ZnO na nano- i mikroskali. U karakterizaciji
prahova korišćene su: rendgenska strukturna analiza, termijska analiza, skenirajuća i
transmisiona elektronska mikroskopija, ramanska i infracrvena spektroskopija.
Pokazano je da su prahovi dobijeni sol-gel sintezom višefazni i da osim ZnO sadrže
cink-hidroksid-acetate. Ustanovljeno je da su kod takvih prahova nanočestice ZnO (dm ≈
4 nm) sa jednakim orijentacijama samouređene u monokristalne domene. Prahovi
dobijeni solvotermalnim postupkom predstavljaju fazno čist ZnO. Njihova morfologija
pri konstantnoj temperaturi i vremenu sinteze (200 °C, 2 h) za pH-vrednosti prekursora
u intervalu od 8 do 12 menja se od pravilnih heksagonalnih prizmi do zaobljenih
nanočestica. Pokazano je da produžavanje vremena sinteze pri konstantnoj temperaturi
(200 °C) i pH-vrednosti (8) uslovljava promenu oblika čestica od heksagonalnih ka
štapićastim, submikronske veličine, uključujući i formiranje mezokristalnih čestica
ZnO. Prah čije čestice pokazuju najveći stepen homogenosti po veličini i obliku
sintetisan je iz slabo kiselog koloidnog prekursora (pH = 6) u solvotermalnoj reakciji na
200 °C tokom 6 h.
VI
Na osnovu rezultata karakterizacije dobijenih prahova predloženi su mehanizmi
rasta nanostuktruktura ZnO za različite uslove sinteza. Pokazano je da se kontrolom
uslova solvotermalne sinteze može u potpunosti kontrolisati morfologija i veličina
čestica ZnO, što omogućava dobijanje prahova unapred zadatih karakteristika. | Zinc oxide is a semiconductor material, which has been found useful in many
applications, such as optoelectronic devices, surface acoustic wave devices, field
emitters, piezoelectric devices, transparent conducting materials, sollar cells, cosmetics
etc., due to its interesting properties, especially wide band gap, chemical and
photochemical stability, optical transparency, piezoelectricity, nontoxicity, antibacterial
and UV-protection properties.
In this work, an influence of sol-gel (modified Spanhel method) and
solvothermal synthetic conditions on structural and microstructural properties of ZnO
powders was studied in detail. Precisely, the effect of pH value in modified Spanhel
procedure, as well as temperature, time and pH value in solvothermal syntheses, on
particle size and morphology was considered. The prepared powders were characterized
by X-ray Diffraction Analysis, Thermal Analysis, Scanning and Transmission Electron
Microscopy, Raman and Infrared Spectroscopy. The results showed that the powders
obtained from sol-gel process are multiphase systems containing ZnO and zinc
hydroxide acetates. These powders consist of ZnO nanoparticles (dm ≈ 4 nm) with the
same orientation, which were self-assembled into single crystal domains. The powders
from solvothermal syntheses are phase-pure ZnO. The morphology of these particles
obtained
at
200 °C,
during
2 h
with
pH
values
in
the
range
8
–
– 12, changes from regular, hexagonal prisms to rounded nanoparticles. At 200 °C and
pH = 8, the extending of reaction time results in changing of particle shape from
hexagonal prisms to rods of submicron scale, including mesocrystal particles. The
powder synthesized from slightly acidic precursor (pH = 6) at 200 °C, during 6 h
revealed the highest homogeneity of particles shape and size.
The most probable growth mechanisms of particles at different reaction
conditions were proposed. It was shown that it is completely possible to control
morphology and size of ZnO particles by adjusting conditions of the solvothermal
VIII
reaction which further enables producing of ZnO powders with predetermined
properties. | true |
Effect of salicylic acid on the expression of heat-shock proteins and morphology of potato (Solanum tuberosum L.) plants under heat stress in vitro | Uticaj salicilne kiseline na ekspresiju proteina toplotnog stresa i morfološke osobine biljaka krompira (Solanum Tuberosum L.) u uslovima toplotnog streasa in vitro | Krompir, Solanum tuberosum L., je četvrta po značaju kulturna biljka u svetu. Najveći
broj sorti krompira je razvijen za gajenje u umerenim klimatskim zonama i izuzetno su
osetljive na visoke temperature. Kao rezultat globalnog zagrevanja u narednih 60
godina, predvidja se značajan pad u prinosu krompira izmedju 18% i 32% koji bi se
mogao smanjiti prilagođavanjem poljoprivredne prakse u smislu promene u vremenu
sadjenja i lokacija, kao i korišćenju sorti otpornih na visoke temperature. Primena
jedinjenja koja pospešuju otpornost krompira na toplotni stres predstavlja drugi način da
se prevaziđu negativni efekti visokih temperatura na rast i prinos ove kulturne biljke.
Osnovni cilj ovog rada bio je ispitivanje uticaja salicilne kiseline (SA) na otpornost
S. tuberosum prema toplotnom stresu. Otpornost krompira na toplotni stres je u
korelaciji sa nivoom akumulacije malih HSP (sHSP), pa je stoga ispitivan efekat SA na
ekspresiju ove grupe proteina, kao i HSP101 i Cu/ZnSOD. Pored toga ispitivan je uticaj
SA na morfološke osobine krompira u uslovima toplotnog stresa. Korišćenje kulture in
vitro omogućilo je izvodjenje eksperimenata pod strogo kontrolisanim uslovima, uz
variranje samo dva faktora – temperature i koncentracije SA. Eksperimentalna
procedura se zasnivala na izlaganju jednonodalnih odsečaka stabla krompira (SNC)
različitim temperaturnim tretmanima, uključujući standardnu temperaturu za uzgoj in
vitro (23 °C, 20 dana) kratkotrajni tretman ekstremno visokom temperaturom (45 °C,
6 h – kratkotrajni toplotni stres) i dugotrajni tretman umereno visokom temperaturom
(35 °C, 20 dana; dugotrajni toplotni stres). Sličan procenat biljaka razvio se iz SNC na
23 °C i tokom perioda oporavka po kratkotrajnom toplotnom stresu, dok je dugotrajni
tretman umereno visokom temperaturom značajno redukovao razvoj biljaka iz SNC
ukazujući da dugotrajno delovanje umereno visoke temperature ima veći uticaj na
razvoj biljaka nego kratkotrajni tretman ekstremno visokom temperaturom. SA
(10-6-10-4 M) je stimulisala razvoj biljaka iz SNC tokom dugotrajnog toplotnog stresa
kod svih ispitivanih sorti krompira, sa izuzetkom sorte Marabel koja nije reagovala na
SA. Dati rezultati ukazuju da SA može pospešiti otpornost krompira na toplotni stres,
ali da postoji značajna razlika u osetljivosti sorti/genotipova na delovanje SA. SA
pretretmani (10-6-10-4 M) nisu imali efekta na inicijaciju i izduživanje internodija u
uslovima standardne temperature gajenja, kao i tremana visokim temperaturama, dok su
SA tretmani stimulatorno delovali na date procese u uslovima kako kratkotrajnog, tako i
dugotrajnog toplotnog stresa. SA pretretmani i SA tretmani (10-6-10-4 M) su značajno
inhibirali inicijaciju i izduživanje korenova u uslovima standarne T gajenja, kao i
kratkotrajnog toplotnog stresa, dok SA tretmani nisu pokazali značajniji inhibitorni
efekat na elongaciju korenova u uslovima dugotrajnog toplotnog stresa. Ovi rezultati
ukazuju na interakciju SA i toplotnog stresa u procesu izduživanja korenova koja zavisi
of dužine toplotnog tretmana, kao i dužine izlaganja biljaka SA. SA pretretman je
stimulatorno delovao na akumulaciju HSP17.6, dok je uočeno smanjenje zastupljenosti
HSP21 u listovima biljaka krompira pri izlaganju temperaturama od 35 °C ili 45 °C 6 h.
Relativno niska koncentracija SA (10-6 M) je stimulisala akumulaciju HSP101
krompira, srednja (10-5 M) nije imala efekta, dok je visoka koncentracija (10-4 M)
smanjila zastupljenost HSP101. Navedeni rezultati ukazuju na značajne razlike u SA-
posredovanoj regulaciji ekspresije HSP17.6, HSP21 and HSP101 u uslovima toplotnog
stresa. Samo pretretman sa 10-5 M SA je imao jak stimulatorni efekat na akumulaciju
Cu/ZnSOD u listovima svih ispitivanih sorti krompira na 23 °C. Stimulatorni efekat
10-5 M SA se pojačao u uslovima 35 °C-toplotnog stresa, dok je mali porast u
zastupljenosti proteina uočen na 45 °C. Dati rezultati ukazuju da stimulatorni efekat SA
na akumulaciju Cu/ZnSOD krompira zavisi kako od temperaturnog tretmana, tako i
primenjene koncentracije SA. | The potato, Solanum tuberosum L., is the fourth most important crop in the world. Most
of the commercial potato cultivars were developed for growing in temperate climate
regions and they are highly sensitive to elevated temperatures. As a result of global
warming over next 60 years, significant potato yield losses in the range of 18 to 32% are
predicted. These losses can be reduced with adaptations in production methods, such as
terms of planting time and use of heat tolerant cultivars. Besides, exploration of
compounds that may enhance potato heat tolerance, such as salicylic acid (SA), is an
alternative approach to cope with adverse effects of high temperatures on potato growth
and yield. The major aim of this study was to investigate effects of SA on the basal heat
tolerance in Solanum tuberosum L. Potato heat tolerance correlates with level of small
heat-shock proteins (sHSP) accumulation, therefore SA effect on this group of HSPs, as
well as HSP101 and Cu/ZnSOD, was investigated. Besides, it was interesting to
investigate SA effects on morphological parameters related to plant growth and
development. Experiments were conducted in vitro since this experimental setup
allowed investigation under strictly controlled conditions, with variation of only
selected factors – temperature and SA concentration. Experimental procedure based on
potato single-node stem cuttings (SNC) exposure to standard growth temperature
(23 °C), excessively elevated temperature (45 °C, 6 h; short-term HS) or prolonged
exposure to moderately elevated temperature (35 °C, 20 days; long-term HS) has been
established. Similar percentage of plants developed from SNC at 23 °C and during
recovery period after exposure to short-term HS, while long-term HS substantially
reduced the number of plantlets developed from SNC implying that in potato prolonged,
moderately elevated temperatures can cause more devastating effects then excessively
elevated ones during short exposure. SA in a range of concentrations from 10-6 M to
10-4 M stimulated development of plants from SNC under long-term HS in all
investigated potato cultivars, with the exception of cultivar ‘Marabel’ which did not
respond on SA treatment. These findings indicate that SA may enhance basal
thermotolerance in potato cultivars/genotypes, as well as variation in genotypes´
sensitivity to SA. SA pretreatments, in the range of concentrations from 10-6 М to 10-4
М, did not affect internode initiation and elongation either under standard or elevated
temperatures, while SA treatments in the same range of the concentrations stimulated
these processes under both short-term and long-term HS. Both SA pretreatments and SA
treatments, in the range of concentrations from 10-6 М to 10-4 М, strongly inhibited root
initiation and elongation at standard growth temperature and after short-term HS, while
SA treatments did not show prominent inhibitory effect on root elongation in the same
range of the concentrations under long-term HS. The results suggest interaction between
SA and HS in the process of root elongation, possibly depending on duration and
intensity of HS, as well as on duration of exposure to SA. SA pretreatment had
stimulatory effect on HSP17.6 accumulation, while all applied SA concentrations
inhibited accumulation of chloroplast HSP21 in investigated potato genotypes when
exposed to 35 °C or 45 °C for 6 h. Relatively low SA concentration (10-6 M) promoted
accumulation of HSP101 in potato, moderate (10-5 M) did not affected abundance, while
high concentration (10-4 M) reduced accumulation of this protein under 45 °C-treatment.
These findings indicate significant differences in SA-mediated regulation of HSP17.6,
HSP21 and HSP101 expression under HS. Pretreatment with only 10-5 M SA had strong
stimulatory effect on Cu/ZnSOD accumulation at 23 °C in leaves of investigated potato
cultivars. Stimulatory effect of 10-5 M SA was accentuated at 35 °C, while slight
increase in abundance of this protein has been observed at 45 °C in most potato
genotypes. These findings indicate that stimulatory effect of SA on Cu/ZnSOD
accumulation in potato depends on both temperature treatment and SA concentration. | true |
Taksonomski status rodova i vrsta Euaphidius mackauer i Remaudierea starý (Hymenoptera, Braconidae, Aphidiinae) na osnovu morfoloških i molekularnih analiza 28S rRNK I COI gena | Taxonomic status of genera and species of Euaphidius mackauer and Remaudierea Starý (Hymenoptera, Braconidae, Aphidiinae) based on morphological and molecular analyses of 28s rRNA and COI genes | Taksonomski status roda Euaphidius Mackauer, sa dve opisane vrste E. cingulatus
(Ruthe) i E. setiger Mackauer, je poslednjih decenija pod znakom pitanja. Ove dve vrste su u
literaturi obično razmatrane u okviru roda Aphidius Nees. Međutim, na osnovu nekih
morfoloških karakteristika, pre svega, tentorijalnog indeksa kao i njihove biologije, Mackauer
(1961) ih je izdvojio u poseban rod, Euaphidius. Rod Remaudierea Starý, sa jednom
opisanom vrstom R. plocamaphidis Starý je sličan vrstama roda Aphidius i Euaphidius, pre
svega po nervaturi krila i obliku petiolusa. Morfološka i genetička varijabilnost tri parazitske
ose E. cingulatus, E. setiger i R. plocamaphidis, kao i njihov taksonomski položaj u odnosu
na vrste roda Aphidius, su analizirani metodama „tradicionalne“ i geometrijske morfometrije,
kao i analizom sekvenci mitohondrijalnog gena za citohrom oksidazu 1 (mt COI) i
varijabilnog D2 regiona gena za veliku ribozomalnu subjedinicu 28S (28S rRNK).
„Tradicionalnom“ morfometrijom utvrđeni su karakteri koji su pokazali najveću varijabilnost
u morfologiji između analiziranih vrsta. To su pokazali karakteri odnos dužine pterostigme i
dužine R1 nerva, broj rinarija na prvom flagelarnom članku, kao i broj dlačica u gornjoj
areoli propodeuma. Analizom oblika prednjih krila uz primenu geometrijske morfometrije
uočeno je da vrste roda Euaphidius imaju kraći R1 nerv i šira krila u centralnom delu u
odnosu na vrste roda Aphidius. Takođe, vrsta E. cingulatus se razlikuje od vrste E. setiger
užim krilom u centralnom delu i dužim distalnim delom prednjeg krila. Molekularnom
analizom na osnovu mtCOI i 28S rRNK gena su utvrđene male razlike između rodova
Euaphidius i Remaudierea, kao i izabranih vrsta roda Aphidius. Kombinacija sve tri
primenjene metode nije potvrdila status roda Eupahidius. Sve analizirane vrste (E. cingulatus,
E. setiger i R. plocamaphidis) su premeštene u rod Aphidius, s tim što se vrste E. cingulatus i
R. plocamaphidis izdvajaju u okviru podroda Euaphidius, a E. setiger u podrod Aphidius. | Taxonomic status of the genus Euaphidius Mackauer with two described species E.
cingulatus (Ruthe) and E. setiger Mackauer has been questionable over past few decades. In
the literature, these two species are considered within the genus Aphidius. However, based on
some morphological characteristics, such as tentorial index, also biology of analysed species,
Mackauer (1961) raised them into the separate genus, Euaphidius. The genus Remaudierea
Starý, which was described by the only known species R. plocamaphidis Starý, resembles
Aphidius and Euaphidius species, primarily by the wing venation pattern and the shape of a
petiole. Morphological and genetic variability of the species E. cingulatus, E. setiger and R.
plocamaphidis, and their taxonomic position in relation to the Aphidius species, were
analysed using traditional and geometric morphometrics, as well as using molecular analysis
of cytochrome oxidase 1 (mtCOI) and the variable D2 region of the ribosomal gene 28S (28S
rRNA). Using traditional morphometrics method, characters that showed the greatest
variability in morphology among analysed species were identified. The significant variability
was detected over ratio values: the length of pterostigma and the length of R1 vein, the
number of olfactory grooves (rhinaria) on the first segment of antenna and the number of
setae in the upper lateral areola of the propodeum. Analysis of the forewing shape using
geometric morphometrics showed that the species from the genus Euaphidius have shorter R1
vein and wider wings in medial part than in species from Aphidius. Likewise, the species E.
cingulatus is discriminated from E. setiger by a narrower forewings in the central part and
generally, they wings are longer in the distal part. On the basis of molecular analysis of the
mtCOI and 28S rRNA genes, small genetic differences among genera Euaphidius and
Remaudierea, and selected Aphidius species were determined. The combination of three
applied methods was not confirmed the status of genus Euaphidius. We have changed the
status of E. cingulatus, E. setiger and R. plocamaphidis including them in the genus Aphidius.
The species E. cingulatus and R. plocamaphidis are joined in the subgenus Euaphidius, while
E. setiger is transferred in the subgenus Aphidius. | true |
Ligninolitički potencijal i medicinska svojstva ekstrakata vrsta roda Trametes fr. | Ligninolytic potential and medicinal properties of extracrs of Trametes fr. species. | Rod Trametes Fr. obuhvata vrste širokog rasprostranjena sa dobro razvijenim ligninolitičkim enzimskim sistemom i značajnim kapacitetom u sintezi raznovrsnih biološki aktivnih jedinjenja, zbog čega sve više nalaze primenu u mnogobrojnim biotehnološkim procesima. Ciljevi ovog istraživanja bili su optimizacija uslova kultivacije za produkciju ligninolitičkih enzima i degradaciju odabranog biljnog otpada sa T. gibbosa, praćenje varijabilnosti u kapacitetu lignocelulolize unutar roda Trametes kao i određivanje antifungalnog, antioksidativnog, genoprotektivnog, citotoksičnog i anti-neurodegenerativnog efekta ekstrakata plodonosnih tela i micelije. Pšenična slama je bila bolji izvor ugljenika nego piljevina bukve za produkciju Mn-oksidujućih peroksidaza i lakaza T. gibbosa koji je maksimalni stepen delignifikacije (48,3%) postigao nakon 19 dana čvrste fermentacije u prisustvu 10,0 mM azota u formi (NH4)2SO4 i 10% pšeničnih mekinja kao dodatnog izvora azota. Prisustvo Fe2+ i Zn2+ u koncentraciji od 0,5 mM, kao i 0,1 mM p-anizidina i 0,5% veratril alkohola je značajno stimulisalo ligninolizu. Značajna inter- i intra-specijska varijabilnost u sintezi proučavanih enzima, degradaciji polimera biljnog ćelijskog zida kao i u selektivnosti delignifikacije je takođe uočena između 6 vrsta i 11 sojeva. T. hirsuta BEOFB 30 je bio najefikasniji i najselektivniji razlagač lignina (56,0% razloženog lignina prema 17,4% hidrolizovane celuloze), posebno u prisustvu p-anizidina koji je stimulisao produkciju Mn-oksidujućih peroksidaza. Dvofaktorskom analizom varijanse je pokazano da su soj, prisustvo indukujućih supstanci kao i njihova interakcija faktori koji značajno utiču na stepen ligninolize. Etanolni ekstrakti bazidiokarpa i micelije T. gibbosa, T. hirsuta i T. versicolor, su imali slab antifungalni potencijal koji se ogledao u slaboj inhibiciji rasta testiranih humanih patogena a jedino je micelijski ekstrakt T. hirsuta pokazao fungicidno dejstvo na Aspergillus glaucus (MFC = 32,0 mg/mL). Ekstrakt plodonosnih tela T. versicolor je pokazao najveći kapacitet neutralizacije DPPH i ABTS radikala (dobijene EC50 vrednosti su 15,22 odnosno 8,46 mg/mL), a glavni nosioci ove aktivnosti su bili fenoli čija je koncentracija bila u pozitivnoj korelaciji sa antioksidativnim kapacitetom... | The genus Trametes Fr. encompasses widely distributed species with strongly developed ligninolytic enzyme system and significant capacity to synthesize diverse biologically active compounds, because of which are increasingly used in numerous biotechnological processes. The aims of this research were the optimization of cultivation conditions for ligninolytic enzyme production and degradation of selected plant residues with T. gibbosa, assessment of the variability in capacity of lignocellulolysis within the genus Trametes and determination of antifungal, antioxidative, genoprotective, cytotoxic and antineurodegenerative effects of basidiocarp and mycelial extracts. In comparison with beach sawdust wheat straw was better carbon source for production of Mn-oxidizing peroxidases and laccase by T. gibbosa which maximum rate of delignification (48.3%) was achieved after 19 day of solid-state fermentation in the presence of 10.0 mM of nitrogen in the form of (NH4)2SO4 and with 10% of wheat bran as additional nitrogen source. Supplementation of wheat straw with Fe2+ and Zn2+ in concentration of 0.5 mM, p-anisidine in concentration of 0.5% and veratryl alcohol in solutions’ concentration of 0.5% significantly stimulated lygninolysis. Significant inter- and intraspecific variability in synthesis of studied enzymes, degradation of plant cell wall polymers and selectivity in delignification was noticed among 6 species and 11 strains. T. hirsuta BEOFB 30 was the most efficient and the most selective lignin degrader (56.0% of degraded lignin vs. 17.4% of hydrolysed cellulose), particularly in the presence of p-anisidine which stimulated Mn-oxidizing peroxidises production. Results of two-way analysis of variance showed that strain, presence of inducers and their interaction represent factors which significantly affect rate of lygninolysis. Ethanolic extracts of basidiocarps and mycelia of T. gibbosa, T. hirsuta and T. versicolor showed weak antifungal potential which reflected in low growth inhibition of tested human pathogens and only mycelial extract of T. hirsuta BEOFB 30 showed fungicidal effect on Aspergillus glaucus (MFC = 32.0 mg/mL). Basidiocarp extract of T. versicolor showed the highest capacity in neutralization of DPPH and ABTS radicals (obtained EC50 values were 15.22 and 8.46 mg/mL, respectively), and the main carriers of this activity were phenolic compounds, which concentration was positively correlated with antioxidative capacity... | false |
Fauna cikada (Hemiptera: Auchenorrhyncha) u vinogradima Makedonije i njihova uloga u epidemologiji ""Candidatus Phytoplasma solani"" | Auchenorrhyncha) in the vineyards of macedonia and their role in the epidemiology of 'Candidatus Phytoplasma solani' | Bois noir (BN), oboljenje koje izaziva stolbur fitoplazma, nanosi velike štete u
vinogradarskim regionima širom Evro-mediteranskog basena. Tokom 2012. i 2013. godine
preduzeta su epidemiološka istraživanja za određivanje potencijalnih insekata vektora i
najvažnijih biljaka rezervoara BN fitoplazme u makedonskim vinogradima, u jugoistočnom
delu zemlje. Istraživanjem diverziteta vrsta iz podreda Auchenorrhyncha utvrđeno je
prisustvo 27 vrsta cikada u okviru 6 familija. Ukupno je registrovana 21 vrsta familije
Cicadellidae, 2 vrste familije Cixiidae i po jedan predstavnik familija Aphrophoridae,
Delphacidae, Dyctiopharidae i Issidae. Tokom istraživanja utvrđena je izrazita učestalost
glavnog vektora stolbur fitoplazme, cikade Hyalesthes obsoletus, dok drugi dokumentovani
vektor BN, Reptalus panzeri, nije zabeležen u makedonskim vinogradima. Molekularna
karakterizacija ‘Candidatus Phytoplasma solani’ u analiziranom materijalu vršena je
analizom genskih regiona tuf, stamp i vmp1 gena, RFLP metodom i sekvenciranjem. Cilj
karakterizacije je da se stekne detaljan uvid u molekularnu raznolikost izolata stolbur
fitoplazme povezanih sa vinovom lozom, potencijalnim biljkama rezervoarima (Urtica
dioica i Convolvolus arvensis) i jedinkama H. obsoletus sakupljenim na ovim biljkama.
Ukupno je analizirano 91 izolat stolbur fitoplazme među kojima je identifikovano 3 tuf, 5
vmp1 i 11 različitih stamp genotipova. Na osnovu sveobuhvatne tuf/vmp1/stamp
genotipizacije utvrđeno je ukupno 12 genotipova stolbur fitoplazme. Najveći diverzitet
genotipova je identifikovan među izolatima iz H. obsoletus sakupljenim na U. dioica, od
kojih je najčešće bio prisutan genotip tuf-ab/V18/M1 (43%). Tuf-b/V2-TA/STOL genotip
je utvrđen u 33% prirodno inficiranih biljaka vinove loze. Dva genotipa stolbur fitoplazme
povezana su sa U. dioica: tuf-ab/V18/M1 (60%) i tuf-a/V3/M4 (40%), dok je samo jedan
genotip (tuf-b/V2-TA/Rqg50) povezan sa C. arvensis. | Bois noir (BN), induced by stolbur phytoplasma, is an important grapevine yellows disease
that causes severe damage in viticultural regions throughout the Euro-Mediterranean basin.
Epidemiological survey to determine potential insect vectors and the main reservoir plants
of BN phytoplasma in Macedonian vineyards was undertaken between 2012 and 2013 in
southeastern part of the country. The study of the species diversity from the suborder
Auchenorrhycha revealed the presence of 27 species, belonging to 6 families. The most
numerous was family Cicadellidae with 21 species, followed by family Cixiidae with 2
species, Aphrophoridae, Delphacidae, Dyctiopharidae and Issidae with only one species
recorded. Our study revealed the high abundance of the principal vector of stolbur
phytoplasma, the planthopper Hyalesthes obsoletus, while the second documented vector of
BN, Reptalus panzeri, was not recorded in Macedonian vineyards. A molecular
characterization performed by sequence and/or RFLP typing of tuf, vmp1 and stamp genes,
was used to gain detailed insight into the molecular diversity of the stolbur phytoplasma
isolates associated with grapevine, tentative reservoir plants (Urtica dioica and
Convolvulus arvensis) and H. obsoletus associated with them. Among 91 stolbur isolates
detected in diverse plant and insect hosts 3 tuf, 5 vmp1 and 11 distinct stamp genotypes
were identified. Overall, twelve comprehensive genotypes of stolbur phytoplasma were
detected according to tuf/vmp1/stamp genotyping. The highest diversity of genotypes was
detected among the isolates from H. obsoletus associated with U. dioica, of which the most
frequent genotype was tuf-ab/V18/M1 (43%). Tuf-b/V2-TA/STOL comprehensive
genotype was found in 33% of naturally infected grapevines. Two stolbur phytoplasma
genotypes were associated with U. dioica: (i) tuf-ab/V18/M1 (60%) and tuf-a/V3/M4
(40%), while only one genotype (tuf-b/V2-TA/Rqg50) was associated with C. arvensis. | true |
Ekspresija i varijante gena za tumorski supresor VHL u papilarnim karcinomima štitaste žlezde | Expression and gene variants of VHL tumour supressor in papillary thyroid carcinoma | Papilarni karcinomi štitaste žlezde (PTC) predstavljaju najučestalije karcinome
endokrinog porekla. Poslednjih decenija, incidenca ovih karcinoma je u stalnom porastu
zbog čega je identifikacija molekularnih markera koji bi doprineli preciznijoj
klasifikaciji bolesnika sa rizikom za progresiju bolesti, a u cilju izbora terapijskog
pristupa, od izuzetnog kliničkog značaja. Gen VHL uključen je u tumorigenezu različitih
tipova karcinoma i u mnogima od njih je povezan sa agresivnijim biološkim
ponašanjem. Uloga i značaj VHL u patogenezi i progresiji PTC do sada nisu ispitivani.
Predmet ove studije bio je ispitivanje nivoa ekspresije VHL u PTC. Ekspresioni profili
VHL iRNK i proteina određeni su metodama PCR u realnom vremenu i
imunohistohemijom. Istovremeno, ispitano je da li su mutacioni događaji u kodirajućoj
sekvenci gena i regulacija koja se odvija na post-transkripcionom nivou a posredovana
je miR-92a, odgovorni za detektovane različite nivoe ekspresije VHL. Sekvenciranjem
sva tri egzona gena VHL nisu detektovane mutacione promene. Nivo ekspresije miR-
92a, iako izmenjen, nije značajno korelisao sa nivoima ekspresije VHL iRNK i proteina.
Ovi rezultati ukazuju na to da su za promene u ekspresiji VHL verovatno odgovorni
neki drugi regulatorni mehanizmi. U odnosu na nivo ekspresije VHL iRNK u PTC i
odgovarajućem neizmenjenom tireoidnom tkivu, utvrđeno je da postoje dve grupe
bolesnika – oni sa sniženom i oni sa povišenom ekspresijom VHL u tumorskom tkivu.
Detektovane promene ekspresionog profila VHL u korelaciji su sa kliničkim i
patohistološkim parametrima PTC. Takođe, utvrđeno je da je niska ekspresija VHL
iRNK povezana sa kliničkim i patohistološkim karakteristikama koje ukazuju na
agresivnije ponašanje tumora, kao i da je povezana sa kraćim trajanjem vremenskog
perioda bez znakova bolesti. Rezultati ove studije sugerišu da bi evaluacija ekspresije
VHL tumorskog supresora mogla imati klinički značaj u prognozi agresivnijeg toka
bolesti kod bolesnika sa PTC. | Papillary thyroid carcinomas (PTC) represent the most prevalent type of endocrine
malignancy. Over the last decades their incidence has been increasing, underlining the
significance of the identification of molecular markers which would improve the risk
stratification and help optimize the therapeutic approach to PTC patients. While the
VHL gene is implicated in tumorigenesis of different types of carcinoma and has been
reported to be associated with more aggressive biological behavior, its importance in the
patogenesis and progression of PTC has not been explored so far. The subject of this
study was to investigate the expression level of VHL in this type of carcinoma. The
expression profiles of VHL mRNA and protein were determined using real time PCR
and immunohistochemistry. In addition, it was investigated whether mutations in the
coding sequence and post-transcriptional expression regulation mediated with miR-92a
were responsible for VHL expression alterations detected in this study. No mutations
were detected by sequencing of all three exons of the VHL gene. The expression level of
miR-92a, although altered, did not show significant correlation with VHL mRNA and
protein levels. These results suggest that probably some other regulatory mechanisms
are responsible for VHL expression alterations. The analysis of VHL mRNA expression
levels in PTC tissues compared to matched non-tumour tissues revealed that there were
two groups of patients – patients with a decreased VHL expression level and patients
with an increased expression level in tumour tissue. The detected VHL expression
profile alterations were in correlation with PTC clinicopathological parameters.
Moreover, low VHL expression was associated with more aggressive tumour features
and with a shorter disease free interval. The results of this study suggest that evaluation
of VHL tumor suppressor expression level might have significance in the prognosis of
more aggressive course of the disease in PTC patients. | true |
Uticaj različitih ekstrakata krastavaca (Cucumis sativus L. cv. Chinese long) na encefalitogeni potencijal T limfocita pacova u eksperimentalnom autoimunskom encealomijelitisu | Effects of different Cucumber exstracts (Cucumis sativus L. cv. Chinese long) on the encephalitogenic potential of T lymphocytes in experimental autoimmune encephalomyelitis | Eksperimentalni autoimunski encefalomijelitis (EAE) je model multiple skleroze (MS),
hronične inflamatorne bolesti centralnog nervnog sistema (CNS). Osnovne patogene
populacije u EAE su efektorski T limfociti koji produkuju interferon-γ (Th1) ili
interleukin-17 (Th17) i aktivirani makrofagi koji dovode do oštećenja CNS. Imajući u
vidu da krastavac (Cucumis sativus) predstavlja bogat izvor sekundarnih metabolita kao
što su polifenoli i kukurbitacini, u ovoj studiji je po prvi put ispitivano dejstvo različitih
ekstrakata krastavca na encefalitogeni potencijal T limfocita, efektorske funkcije
makrofaga i antigen-prezentujuću aktivnost dendritskih ćelija (DĆ). Rezultati su
pokazali da ekstrakt lista krastavca (ELK) ostvaruje najpotentnije dejstvo, inhibirajući
produkciju IFN-γ i IL-17 od strane encefalitogenih T limfocita, modulišući signalne
puteve Nf-κB i p38 (MAPK). Pored toga, ELK je ostvario inhibitorno dejstvo na
efektorske funkcije makrofaga i antigen-prezentujuću aktivnost DĆ u koncentracijama
koje nisu pokazale citotoksičnost na embrione zebrice (Danio rerio) in vivo. Dalje, ELK
je uspešno inhibirao encefalitogene T limfocite nakon in vivo primene u EAE.
Kvantitativna i kvalitativna analiza ELK metodom masene spektrometrije potvrdila je
prisustvo 37 različitih jedinjenja od kojih su kukurbitacini bili najzastupljenija
jedinjenja. Shodno tome, ispitana su i imunomodulacijska dejstva kukurbitacina B i
kukurbitacina E na glavnim imunskim ćelijama uključenim u patogenezu EAE. Ova
ispitivanja su potvrdila dejstva ELK, sugerišući da su kukurbitacini, kao glavni
konstituenti ovog ekstrakta, barem delimično odgovorni za ostvarena dejstva ELK.
Rezultati ove doktorske disertacije ukazuju na to da ekstrakt lista krastavca i
kukurbitacini ostvaruju snažne antiencefalitogene efekte u modelu EAE i ukazuju na
važnost dodatnih istraživanja u cilju pronalaženja odgovarajućih tretmana autoimunosti
CNS. | Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple
sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). T
lymphocytes, which produce interferon-γ (Th1) or interleukin-17 (Th17), and activated
macrophages, that lead to CNS damage, are the major pathogenic cell populations in
EAE. Having in mind that cucumber (Cucumis sativus) is a rich source of secondary
metabolites such as polyphenols and cucurbitacins, in this study, for the first time, the
effects of various cucumber extracts were investigated on encephalytogenic T
lymphocytes, effector function of macrophages and antigen-presenting activities of
dendritic cells (DC). The results show that cucumber leaf extract (CLE) expresses the
most potent effect by inhibiting the production of IFN-γ and IL-17 in encephalytogenic
T lymphocytes, through modulation of signaling pathways Nf-κB and p38 (MAPK).
Additionally, CLE imposes an inhibitory effect on the effector functions of
macrophages and antigen-presenting activity of DC in concentrations that do not show
cytotoxicity in zebrafish (Danio rerio) in vivo. Also, CLE inhibits generation of
encephalitogenic cells in vivo. Phytochemical analysis of CLE, characterized by mass
spectrometry, confirmed the presence of 37 different compounds, among which
cucurbitacins were the most abundant compounds. Hence, the immunomodulatory
effects of cucurbitacin B and cucurbitacin E were also examined on the major immune
cells involved in EAE pathogenesis. The obtained results confirm the effects of CLE,
suggesting that cucurbitacins are, at least partially, responsible for its effects. The
results of this doctoral dissertation indicate that the cucumber leaf extract and its major
constituents cucurbitacins have anti-encephalitogenic effects in the EAE model,
Moreover, they suggest the importance of additional research towards novel highly
efficient CNS autoimmunity treatments. | true |
Ispitivanje antitumorskog delovanja 6-[trifenilstanil]-1-heksanola vezanog za mezoporozni nanomaterijal SBA-15 u modelu mišjeg melanoma in vitro i in vivo | Evaluation of antitumor activity of 6-(triphenylstannyl)hexan-1-ol loaded in SBA-15 mesoporous nanostructured material in the mouse model of melanoma in vitro and in vivo | Uprkos razvoju imunoterapije i ciljanih terapija, globalna incidenca melanoma nastavlja
da raste. Konvencionalna hemoterapija, naţalost, pokazuje ograniĉenu efikasnost u
leĉenju metastatskog melanoma. Primenu najĉešće korišćenog hemoterapeutskog leka
cisplatine, ograniĉava uroĊena rezistencija ovog tipa tumora, što sve naglašava potrebu
za razvojem efikasnije hemoterapije melanoma. Progres u medicinskoj organometalnoj
hemiji omogućio je racionalni dizajn novih, nekonvencionalnih platinskih kompleksa,
kao i neplatinskih antitumorskih jedinjenja, ukljuĉujući organokalajna jedinjenja, ĉija
aktivnost ne poĉiva na direktnoj interakciji sa DNK. MeĊutim, nedovoljna rastvorljivost
i toksiĉnost organokalajnih jedinjenja ograniĉava kompleksnija istraţivanja i ulazak u
kliniĉka ispitivanja. U tom kontekstu, primena nanotehnologije u svrhu povećanja
rastvorljivosti lekova i njihove selektivnosti prema maligno transformisanim klonovima
u hemoterapiji kancera ima vodeću ulogu. Inkapsulacija hidrofobnih antitumorskih
lekova u mezoporozne nanomaterijale silike, predstavlja najperspektivniju strategiju u
ovoj oblasti. Zbog visokog potencijala za adsorpciju, nosaĉ SBA-15 je najinteresantniji
mezoporozni silikat za ciljanu isporuku lekova. U skladu sa tim, u ovoj studiji je po prvi
put ispitivan antitumorski potencijal organokalajnog(IV) jedinjenja [Ph3Sn(CH2)6OH]
skladištenog u mezoporozni materijal SBA-15 (SBA-15pSn) in vitro i in vivo u modelu
mišjeg melanoma. Imajući u vidu izuzetnu heterogenost melanoma, ispitivan je i njihov
uticaj na visoko invazivnu liniju humanog melanoma, A375. Analizirani su ćelijski i
molekulski mehanizmi pokrenuti u odgovoru na tretman eksperimentalnim agensima u
ćelijama melanoma, kao i mehanizam preuzimanja ĉestica SBA-15pSn u humane ćelije
melanoma u sistemu in vitro.
[Ph3Sn(CH2)6OH] u slobodnoj formi ali, mnogo potentnije, uskladišten u SBA-15
smanjio je vijabilitet ćelijskih linija melanoma poreklom od miša B16, i ĉoveka A375.
Isti trend je zapaţen u singenom modelu mišjeg melanoma in vivo. Pokazalo se da
A375 ćelije melanoma preuzimaju SBA-15pSn pasivnim putem i makropinocitozom. U
osnovi antitumorskog delovanja eksperimentalnih jedinjenja bila je indukcija apoptoze i
zastoj u proliferaciji ćelija melanoma. MeĊutim, [Ph3Sn(CH2)6OH] skladišten u SBA-
15, ali ne i u slobodnoj formi, aktivira proces melanogeneze u B16 ćelijama melanoma,
koji je bio praćen indukcijom prevremene ćelijske senescencije. Senescentni fenotip
B16 ćelija, bio je povezan sa aktivacijom signalnog puta mitogenom-aktivirane protein
kinaze p38 (p38 MAPK), dok je povećanje ekspresije markera diferencijacije mišjih
ćelija melanoma bilo udruţeno sa trajnom inhibicijom signalnog puta fosfatidilinozitol
3-kinaze (PI3K). Paralelno, proces senescencije pokrenut SBA-15pSn u A375 ćelijama,
bio je usklaĊen sa njihovom morfološkom transformacijom i povećanom zastupljenošću
subpopulacije koja eksprimira fenotipski marker najranijih stadijuma razvića Švanovih
ćelija - fenomen koji odlikuje terminalno diferentovane melanocite u nevusima. Analiza
Wnt/β-katenin i Notch signalnih puteva, glavnih regulatora migracije melanocita tokom
razvića, koji su ĉesto reaktivirani u melanomu, je ukazala da je izmenjeni fenotip A375
ćelija tretiranih SBA-15pSn bio u korelaciji sa oslabljenim metastatskim potencijalom
humanih ćelija melanoma.
U celini, rezultati ove disertacije pokazuju da skladištenje hidrofobnih organokalajnih
jedinjenja u ureĊene mezoporozne nanoĉestice silike unapreĊuje njihova svojstva, ĉime
je omogućen nastavak prekliniĉkih ispitivanja sa krajnjim ciljem kreiranja uspešnije
terapije melanoma. | Despite rapid development in immunotherapy and targeted therapies, the global
incidence of melanoma continues to increase. Besides, conventional chemotherapy
demonstrates only limited efficacy in the treatment of advanced melanoma. Clinical
application of widely used chemotherapeutic drug cisplatin, is hampered by the intrinsic
resistance of this type of tumor, indicating the urgent need for the development of more
effective chemotherapies for melanoma. The rapid progress in medicinal organometallic
chemistry in the past few years allows rational design of novel platinum compounds, as
well as nonplatinum metal-based antitumor agents, including organotin compounds,
whose activity is not based on unrepairable interaction with DNA. Poor solubility and
toxicity of organotin compounds, however, restrains more complex studies and further
development in the preclinical and subsequently clinical setting. Nanotechnology now
becomes an important part of pharmaceutical research and development, for improving
the solubility and selective toxicity of cancer therapeutics. Encapsulating hydrophobic
antitumor agents in ordered mesoporous silica nanomaterials is an emerging strategy to
enhance drug dissolution. Due to its high drug loading capacity, SBA-15 is the most
interesting mesoporous silicate for targeted drug delivery. Antitumor potential of
organotin(IV) compound [Ph3Sn(CH2)6OH] grafted on nanostructured material SBA-15
(SBA-15pSn) in vitro and in vivo in the mouse model of melanoma, was tested for the
first time in this study. Considering the enormous heterogeneity of this type of tumor, it
was of interest to test these experimental therapeutics in a highly invasive human A375
melanoma cell line. Given the fact that SBA-15pSn nanoparticles are relatively large,
the mechanism of cellular uptake was defined. At the molecular level, main intracellular
events triggered by the treatment, as well as the expression of key signaling pathways
mediating tumor cell response, were analyzed.
[Ph3Sn(CH2)6OH], as a free compound, but more potently when grafted on SBA-15
material, reduced the growth of B16 and A375 melanoma cell lines in vitro. Tested
compounds inhibited B16 melanoma tumor growth in syngeneic C57BL/6 mice. SBA-
15pSn particles were taken up by macropinocytosis and nonspecific cellular uptake in
A375 melanoma cells. [Ph3Sn(CH2)6OH] and SBA-15pSn triggered caspase dependent
apoptosis and cell cycle arrest in both melanoma cell lines. However, [Ph3Sn(CH2)6OH]
grafted on SBA-15, but not as a free compound, stimulated melanin synthesis in B16
cells, followed by induction of premature cellular senescence, associated with activation
of p38 signaling pathway. High expression of differentiation markers in SBA-15pSn
treated B16 cells, was well synchronized with permanent inhibition of PI3K signaling
pathway. In parallel, senescence triggered by SBA-15pSn in A375 melanoma cells, was
associated with morphological transformation and enhanced percentage of Schwann-
like cell subpopulation. This molecular signature characterizes terminally differentiated
melanocytic nevus cells in the dermis. Downregulation of Wnt/β-catenin and Notch
signaling pathways, critical regulators of melanocyte lineage development, which are
often reactivated in melanoma, indicates that morphological changes of SBA-15pSn
treated A375 cells correlated with lower metastatic potential of human melanoma cells.
Our findings demonstrate that encapsulating cytotoxic antitumor metal-based drugs in
ordered mesoporous silica nanoparticles is a promising targeting strategy in an effort to
diminish deleterious side-effects of chemotherapy and a creative approach to the design
of novel anticancer therapeutics. | true |
Uloga Sonic Hedgehog signalnog puta u regulaciji ekspresije SOX18 gena u HeLa ćelijama, kao model sistemu karcinoma grlića materice | The role of Sonic Hedgehog signaling pathway in the regulation of SOX18 gene expression in HeLa cells, model system of cervical carcinoma | Sonic Hedgehog (SHH) signalni put ima važnu ulogu u procesima koji se
odvijaju tokom embrionalnog razvića u kojima kontroliše proliferaciju i diferencijaciju
ćelija i učestvuje u održavanju polarnosti tkiva. Poslednjih godina mnogobrojni
literaturni podaci pokazuju da promena u regulaciji SHH signalnog puta dovodi do
nastanka i progresije različitih vrsta tumora kod čoveka. SHH signalni put je povezan sa
meduloblastomom, leukemijom, karcinomom bazalnih ćelija, tumorima pluća, prostate,
pancreasa, dojke i jajnika. Takođe, povećana ekspresija komponenti SHH signalnog
puta je primećena u premalignim lezijama i ćelijama karcinoma grlića materice.
SOX18 gen pripada familiji SOX gena koji kodiraju transkripcione faktore
uključene u kontrolu različitih procesa tokom embrionalnog razvića. SOX18 protein
ima važnu ulogu u razviću vaskularnog sistema kao i u adultnoj neovaskularizaciji.
Uloga SOX18 proteina u vaskularnom razviću otkrivena je na osnovu poremećaja u
razvoju vaskularnog sistema koji su uočeni kod prirodnih mutanata miša i čoveka. Osim
toga, kod adultnog organizma SOX18 protein učestvuje u regulaciji angiogeneze i
limfangiogeneze kako u fiziološkim stanjima organizma, tako i tokom patofizioloških
promena kao što su zarastanje rana i tumorska angiogeneza. Takođe, pokazano je da se
inhibicijom SOX18 funkcije utiče na limfangiogenezu čime se sprečava metastaza ćelija
tumora. Najnoviji podaci pokazuju da je ekspresija SOX18 gena, detektovana u ćelijama
invazivnog karcinoma dojke i tumora jajnika, a nivo ekspresije je u direktnoj korelaciji
sa stadijumom tumora i može predstavljati prognostički marker.
Predmet istraživanja prikazanog u ovom radu je bila analiza uloge SHH
signalnog puta u regulaciji ekspresije SOX18 gena u HeLa ćelijama, in vitro model
sistemu karcinoma grlića materice. Prvo je analizirana uloga GLI regulatornih proteina,
finalnih efektora SHH signalne kaskade, u regulaciji transkripcije SOX18 gena.
Prikazani rezultati su pokazali da su GLI1 i GLI2 pozitivni regulatori promotorske
aktivnosti SOX18 gena kao i da povećavaju endogenu ekspresiju SOX18 gena, dok
GLI3 transkripcioni faktor nije uticao na aktivnost SOX18 promotorskog konstrukta niti
na nivo endogene ekspresije SOX18 gena u HeLa ćelijama. U eksperimentima smanjene
elektroforetske pokretljivosti pokazano je da se GLI1 transkripcioni faktor specifično
vezuje za tri od ukupno sedam identifikovanih potencijalnih vezivnih mesta za GLI
proteine u okviru promotora SOX18 gena. Rezultati su takođe pokazali da promena
aktivnosti SHH signalnog puta upotrebom specifičnih aktivatora i inhibitora dovodi do
promena u nivou proliferacije, vijabilnosti i migracije HeLa ćelija. Najbitnije,
modulacija aktivnosti SHH signalnog puta dovodi do promena u nivou ekspresije
SOX18 gena, tako što se nivo ekspresije smanjuje tokom inhibicije SHH signalne
kaskade, odnosno povećava tokom njegove aktivacije. Osim toga, u radu je analiziran
uticaj povećane ekspresije SOX18 gena u HeLa ćelijama i pokazano je da nema uticaja
na proliferaciju i vijabilnost, dok je zapažen značajan uticaj na migraciju HeLa ćelija.
Takođe, rezultati pokazuju da SOX18 smanjuje ekspresiju GLI1, GLI2, GLI3
transkripcionih faktora i SHH proteina.
SOX18 gen je identifikovan kao novi ciljni gen čiju ekspresiju reguliše SHH
signalna kaskada u HeLa ćelijama. Prikazani rezultati omogućiće bolje razumevanje
molekularnih mehanizama uključenih u regulaciju transkripcije SOX18 gena. Na osnovu
prikazanih rezultata možemo zaključiti da njegova uloga u HeLa ćelijama može biti u
regulisanju procesa migracije gde pokazuje promigratorni potencijal. Modulacija
aktivnosti SHH signalnog puta malim, aktivnim molekulima za posledicu ima i
promene u regulaciji SOX18 ekspresije, čime se otvara mogućnost za potencijalnu
farmakološku manipulaciju ekspresijom SOX18 gena u tumorskim ćelijama. | The Sonic Hedgehog (SHH) signaling pathway plays important role in
embrionic development directing cell proliferation and diferentiation and maintaing
tissue polarity. In adults, this signaling pathway is rather suppressed. SHH signaling
pathway has been associated with medulloblastoma, leukemia, basal cell carcinoma and
lung, prostate, pancreatic, breast and ovarian cancers. In the last years, numerous data
show that deregulation of SHH signaling pathway has been associated with onset and
progression of various types of human cancer. It was shown that increased expression of
HH-signaling molecules was seen in precancerous lesions and in cervical cancer.
SOX18 gene is a member of SOX gene family that encodes transcription factors
implicated in the control of various developmental processes. SOX18 protein plays
important roles in vascular development and postnatal neovascularization. The
functional importance of SOX18 protein in vascular development is revealed by the
vascular defects caused by mutation in mice and humans. Also, in adults SOX18 is
involved in the regulation of angiogenesis and lymphangiogenesis in physiological and
pathophysiological condition such in wound healing or tumor growth. Furthermore, it
has been reported that interfere with SOX18 function impairs tumor lymphangiogenesis
which decreases the rate of cancer cell metastasis. In past two years, literature data show
SOX18 expression in invasive ductual breast carcinoma and ovarian carcinoma and that
level of expression correlates with poor prognosis suggesting that a SOX18 expression
may serve as a prognostic marker.
The aim of this study was to investigate the role of Sonic Hedgehog signaling
pathway in the regulation of SOX18 gene expression in HeLa cells, in vitro model
system of cervical cancer . We were analyzing the effect of GLI regulator proteins, final
effectors of SHH signaling pathway, on transcriptional regulation of SOX18 gene
expression. Presented results have shown that GLI1 and GLI2 are potent activators of
SOX18 promoter activity as well as SOX18 expression in HeLa cells, while GLI3 had no
effect. By in vitro binding assays we show binding of GLI1 protein to three out of seven
predicted binding sites within SOX18 promoter. Presented results also show that
modulation of pathway using inhibitors and activators of SHH signaling pathway
affected cell proliferation, viability and migration of HeLa cells. More importantly,
modulation of SHH signaling pathway affected SOX18 gene expression, presenting that
cyclopamine thretman of HeLa cells decreased SOX18 gene expression while
purmorphamine thretman of HeLa cells increased SOX18 gene expression. Furthermore,
we have shown that overexpression of SOX18 caused no significant change in cell
proliferation and viability while had considerable effect on HeLa cell’s migratory
potential. Also, SOX18 overexpression resulted in down regulation of GLI1, GLI2,
GLI3 and SHH gene expression.
SOX18 was identified as a novel target gene regulated by SHH signaling in
HeLa cells. Presented results will help in better understanding of molecular mechanisms
involved in transcriptional regulation of SOX18 gene expression. Presented results also
show that SOX18 is involved in the regulation of HeLa cells migration. Applying small
molecules inhibitors and activators for SHH signaling, it was possible to affect SOX18
gene expression, opening a new possibilities for pharmacological manipulation with
SOX18 gene expression in cancer cells. | true |
Povezanost ekspresije PTEN proteina i prognoze bolesti kod žena obolelih od ranog karcinoma dojke lečenih ovarijalnom ablacijom | The relationship between PTEN protein expression and prognosis in women with early breast cancer treated with ovarian ablation | Sve pacijentkinje sa detektabilnom ekspresijom hormonskih receptora (HR) za
karcinom dojke (KD) trebalo bi da se leče endokrinom terapijom (ET). Za
premenopauzne pacijentkinje, tamoksifen i/ili ovarijalna supresija (OvS) su adekvatna
terapija. Promene u fosfataza i tenzin homolog (PTEN) signalnom putu bi mogle da
budu jedan od mogućih mehanizama rezistencije na antiestrogenu terapiju. Cilj ovog
istraživanja je ispitivanje povezanosti ekspresije PTEN proteina sa prognostičkim
faktorima [(histologija i gradus tumora, status receptora za estrogen (ER) i receptora za
progesteron (PgR), status receptora za humani epidermalni faktor rasta (HER2)] i sa
odgovorom na terapiju kod premenopauznih pacijentkinja sa HR pozitivnim ranim KD
lečenih adjuvantnom ovarijalnom ablacijom (OA).
Pacijenti i metode: Analizirali smo grupu premenopauznih pacijentkinja sa ranim
(stadijum I/II) HR pozitivnim KD koji su lečeni radikalnom mastektomijom a zatim
adjuvantnom terapijom u vidu OA. Sadržaji ER i PgR su određeni klasičnom
biohemijskom metodom [engl. Classical biochemical dextrane-coated charcoal (DCC)
method], HER2 status je određen hromogenom in situ hibridizacijom (CISH metoda) i
PTEN status je određen imunohistohemijskom metodom (IHH).
Rezultati: U ovu analizu je uključeno 66 premenopauznih pacijentkinja. Medijana
praćenja je 17 godina (opseg 1-29 godina). U poređenju sa PTEN pozitivnim KD,
PTEN negativni KD su bili značajno više povezani sa lobularnim histološkim tipovima
tumora (p<0.05) i sa višim sadržajem ER (p<0.05). Pacijentkinje sa PTEN negativnim
KD su imali značajno kraće preživljavanje bez progresije bolesti (DFS) (p<0.01) i
ukupno preživljavanje (OS) (p<0.01) u poređenju sa pacijentkinjama sa PTEN
pozitivnim KD.
Zaključak: Naši rezultati mogu da znače da PTEN status određen kao ekspresija PTEN
proteina može da razdvoji premenopauzne HR pozitivne pacijentkinje sa KD lečene OA,
na podgrupe sa dobrom i lošom prognozom bolesti. | All breast cancer (BC) patients with detectable hormone receptors (HR)
expression should be offered endocrine therapy (ET). For premenopausal patients,
tamoxifen and/or ovarian suppression (OvS)/ ablation (OA) may improve disease
outcome. Alteration of phosphatase and tensin homolog (PTEN) signaling pathway
could be one of possible mechanisms of resistance to antiestrogen therapy. The aim of
this study was to investigate the association of PTEN protein expression with prognostic
factors [tumor histology and grade, estrogen receptor (ER) and progesterone receptor
(PgR) status, human epidermal growth factor receptor 2 (HER2) status] and disease
outcome in premenopausal patients with HR-positive early BCs treated with adjuvant
OA.
Methods: We analyzed a group of premenopausal early (stages I/II) HR-pos BC
patients who had undergone radical mastectomy followed with adjuvant OA by
irradiation only. ER and PgR contents were determined by classical biochemical
dextrane-coated charcoal (DCC) method, HER2 status by chromogen in situ
hybridization (CISH) analysis and PTEN status by immunohistochemistry (IHC).
Results: Sixty-six premenopausal patients included into this analysis were followed for
a median of 17 years (range 1-29). Compared to PTEN-pos BCs, PTEN-neg BCs were
significantly more frequently associated with lobular tumor histology (p<0.05) and a
higher ER content (p<0.05). Patients with PTEN-neg BC had significantly decreased
disease-free survival (DFS) and overall survival (OS) (p<0.01 for both) compared to
patients with PTEN-pos BCs.
Conclusions: It seems that PTEN status determined by protein expression may
discriminate between subgroups with poor and good prognosis in premenopausal HR-
pos BC patients receiving adjuvant OA. | true |
Efekti fluoksetina i klozapina na antioksidativni sistem i parametre inflamacije u mozgu i jetri pacova u uslovima hronične izolacije | The effects of fluoxetine and clozapine on antioxidative system and inflammation parameters in the brain and liver of chronically isolated rats | Depresija je visokoprevalentno, multifaktorijalno i heterogeno psihijatrijsko
oboljenje kompleksne neurobiološke osnove. S obzirom da veliki broj pacijenata (30–
40%) ne postiže remisiju bolesti uprkos antidepresivnoj terapiji, patofiziologija
depresije i mehanizmi delovanja antidepresiva i danas su aktuelne problematike iako se
intenzivno istražuju decenijama unazad. Brojne studije ukazuju da hronični
psihosocijalni stres, vodeći sredinski faktor rizika za razvoj depresije, izaziva
oksidativni stres i inflamaciju u mozgu, procese koji imaju važnu ulogu u
etiopatofiziologiji ovog oboljenja. Pored toga, sve je više podataka koji pokazuju da je
disregulacija γ-aminobuterna kiselina (GABA) sistema u mozgu povezana sa
depresijom.
U radu su analizirani efekti antidepresiva fluoksetina i atipičnog antipsihotika
klozapina na glutation (GSH)-zavisni antioksidativni sistem i parametre inflamacije u
hipokampusu i prečeonoj zoni kore cerebruma, moždanim regionima posebno osetljivim
na stres. Takođe, ispitan je uticaj ovih lekova na GABA signalizaciju u medijalnoj
prečeonoj zoni kore cerebruma, kao i njihova hepatotoksičnost. Ovi efekti su istraživani
na životinjskom modelu depresije koji se zasniva na hroničnom izlaganju
psihosocijalnom stresoru. Odrasli mužjaci pacova Wistar soja su podvrgavani 21-
dnevnoj izolaciji, a fluoksetin i klozapin su davani stresiranim pacovima tokom
izolacije, kao i nestresiranim (kontrolnim) pacovima u periodu od 21 dana.
Hronična izolacija je dovela do promena u ponašanju koje nalikuju depresivnom
i anksioznom, kompromitovala antioksidativnu odbranu posredovanu glutation
peroksidazom i izazvala porast nivoa faktora nekroze tumora α (TNF-α) u
hipokampusu. Takođe, izolacija je uzrokovala smanjenje koncentracije GSH, aktivaciju
nuklearnog faktora-κB i porast nivoa medijatora inflamacije ciklooksigenaze-2 (COX-
2), interleukina-1β i TNF-α u prečeonoj zoni kore cerebruma. Fluoksetin i klozapin su
onemogućili razvoj promena u ponašanju i proinflamatorne procese u obe ispitivane
moždane strukture kod izolovanih pacova. Što se tiče GSH-zavisnog sistema, fluoksetin
je pokazao protektivni efekat ali samo u prečeonoj zoni kore cerebruma pacova. Ovi
rezultati pokazuju ulogu proinflamatornih molekularnih promena u razvoju simptoma
depresije, kao i antiinflamatornih efekata fluoksetina i klozapina u sprečavanju istih.
Uticaj izolacije, kao i potencijalni protektivni efekti fluoksetina i klozapina na
broj parvalbumin pozitivnih (PV+) ćelija ispitani su u medijalnoj prečeonoj zoni kore
cerebruma, moždanom regionu za koji je pokazano da je veoma značajan sa aspekta
razvoja simptoma depresije. Utvrđeno je da je izolacija dovela do značajnog smanjenja
broja PV+ ćelija u cingulatnoj kori 1, prelimbičkom (PrL) i infralimbičkom (IL)
podregionu, kao i dorzalnoj pedunkularnoj kori medijalne prečeone zone kore
cerebruma. Fluoksetin i klozapin su onemogućili izolacijom izazvano smanjenje u PrL i
IL, što ukazuje na značaj koji GABA signalizacija u ovim podregionima ima za
promene ponašanja u uslovima hronične izolacije.
Hronični tretmani fluoksetinom i klozapinom su doveli do oksidativnih
oštećenja proteina i lipida u jetri, kako izolovanih, tako i nestresiranih pacova.
Oksidativna oštećenja su uočena i u jetri izolovanih pacova koji nisu tretirani lekom. U
jetri ovih pacova je uočena povećana proizvodnja azot-monoksida i COX-2, koja je
onemogućena hroničnim tretmanom fluoksetinom. Fluoksetin nije značajno uticao na
strukturu tkiva jetre za razliku od klozapina koji je izazvao brojne makrovezikularne
masne promene i fokalne nekroze, naročito kod izolovanih pacova.
Studija opisuje molekularne promene uključene u patofiziologiju stanja nalik
depresiji hronično izolovanih pacova. Te promene obuhvataju kompromitovanje GSH-
zavisnog sistema, povećanje ekspresije medijatora inflamacije u hipokampusu i
prečeonoj zoni kore cerebruma, kao i smanjenje broja PV+ ćelija u medijalnoj
prečeonoj zoni kore cerebruma. Takođe, studija proširuje znanja o mehanizmima
delovanja fluoksetina i klozapina izvan okvira serotoninske i dopaminske signalizacije.
Antidepresivni i anksiolitički efekti ovih lekova praćeni su inhibicijom proinflamatornih
promena u obe moždane strukture, kao i prevencijom smanjenja broja PV+ ćelija u PrL
i IL podregionima prečeone zone kore cerebruma pacova u uslovima hronične izolacije.
Rezultati studije jasno ukazuju i da klozapin ispoljava više štetnih efekata na jetru od
fluoksetina. | Depression is a highly prevalent, multifactorial and heterogeneous disorder with
complex neurobiology. Large numbers of patients (30–40%) do not achieve disease
remission despite receiving antidepressive therapy. Thus, despite the decades of
research, the pathophysiology of depression and mechanisms of antidepressants’ actions
are ongoing and widespread issues. A growing body of evidence indicates that
psychosocial stress, the leading environmental risk factor, causes oxidative stress and
inflammation in the brain and that these processes have important role in the
pathophysiology of depression. In addition, dysregulation of γ-aminobutyric acid
(GABA)-ergic system is becoming increasingly associated with this disorder.
In this study, we analyzed the effects of antidepressant fluoxetine and atypical
antipsychotic clozapine on the glutathione (GSH)-dependent antioxidative system and
parameters of inflammation in hippocampus and prefrontal cortex, brain regions
particularly sensitive to stress. Besides, we examined the effects of drugs on the GABA-
ergic signalization, as well as their hepatotoxicity. These effects were investigated in an
animal model of depression based on chronic psychosocial stress. Adult male Wistar
rats were isolated for 21 days and fluoxetine and clozapine were applied to the stressed
rats during isolation, as well as to non-stressed rats for 21 days.
Chronic isolation caused depressive- i anxiety-like behaviors, compromised
glutathione peroxidase-mediated antioxidative defense and caused increase in tumor
necrosis factor α (TNF-α) levels in hippocampus. Besides, isolation decreased GSH
levels, activated nuclear factor-κB and elevated cyclooxygenase-2 (COX-2),
interleukin-1β i TNF-α levels in prefrontal cortex. Fluoxetine and clozapine prevented
behavioral changes and proinflammatory events in both examined brain structures in
isolated rats. Regarding GSH-dependent defense, fluoxetine showed protective effects,
which were restricted to the prefrontal cortex. These results indicate the significant role
of proinflammatory molecular changes in the development of depressive symptoms, as
well as the importance of the anti-inflammatory properties of fluoxetine and clozapine
in their prevention.
The effects of isolation, as well as potential protective effects of fluoxetine and
clozapine on the number of parvalbumin positive (PV+) cells were examined in medial
prefrontal cortex, a brain region highly implicated in the development of depressive
symptoms. Decreased number of PV+ cells in cingulate cortex area 1, prelimbic area
(PrL), infralimbic area (IL) and dorsal peduncular cortex of medial prefrontal cortex
were found in isolated rats. Fluoxetine and clozapine prevented this effect in PrL and IL
subregions, which indicates the importance of intact GABA-ergic signalization in these
areas for resistance against isolation-induced behavioral changes.
Chronic fluoxetine and clozapine treatment caused oxidative damages of
proteins and lipids in the rat liver in both non-stressed and isolated rats. The chronic
isolation itself also provoked oxidative damage. Besides, isolation increased nitrogen
monoxide and COX-2 production in the liver, which were prevented by fluoxetine
treatment. Fluoxetine did not significantly affect the hepatic architecture while
clozapine caused numerous macrovesicular fatty changes and focal necrosis,
particularly in isolated rats.
This study describes molecular changes involved in the pathophysiology of
depressive-like state in chronically isolated rats. These changes include compromised
GSH-dependent defense, elevated expression of proinflammatory mediators in
hippocampus and prefrontal cortex, as well as decreased number of PV+ cells in medial
prefrontal cortex. Furthermore, this study expands the knowledge about the fluoxetine
and clozapine action beyond serotonergic and dopaminergic signaling. The
antidepressive and anxiolytic effects of these drugs paralleled the inhibition of
hippocampal and prefrontal cortical inflammation, as well as the prevention of decrease
in PV+ cells number in PrL and IL. Finally, results of this study reveal that clozapine is
more harmful for the liver, compared to fluoxetine. | true |
Vijabilnost populacija tekunice Spermophilus citellus (L., 1766) na području Srbije | Viability of European ground squirrel Spermophilus citellus (L., 1766) populations in Serbia | Evropska tekunica (Spermophilus citellus, L. 1766) je endemična vrsta za područje
Centralne i Jugoistočne Evrope. Recentni areal je karpatskim masivom podeljen na dva
dela. Severni deo se karakteriše uniformnim stepskim staništima koja se javljaju na malim
nadmorskim visinama, dok se južni deo areala nalazi na Balkanu, gde su prisutni različiti
tipovi staništa sa različitim ekološkim uslovima i na različitim nadmorskim visinama.
Najveći broj populacija na području Srbije nalazi se u njenom severnom delu, tj.
Vojvodini, dok je svega nekoliko malih i izolovanih planinskih populacija prisutno u
istočnom i jugoistočnom delu zemlje
Tekunice naseljavaju isključivo stepska staništa, livade i pašnjake koji su obrasli
niskom travom. Međutim, usled intenzivnog pretvaranja stepskih predela u poljoprivredno
zemljište tokom proteklih nekoliko decenija, nestala su široka prostranstva na kojima je
tekunica bila kontinuirano rasprostranjena. Danas vrsta opstaje samo na jako izolovanim
stepsko-livadskim fragmentima koji su sačuvani unutar dominantnog poljoprivrednog
matriksa.
S obzirom da broj populacija konstantno opada na celom arealu i da je budući
opstanak vrste doveden u opasnost, tekunica je od 2008. godine na IUCN Crvenoj listi
ugroženih vrsta svrstana u kategoriji ranjiva (VU). Zakonski status tekunice u Evropi
regulišu EU Direktiva o staništima (Aneks II i IV) i Konvencija o očuvanju evropske
divlje flore i faune i prirodnih staništa, poznatija kao Bernska konvencija (Aneks II).
Unutar mreže Natura 2000 do sada je u režim zaštite uključeno 414 staništa koja naseljava
tekunica. Na nacionalnom nivou tekunica je zaštićena zakonom kao strogo zaštićena
vrsta.
U cilju analize genetičkog diverziteta i strukture populacija tekunice na teritoriji
Srbije, u periodu 2007-2009. godine, prikupljeno je ukupno 180 uzoraka tkiva sa 11
lokaliteta (Krušedol, Neradin, Mala Remeta, Sremska Mitrovica, Banatska Palanka,
Šušara, Mokrin, Lok i Tomislavci sa teritorije Vojvodine i po jedan lokalitet sa Vlasine i
Stare planine). Procena gustine i brojnosti populacija tekunice vršena je u periodu 2004-
2014. godine na lokalitetima Krušedol i Neradin koji su smešteni na obodu fruškogorskog
masiva, kao i lokalitetu Banatska Palanka koji se nalazi na obodu Deliblatske peščare. Na
kraju je urađena procena minimalne vijabilne veličine populacije (MVP) uz pomoć
programa Vortex.
Rezultati dobijeni ovim istraživanjima nedvosmisleno ukazuju da je genetički
diverzitet populacija tekunice sa teritorije Vojvodine značajno viši u odnosu na one sa
periferije areala, kao i da je sličan genetičkom diverzitetu panonskih populacija iz
okruženja koje zauzimaju centralni položaj u okviru severne filogeografske grupe. Sa
druge strane, analize pokazuju da je genetički diverzitet planinskih populacija sa Vlasine i
Stare planine znatno niži u odnosu na populacije iz Vojvodine. Sve ispitivane populacije
tekunica iz Srbije su pokazale visok stepen genetičke diferencijacije, što ukazuje na
međusobnu izolovanost i odsustvo protoka gena između populacija.
Klaster analize su pokazale da je jedanaest ispitivanih populacija iz Srbije
najoptimalnije grupisati u četiri klastera. U prva tri klastera su raspoređene populacije iz
Vojvodine, dok su udaljene planinske populacije sa Vlasine i Stare planine izdvojene u
četvrti klaster. Raspored vojvođanskih populacija po klasterima ukazuje da Dunav, za
razliku od Tise, predstavlja značajnu barijeru za protok gena između posmatranih
populacija.
Gustina i brojnost posmatranih populacija tekunice su jako varirale tokom
posmatranog perioda od jedanaest godina i to kako na istom lokalitetu, tako i između
lokaliteta. Tokom posmatranog perioda pozitivan rast populacije registrovan je samo na
lokalitetu Neradin, dok su na lokalitetima Krušedol i Banatska Planka zabeležene velike
fluktuacije, kao i pad u brojnostima i gustinama ispitivanih populacija.
Analiza MVP je pokazala da je za opstanak izolovane populacije tekunica, uz
očuvanje 90 % genetičkog diverziteta i izbegavanje štetnog efekta inbridinga, neophodno
obezbediti prisustvo bar 300 jedinki, posmatrano za period od 50 godina. Međutim, sa
povećanjem demografske i sredinske varijabilnosti, veličina MVP se drastično povećava.
Ovo ukazuje na potrebu uspostavljanja što optimalnijih uslova na postojećim staništima
tekunice, čime bi se smanjile velike fluktuacije u brojnosti izazvane nepovoljnim
faktorima i povećala verovatnoća opstanka populacija.
Gubitak pogodnih staništa, kao i stvaranje barijera koje onemogućavaju migraciju
jedinki i protok gena između populacija, trenutno predstavljaju najznačajnije faktore koji
direktno i indirektno utiču na opstanak populacija tekunice.
Dobijeni rezultati predstavljaju dobru konzervacionu osnovu i daju smernice za
aktivan pristup zaštiti i očuvanju tekunice u Srbiji, izradu akcionog i plana upravljanja
populacijama tekunice u Srbiji, kao i osnovu za dalja fundamentalno-ekološka i
konzervaciona istraživanja ove krajnje ugrožene vrste kod nas. Buduće očuvanje tekunice
na našim prostorima nema samo nacionalni značaj. Naime, među populacijama od
najvećeg konzervaciong značaja za očuvanje evropske tekunice su upravo populacije sa
područja Bugarske i Srbije, tj. sa prostora koji je u više navrata u prošlosti služio kao
refugijum iz kog se vrsta tokom povoljnih klimatskih uslova širila ka Centralnoj Evropi.
Samim time imamo veliku obavezu očuvanja vijabilnih populacija tekunice uz minimalni
gubitak postojećeg genetičkog diverziteta. | The European ground squirrel (Spermophilus citellus, L. 1766) is endemic species
to the Central and Southeastern Europe. Its recent range is divided in two main parts by
the Carpathian Mountains. The northern part of the range is characterized by uniform
steppe habitats at low altitudes, while the southern part is situated on the Balkan
Peninsula, where different types of habitats, with different environmental conditions and
at different altitudes are present. The majority of Serbian populations is located in the
northern part of the country, i.e. in Vojvodina, while only a few small and isolated
mountain populations are present in the east and southeastern part of the country.
The European ground squirrel inhabits steppes, meadows and pastures covered with
short grass. These once widely distributed habitats, which were commonly inhabited by
European ground squirrels, had disappeared due to their intensive transformation into
agricultural land during the past few decades. Today, species survives only in very
isolated steppe-meadow fragments which are preserved within the dominant agricultural
matrix.
Given that a number of populations is continually declining throughout the range
and that the future survival of the species is put in danger, the European ground squirrel
has been protected since 2008 under the IUCN Red List of threatened species as a
vulnerable species (VU). Its status in Europe is governed by the EU Habitats and Species
Directive (Annexes II and IV) and the Convention on the Conservation of European
Wildlife and Natural Habitats, known as the Bern Convention (Annex II). Also, 414 of its
habitats are under protection regime within the Natura 2000 network. At the national
level, European ground squirrel is protected by law as a strictly protected species.
In order to analyze the genetic diversity and the structure of the European ground
squirrel in Serbia, a total of 180 tissue samples from 11 localities (Krušedol, Neradin,
Mala Remeta, Sremska Mitrovica, Banatska Palanka, Šušara, Mokrin, Lok and
Tomislavci from area of Vojvodina, one locality from Vlasina and one from Stara Planina
Mountain were collected during the 2007-2009. Density and abundance of the European
ground squirrel populations were estimated during the period 2004-2014 on the localities
Krušedol and Neradin, which are located on the periphery of Fruška Gora Mountain, as
well as on the locality Banatska Palanka, which is located on the periphery of the
Deliblato Sands. Finally, the minimum viable population size (MVP) was estimated using
the Vortex software.
The results gained in this study clearly indicated that the genetic diversity of
populations from Vojvodina is significantly higher than those from the periphery of the
species range, and similar to the genetic diversity of surrounding Pannonian populations
which are occupying a central position within the Northern phylogenetic group. On the
other hand, the results showed that the genetic diversity of mountain populations from
Vlasina and Stara Planina Mountain is significantly lower compared to the populations
from Vojvodina. All analyzed populations from Serbia showed high level of genetic
differentiation, indicating the presence of isolation and the absence of gene flow between
existing populations.
According to the cluster analysis, eleven studied populations from Serbia are
optimally grouped into four clusters. Populations from Vojvodina made first three
clusters, while two far mountainous populations from Vlasina and Stara Planina made the
fourth cluster. Arrangement of populations from Vojvodina by clusters indicates that the
Danube River, but not the Tisza River, represents a significant barrier to gene flow among
populations.
Observed populations showed high fluctuations in density and abundance during the
eleven studied years, both within and between localities. During this period, positive
population growth was observed only in the locality Neradin, while large fluctuations
were recorded on the localities Krušedol and Banatska Planka, followed by dramatic
decline in density and abundance.
According to the MVP analysis, for the survival of isolated populations of European
ground squirrel, preservation of 90% of genetic diversity and avoidance of the harmful
effects of inbreeding, it is necessary to ensure the presence of at least 300 individuals over
50 years. However, if we take into account different levels of demographic and
environmental stochasticity, MVP would be considerably larger. This pointed out the
need to establish more optimal habitat conditions and thus minimize large fluctuations in
the abundance caused by extremely adverse factors and increase the likelihood of
population survival.
The loss of suitable habitats, as well as the formation of barriers that prevent the
migration and gene flow between populations, currently represent the most important
factors that directly and indirectly affect the survival of populations of the European
ground squirrel.
The results from this study represent a good conservation basis and provide
guidance for an active approach to the protection and preservation of the European ground
squirrel in Serbia, development of the Action and Management Plans, as well as a basis
for the future fundamental ecological and conservation research of this endangered
species. Future conservation of the European ground squirrel in Serbia has not only the
national relevance. Namely, populations from Serbia and Bulgaria are of key importance
for the future preservation of the whole species, as these areas served as refugia from
which the species spread to the Central Europe during favorable climatic conditions in the
past. Therefore, we have a great obligation to preserve viable populations of the European
ground squirrel with a minimum loss of the existing genetic diversity. | true |
Uloga mTOR i MAPK signalnih puteva u rezistenciji karcinoma štitaste žlezde na hemioterapiju | The role of mTOR and MAPK signaling pathways in resistance of thyroid carcinoma to chemotherapy. | Karcinomi štitaste žlezde su najčešći maligniteti endokrinog sistema.
Klasifikacija ovih maligniteta je izvršena na osnovu njihovih histopatoloških
karakteristika na papilarni, folikularni medularni i anaplastični karcinom (ATC).
Većina karcinoma štitaste žlezde je dobro diferencirana i ima odličnu prognozu
(papilarni i folikularni), dok anaplastični karcinom predstavlja agresivni tip sa izrazito
lošom prognozom uprkos različitim terapijskim pristupima u njegovom lečenju.
Promene u PI3K/AKT/mTOR i RAS/MAPK/ERK signalnim putevima su
karakteristične za nastanak karcinoma štitaste žlezde, koji su urođeno rezistentni na
klasičnu hemioterapiju. Upravo promene u aktivnosti PI3K/AKT/mTOR i
RAS/MAPK/ERK signalnih puteva mogu dovesti i do rezistencije na klasične
hemioterapeutike. Još jedan od mogućih uzroka neuspeha hemioterapije je i pojava
višestruke (engl. multi-drug, MDR) rezistencije. Najčešći uzrok MDR-a je povišena
ekspresija P-gp i BCRP transportnih pumpi.
Cilj
ove
studije
je
bio
ispitivanje
uloge
ključnih
komponenti
PI3K/AKT/mTOR i RAS/MAPK/ERK signalnih puteva u patogenezi i rezistenciji
ATC. Analizirane su promene na genskom i proteinskom nivou u uzorcima pacijenata
obolelih od ovog tipa karcinoma, kao i efekat inhibicije komponenti signalnih puteva
kod humanih ATC ćelijskih linija. Pored toga, ispitana je i uloga P-gp i BCRP pumpi
u rezistenciji ATC.
Pokazano je da su i PI3K/AKT/mTOR i RAS/MAPK/ERK signalni putevi
važni za genezu ATC, kao i da se pritom međusobno isključuju. NRAS onkogen i p53
tumor supresor su izmenjeni u ispitivanim tumorskim uzorcima sa visokom
učestalošću. Najčešće je izmenjen NRAS gen što ukazuje na njegovu ključnu ulogu u
razvoju ATC. Sve otkrivene mutacije u NRAS genu i dve mutacije u p53 genu su po
prvi put prijavljene kod ATC.
In vitro studije su pokazale da se inhibicijom komponenti RAS/MAPK/ERK i
PI3K/AKT/mTOR signalnih puteva povećava senzitivnost humanih ATC ćelija na
klasičnu hemioterapiju. Najefikasnijim se pokazao dvostruki mTOR inhibitor
AZD2014, kako u pojedinačnim tretmanima tako i u kombinaciji sa paklitakselom
(PTX) i doksorubicinom (DOX).
Imunohistohemijska analiza P-gp i BCRP pumpi pokazala je njihovo značajno
prisustvo kod ATC pacijenata što ukazuje na učešće ovih proteina u rezistenciji ATC.
Sortiranjem ATC ćelija sa smanjenom akumulacijom rodamina 123 (Rho123),
poznatog P-gp supstrata, uspostavljena je nova ATC ćelijska linija. Na taj način,
dobijen je model koji više odgovara fenotipu uočenom kod ATC pacijenata nego
komercijalne ATC ćelijske linije korišćene u ovoj studiji.. Na ovom modelu je
ispitana efikasnost kombinovanog tretmanadvostrukim mTOR inhibitorom AZD2014
i PTX-om. Pokazano je da AZD2014 ne samo da povećava osetljivost ATC ćelija na
PTX, već u kombinaciji sa ovim citostatikom efikasno inhibira i migraciju i invaziju
ATC ćelija. Imajući u vidu da su rezistentnost i invazivnost ATC glavni uzroci loše
prognoze, terapija kombinacijom dvostrukog mTOR inhibitora i PTX-a bi mogla
doprineti efikasnijem lečenju pacijenata obolelih od ovog karcinoma. | Thyroid carcinoma is the most common malignancy of the endocrine system.
Thyroid malignancies are classified according to their histopathological characteristic
as papillary, follicular, medullary and anaplastic thyroid carcinoma (ATC). Most
thyroid malignancies (papillary thyroid carcinoma and follicular thyroid carcinoma)
are well differentiated and have favorable prognosis. On the other hand, ATC is one
of the most aggressive human cancers, with an intrinsic resistance and dismal
prognosis despite various therapeutic modalities. Changes in components of
RAS/MAPK/ERK and PI3K/AKT/mTORpathways are common in thyroid cancer
genesis which are resistant to classic chemotherapy agents. Changes in the activity of
RAS/MAPK/ERK and PI3K/AKT/mTORsignaling pathways can lead to drug
resistance. Besides these changes, possible cause of chemotherapy resistance is also
multi-drug resistance (MDR). The most common cause of MDR is high expression of
P-gp and BCRP proteins.
The aim of this study was to investigate the role of the key components of
RAS/MAPK/ERK and PI3K/AKT/mTOR pathways in the pathogenesis and
chemoresistance of ATC. We analyzed gene and protein changes in set of ATC
patient samples. We also investigated the role of inhibition of RAS/MAPK/ERK and
PI3K/AKT/mTOR pathways in ATC chemosensitization using human ATC cell lines.
The role of P-gp and BCRP proteins in ATC chemoresistance was also investigated.
Analysis of alterations in RAS/MAPK/ERK and PI3K/AKT/mTOR pathways
in ATC patients indicated that both pathways cooperate in the development of ATC.
Our results revealed a negative correlation between the activity of RAS/MAPK/ERK
and PI3K/AKT/mTOR pathways in the samples of ATC patients. NRAS oncogene and
p53 tumor suppressor are mutated with high frequency in our set of ATC samples.
NRAS is dominantly mutated gene, indicating the importance of this gene in ATC
development. All detected mutations in NRAS gene, and two mutations in p53 gene,
have never been reported in ATC genesis before.
In vitro results suggest that the inhibition of either RAS/MAPK/ERK or
PI3K/AKT/mTOR components may confer sensitivity of ATC cells to classic
chemotherapeutics. Treatment with dual mTOR inhibitor, AZD2014, alone or in
combination with paclitaxel (PTX) or doxorubicin (DOX) was shown to be the most
effective.
Immunohistochemical analysis showed high P-gp and BCRP expression in
our ATC samples, which indicates the role of these proteins in ATC chemoresistance.
We sorted ATC cells with the low Rhodamin123 (Rho123) accumulation which is
substrate of P-gp protein and established new ATC cell line. In this way we obtained
in vitro model system more similar to the patients’ phenotype, then comercial ATC
cell lines used in this study. We investigated the potential of dual mTOR inhibitor,
AZD2014 combined with PTX to sensitize this new ATC cell line. It was showed that
treatment with AZD2014 not only sensitizes ATC cells to PTX, but also combined
with this cytostatic, efficiently inhibits ATC cell migration and invasion. Taking into
account that chemoresistance and invasiveness of ATC are the main causes of poor
outcome, the application of dual mTOR inhibitor combined with PTX, seems to be a
logical therapeutic strategy for patients with ATC. | true |
Efekat estradiola u modulaciji apoptotskih signala u moždanoj kori i hipokampusu pacova tokom hronične moždane hipoperfuzije | Effects of estradiol on modulation of apoptotic signals in cerebral cortex and hippocampus of rats during chronic cerebral hypoperfusion | Iako se ishemijske bolesti mozga, koje se karakterišu pogoršanjem motornih i
kognitivnih funkcija, uključujući smanjenje sposobnosti učenja i memorije, intenzivno
proučavaju poslednjih 20 godina malo je poznato o molekulskim mehanizmima
njihovog nastanka, kao i odgovarajućim terapijama. Osnovni cilj ove doktorske
disertacije je bio da se ispita efekat hroničnog tretmana polnim hormonom, estradiolom,
na apoptotske puteve u kortikalnim i hipokampalnim ćelijama polno zrelih mužjaka
pacova Wistar soja u stanju nametnute hronične moždane hipoperfuzije. Tome je
predhodilo proučavanje efekta moždane hipoperfuzije na složenu kaskadu apoptotskih
signalnih puteva u kori prednjeg mozga i hipokampusu pacova u različitim vremenskim
tačkama.
Za potrebe prvog dela eksperimenta jedna grupa pacova je lažno operisana, dok je druga
grupa podvrgnuta operaciji trajnog podvezivanja zajedničkih karotidnih arterija. Jedinke
obe grupe su ostavljene na preživljavanju 3, 7 ili 90 dana. Za potrebe drugog dela
eksperimenta pacovi su podeljeni u dve grupe: životinje kojima su trajno podvezane
zajedničke karotidne arterije, tretirane 7 dana lanenim uljem i jedinke podvrgnute
operaciji trajnog podvezivanja zajedničkih karotidnih arterija i tretirane 7 dana
estradiolom (33.3 g/kg/dan, rastvoren u lanenom ulju). Praćeni su sledeći parametri:
obim neuralne smrti, fragmentacija DNK koja je karakteristična za apoptozu, kao i
promene u ekspresiji proteina i iRNK markera procesa ćelijske smrti odnosno
preživljavanja ćelija.
Rezultati ove doktorske disertacije pokazuju da se u zadatim eksperimentalnim
uslovima, u kori prednjeg mozga u ranim vremenskim tačkama javljaju
neurodegenerativne
promene
koje
prate
ekspresiju
apoptotskih
molekula
u
neprečišćenoj membranskoj frakciji, dok se u kasnije aktiviraju adaptivni i
kompenzatorni mehanizmi. U hipokampusu je uočen specifičan vremenski-zavisan
trend ekspresije analiziranih pro- i anti-apoptotskih molekula koji dovode do pojave
odložene ćelijske smrti. Na osnovu proučavanja efekata hroničnog tretmana malim
dozama estradiola u stanju nametnute moždane hipoperfuzije može se zaključiti da ovaj
potentni polni hormon ima kapacitet da oblikuje odgovor ispitivanih moždanih
struktura. Naime, anti-apoptotski efekat delovanja estradiola se ogleda u modulaciji i
nishodnoj regulaciji ispitivanih markera mitohondrijskog puta apoptoze. Takođe, na
osnovu dobijenih rezultata za neprečišćenu membransku, citosolnu, mitohondrijsku i
jedarnu frakciju obe ispitivane moždane strukture može se pretpostaviti da su
protektivni efekti estradiola manje izraženi u neprečišćenoj membranskoj frakciji ili da
su promene nastale pod uticajem ishemije znatnije u ovoj ćelijskoj frakciji, stoga
primenjeni tretman nije dovoljan da ublaži efekte povrede.
Dodatna istraživanja delovanja estradiola unutar mozga mogla bi omogućiti razvoj
novih terapeutskih strategija za modulaciju odgovora specifičnih ćelija i moždanih
struktura uključenih u kogniciju, formiranje memorije i učenje kod starih osoba, kao i
obolelih od progresivnih neurodegenerativnih bolesti, čime bi se znatno poboljšao
kvalitet njihovih života. | Although ischemic brain diseases, characterized by decline of motor and cognitive
functions, including reducing the ability of learning and memory, have been intensively
studied over the past 20 years, the molecular mechanisms of their generation, as well as
appropriate treatments are still matter of controversy. The main goal of this PhD thesis
was to investigate the effects of repeated estradiol treatment on apoptotic pathways in
cortical and hippocampal cells of adult male Wistar rats in a state of imposed chronic
cerebral hypoperfusion. This was preceded by the study of the effects of cerebral
hypoperfusion on complex cascade of apoptotic signalling pathways in the cerebral
cortex and hippocampus of rats at different time points.
For the purposes of the first part of the experiment, one group of rats was sham
operated, while the other group was subjected to permanent ligation of common carotid
arteries. Animals were sacrificed 3, 7 or 90 days following the insult. For the second
part of the experiment, the rats were divided into two groups: animals that underwent
permanent common carotid artery occlusion, treated for 7 days with linseed oil and rats
subjected to permanent ligation of the common carotid arteries, treated for 7 days with
estradiol (33.3 µg/kg/day, dissolved in linseed oil). Parameters that were monitored are:
volume of neuronal death, DNA fragmentation typical for apoptosis, as well as changes
in mRNA and protein expressions of cell death or survival markers.
The results obtained in this dissertation indicate that in cerebral cortex at two early
investigated time points, the expression of apoptotic molecules in synaptosomal fraction
is accompanied with neurodegenerative changes, while later adaptive and compensatory
mechanisms are activated. In hippocampus, a specific time-dependent expression of
analyzed pro- and anti-apoptotic molecules is associated with delayed apoptotic death.
Furthermore, according to gained data, repeated estradiol treatment in state of imposed
chronic cerebral hypoperfusion exerts a potent capacity to profile the response of
investigated brain structures, through modulation of expression of mitochondrial
apoptotic pathway markers. Also, results for synaptosomal, cytosolic, mitochondrial and
nucleus fractions of both investigated brain structures, point that protective effects of
estradiol are either less pronounced in synaptosomal fraction or ischemic changes in this
cell fraction are significant, thus applied treatment is not sufficient to compensate the
effects of the injury.
Additional research of estradiol activity in the brain could enable the development of
new therapeutic strategies to modulate the response of specific cells and brain structures
involved in cognition, memory formation and learning in the elderly and patients with
progressive neurodegenerative disease, which would significantly improve the quality
of their lives. | true |
Molekularna i biohemijska osnova dijapauze kukuruznog plamenca Ostrinia nubilalis (Hbn.) (Lepidoptera: Pyralidae) | Molecular and biochemical basis of diapause of European corn borer Ostrinia nubilalis (Hbn.) (Lepidoptera: Pyralidae) | Dijapauza je period zaustavljenog razvoja kod insekata, drugih zglavkara i
određenih vrsta riba, a koja se javlja na određenom, za svaku vrstu
karakterističnom, razvojnom stadijumu. Dijapauzu, kao dugotrajnu i „skupu“ fazu
mirovanja, karakteriše prilagođavanje organizma na molekularnom, fiziološkom,
morfološkom i bihevioralnom nivou. Promene koje karakterišu dijapauzu većine
vrsta su supresija metabolizma, zaustavljanje ili usporavanje razvića, izmenjena
ekspresija gena, sinteza zaštitnih proteina i metabolita koji vode ka povećanju
otpornosti organizma. Sposobnost insekata da prežive dugotrajne nepovoljne
periode u stanju dijapauze omogućila im je da koriste sezonski uslovljene resurse,
kolonizuju raznovrsna staništa, smanjuju ukrštanje u srodstvu i povećavaju
genetičku raznovrsnost. S obzirom na ogroman značaj dijapauze u životnom
ciklusu insekata, ali i drugih organizama, bilo kakvo odlaganje ili njeno prerano
prekidanje može imati negativne posledice na njihovo preživljavanje. Iz tog
razloga, bolje poznavanje biološke osnove dijapauze može omogućiti unapređenje
kontrole insekatskih vrsta koje nanose ekonomsku štetu u poljoprivredi, ali i
poboljšanje metoda gajenja vrsta koje su korisne (bumbari, pčele, svilena buba,
bubamare, parazitske osice i druge). Takođe, razumevanje mehanizama otpornosti
i zaštite od stresa, npr. na niske temperature, mogu doprineti razvoju novih
tehnika za krioprezervaciju organa i tkiva. Na kraju, insekti koji miruju, zbog
specifičnosti metaboloma, mogu biti potencijalan izvor novih lekova, ali i dobri
modeli za istraživanje starenja, gojaznosti i dijabetesa.
Kukuruzni plamenac Ostrinia nubilalis (Hübner 1796) je vrsta moljca čije su larve
polifagne herbivore koje se hrane na preko 200 biljnih vrsta, uključujući važne
poljoprivredne useve, povrće i voće. Krajem leta i početkom jeseni larve 5. stupnja
ulaze u fakultativnu dijapauzu, preživljavajući oštre kontinentalne zime u fazi
mirovanja. Tokom dijapauze gusenice O. nubilalis razvijaju otpornost na niske
temperature i sposobnost da prežive zamrzavanje vanćelijskih telesnih tečnosti,
VIII
što je omogućilo ovoj vrsti da nastani ogromna prostranstva Evroazije, ali i da
nepažnjom čoveka nasele Severnu Ameriku. Cilj ove doktorske disertacije je bio da
se doprinese boljem razumevanju molekularno-biološke i biohemijske prirode
dijapauze kukuruznog plamenca O. nubilalis.
Larvalna dijapauza je inicirana u laboratorijskim uslovima pod ravnodnevnim
svetlosnim režimom (12:12=svetlo:tama) i na temperaturi od 22°C, dok su
nedijapauzirajuće larve (ND) i lutke gajene u dugom fotoperiodu koji pogoduje
aktivnom razvoju (18:6=svetlo:tama) i na temperaturi od 22°C. Predijapauzirajuća
grupa (PD), larve 5. stupnja koje su prestale da se hrane i ušle u fazu lutanja,
podeljena je u dve podgrupe – gusenice koje su ostavljene da uđu u dijapauzu na
22°C (D2t grupa) i gusenice koje su postepeno aklimatizovane tokom dve nedelje
do temperature od 5°C, na kojoj su čuvane dva, četiri ili šest meseci (D2, D4, D6).
Metaboličke promene – sadržaj nukleotida i koenzima, aktivnost citohrom c
oksidaze (COX), sadržaj i promet vode određeni su kapilarnom elektroforezom,
spektroskopskom metodom i diferencijalnom skenirajućom kalorimetrijom.
Ekspresija odabranih gena je praćena metodom kvantitativne lančane polimeraze
u
realnom
vremenu
(Q-PCR).
Apoptoza
je
analizirana
fluorescentnom
mikroskopijom (DAPI bojenje), esejom fragmentisanosti DNK i fluorimetrijskim
određivanjem aktivnosti kaspaza u homogenatima larvi i lutki.
Rezultati kapilarne elektroforeze, aktivnosti COX, kao i ekspresije odabranih gena
za subjedinice respiratornog lanca pokazali su da je metabolizam intenzivniji kod
gusenica koje su gajene na 22°C (ND, PD i D2t), a niži kod onih koje su bile
aklimatizovane na 5°C, naročito u početnim mesecima dijapauze. Sa druge strane,
rezultati gravimetrijske analize i diferencijalne skenirajuće kalorimetrije pokazali
su da je gubitak vode manji u larvama koje su u dijapauzi na nižoj temperaturi, u
odnosu na larve u dijapauzi na višoj temperaturi, kao i da u tkivima opada sadržaj
osmotski aktivne, a raste sadržaj osmotski neaktivne vode. Ekspresija većine
analiziranih gena u vezi sa odgovorom na stres (različiti proteini toplotnog stresa,
feritin, metalotionein, glutaredoksin) povećava se u dijapauzi, sem na samom
početku dijapauze, kada je najčešće ili nepromenjena ili smanjena u odnosu na
nedijapauzirajuću grupu. Bojenje fluorescentnom DAPI bojom nije pokazalo
IX
apoptotske morfološke promene u tkivima dijapauzirajućih larvi. Nasuprot tome,
Sa druge strane, fluorimetrijska merenja su utvrdila postojanje a) umerene
aktivnosti enzima sličnih inicijatorskim kaspazama 9 i 8 u dijapauzirajućim
larvama, b) visoku aktivnost kaspazi 8 sličnih enzima u nedijapauzirajućim
larvama i c) veoma visoku aktivnost izvršne kaspaze 3 u lutkama.
Na osnovu dobijenih rezultata može se zaključiti da se dužina trajanja dijapauze i
temperatura na kojoj su gusenice aklimatizovane odražavaju na promene u
energetskom metabolizmu, prometu vode, ekspresiji gena odgovornih za zaštitu od
stresa i inicijaciji apoptoze. Najintenzivnije promene se događaju u početnim
mesecima dijapauze i ogledaju se u supresiji intenziteta metabolizma, razvoju
mehanizama za očuvanje vode i otpornost na temperaturni stres. Gusenice koje su
u dijapauzi na nižoj temperaturi su bolje adaptirane za preživljavanje dužeg
perioda mirovanja, jer imaju veću supresiju metabolizma, razvijaju zaštitne
mehanizme koji im omogućavaju bolju kontrolu prometa vode, a verovatno
pomažu i u regulaciji ćelijske smrti. Sa druge strane, gusenice koje su u dijapauzi na
visokoj temperaturi su pod intenzivnijim stresom, imaju previše intenzivan
metabolizam za fazu mirovanja, prebrzo troše metaboličke rezerve i gube vodu, što
dovodi do povećane smrtnosti i preranog okončanja dijapauze. Na osnovu ovoga
može se zaključiti da je izlaganje niskim temperaturama neophodno za dugoročno
preživljavanje tokom dijapauze kukuruznog plamenca. | Diapause is a carefully regulated state of arrested development that occurs at a
species-specific life stage of insects, other arthropods and some fish species. It
engages a plethora of molecular, physiological, morphological and behavioral
changes that altogether increase stress tolerance and the ability to survive. Thus,
diapause enables insects to exploit seasonally variable food resources and colonize
new habitats, as well as decrease inbreeding and increase mating of temporally
separated genotypes in a population. Due to the tremendous importance of
diapause for survival, any factors that delay or perturb diapause can potentially
compromise the fitness of an organism. Thus, deeper knowledge of the biology of
diapause could help improve or develop new means of pest control for insect
populations, as well as to advance current rearing techniques of beneficial species
(bees, ladybirds, parasitic wasps, silkworm etc.). Additionally, defining the
molecular background of stress tolerance during diapause, such as cold hardiness,
could improve techniques for cryopreservation of organs, tissues and cells. Also,
because of the specificity of the metabolom, diapausing insects could be regarded
as a potentially rich source of new pharmaceutical agents, or could be used as
model organisms to study ageing, obesity and diabetes.
The European corn borer (ECB), Ostrinia nubilalis (Hübner 1796), is a pest moth
species whose polyphagous larvae feed on more than 200 plants, including
important crops, vegetables and fruits. In late August or the beginning of
September, because of a critical shortening of the photoperiod, 5th instar larvae of
ECB stop feeding and enter into diapause. Diapausing larvae gradually develop
freeze tolerance and become cold hardy, which has enabled this species to
successfully colonize temperate regions. The aim of this dissertation was to
improve our understanding of the molecular and biochemical mechanisms
governing diapause of O. nubilalis.
XI
Non-diapausing larvae and pupae were reared at 22°C under a long photoperiod of
18L:6D, while diapause was induced at 22°C, but under a photoperiod of 12L:12D.
Pre-diapausing larvae (PD) that entered into the 5th Instar wandering stage were
split into two groups – one left to diapause at 22°C (D2t) and another that was
gradually acclimated to 5°C and left to diapause for two, four and six months (D2,
D4, D6). Molecular and biochemical changes during diapause were assessed by
measuring: a) the content of nucleotides and co-enzymes by capillary
electrophoresis, b) activity of cytochrome-c oxidase by spectrophotometry, c)
water content and supercooling points using differential scanning calorimetry
(DSC) and d) gene expression using quantitative PCR (Q-PCR). Apoptosis was
examined using fluorescence microscopy (DAPI staining), DNA laddering assay and
measuring the activity of selected caspases using fluormetric substrates.
Results show little or no difference between non-diapausing or pre-diapausing
groups, while diapausing groups did differ, depending on the temperature and
period of diapause. Generally, metabolism was high in groups reared at 22°C (ND,
PD, D2t) and more suppressed in groups reared at 5°C (D2-6), especially in the
beginning of diapause (D2). On the contrary, water loss was lower in cold-
acclimated diapausing larvae (D2, D4, D6) in comparison to the warm acclimated
group (D2t). In addition, the content of osmotically active (OA) water was
decreased and osmotically inactive (OI) water was increased in larval tissues
during diapause. Expression of stress-related genes was mostly up-regulated in the
D2t group, suppressed in the D2 group and was gradually increased during
diapause, from D2 to D6. DAPI staining did not reveal morphological signs of
apoptosis in tissues of diapausing larvae. On the other hand, fluorimetric analysis
of selected caspases showed: a) the presence of moderate activity of initiator
caspases (casp-9 and -8) in diapausing larvae, b) high activity of caspase-8 in non-
diapausing larvae and c) very high activity of effector caspase-3 in pupae.
The following conclusions can be drawn from the study of larval diapause of
Ostrinia nubilalis: a) diapause in this species is not static but rather a dynamic
event encompassing various biochemical and molecular mechanisms b) energy
metabolism, water management, gene expression and apoptotic processes differ
XII
depending on the temperature and period larvae spend in diapause, c) the most
dramatic changes occur in the beginning of diapause and differ tremendously
among cold and warm acclimated diapausing larvae, d) larvae that were cold
acclimated suppressed metabolism more significantly, efficiently preserved energy
and water, and e) warm acclimated larvae less successfully suppressed
metabolism, lose energy and water faster, which altogether leads to a state of
increased stress, high mortality and premature exit from diapause in this group.
Based on these results, low temperature exposure of O. nubilalis larvae in diapause
initiates a set of molecular and biochemical processes that are vital for diapause
endurance as well as successful resumption of further development. | true |
Struktura segmentalne duplikacije 5q13.2 kao modifikator fenotipa spinalne mišićne atrofije i amiotrofične lateralne skleroze | Structure of the 5q13.2 segmental duplication as a modifier of the phenotype of spinal muscular atrophy and amyotrophic lateral sclerosis | Spinalna mišićna atrofija (SMA) i amiotrofična lateralna skleroza (ALS) su bolesti
motoneurona. SMA je najčešći monogenski uzročnik smrtnosti dece, dok je ALS adultna,
neizlečiva bolest sa vrlo progresivnim tokom. SMA je uzrokovana homozigotnim odsustvom
gena SMN1 u telomernom delu segmentalne duplikacije 5q13.2, dok se u njenom
centromernom delu nalazi gotovo istovetan gen SMN2 koji daje ~10% funkcionalnog
proteina SMN. Klinička prezentacija SMA je izuzetno varijabilna i kreće se od najtežeg
oblika kod novorođenčadi (tip I) do blagog oblika sa adultnim početkom (tip IV).
Homogenost uzročne mutacije i izražena fenotipska heterogenost ukazuju na postojanje
genetičkih, epigenetičkih i sredinskih faktora koji modifikuju fenotip SMA. Visoka stopa
nejednakih hromozomskih rearanžmana u segmentalnoj duplikaciji 5q13.2 dovodi do
varijacija u broju kopija i strukturnih promena ne samo gena SMN, već i drugih prisutnih
gena, poput SERF1 i NAIP, i one mogu uticati na tok i težinu bolesti. Dosadašnji podaci
ukazuju na to da bi jednim delom uzrok fenotipskih razlika kod SMA mogao biti različit
stepen metilacije promotora SMN2. Sporadična ALS (SALS) čini oko 90% svih slučajeva
ALS i njena etiologija nije razjašnjena. Iako se genetička osnova, vreme pojave simptoma i
tok SMA i SALS razlikuju, usled sličnosti patoloških promena i učešća proteina SMN i
proteina asociranih sa ALS u zajedničkim molekularnim putevima, među genima
kandidatima za nastanak i progresiju SALS velika pažnja usmerena je upravo na gene SMN.
Cilj ove doktorske disertacije bio je ispitivanje genomske strukture, prirode rearanžmana i
epigenetičkih oznaka u segmentalnoj duplikaciji 5q13.2 kod SMA bolesnika. Takođe, cilj je
bio ispitivanje asocijacije broja kopija gena SMN1, SMN2, SERF1 i NAIP sa fenotipom dečije
SMA, rizikom za nastanak SALS i preživljavanjem SALS bolesnika.
U grupi od 99 SMA bolesnika i 122 roditelja bolesnika metodom umnožavanja većeg broja
ligiranih proba (MLPA) analizirana su 442 hromozoma i identifikovano je 20 različitih alela
telomernog dela duplikacije 5q13.2. Između broja kopija gena SMN2, SERF1 i NAIP i tipa
dečije SMA uočena je statistički značajna obrnuto srazmerna korelacija (Spirmanov test
korelacije, SMN2 p=2,2e-16, SERF1 p=4,264e-10, NAIP p=2,722e-8). Generalizovanim
linearnim modelom i selekcijom unazad, počevši od punog modela koji je obuhvatio broj
kopija SMN2, SERF1 i NAIP i njihove dvostruke interakcije, dobijen je najbolji minimalni
model koji objašnjava fenotipsku varijabilnost dečije SMA i obuhvata broj kopija SMN2
(p<2e-16) i SERF1 (p<2e-16) i njihov udruženi efekat (p=0,026). Ispitivanje stepena
metilacije dela promotora gena SMN2 kod bolesnika sa SMA tipovima I, II i III primenom
metode analize krivih topljenja visoke rezolucije (HRM) ukazalo je na njegovu heterogenu
metilaciju, što je potvrđeno molekularnim kloniranjem i Sangerovim sekvenciranjem. U grupi
od 153 SALS bolesnika iz populacije Srbije nije pokazana statistički značajna asocijacija
između rizika za pojavu bolesti i broja kopija gena SMN1 (p=0,650, Fišerov test tačnosti),
SMN2 (p=0,369), SERF1 (p=0,257) i NAIP (p=0,908). Krive preživljavanja zasnovane na
rezultatima multivarijabilne Koksove regresione analize pokazale su da tri kopije gena SMN1
(HR=10.276, 95% IP 3.106-33.998), niži zbir revidirane skale funkcionalne procene
(ALSFRS-R) u trenutku dijagnoze (HR=0.885, 95% IP 0.834-0.938), brži pad
funkcionalnosti (ΔALSFRS) (HR=2.193, 95% IP 1.618-2.972) i kraće dijagnostičko
kašnjenje (HR=0.939, 95% IP 0.888-0.994) rezultuju kraćim preživljavanjem bolesnika sa
SALS iz Srbije.
Rezultatima ove studije pokazana je izuzetno visoka inherentna nestabilnost regiona 5q13.2.
Delecije u telomernom delu regiona 5q13.2 karakteristične su pretežno za bolesnike sa SMA
tipom I, dok je genska konverzija gena SMN1 u SMN2 uglavnom osobina bolesnika sa SMA
tipovima II i III. Broj kopija gena SMN2, SERF1 i NAIP utiče na kliničku prezentaciju dečije
SMA i najboji minimalni model koji objašnjava njenu fenotipsku varijabilnost uključuje broj
kopija gena SMN2 i SERF1 i njihov udruženi efekat. Kod bolesnika sa SMA tipom I, II i III
ispitivani deo promotora gena SMN2 odlikuje se metilacijom izuzetno visoke heterogenosti
za čije ispitivanje se metoda HRM pokazala kao neadekvatna. Veći broj kopija gena SMN1
pokazao se kao negativan prognostički faktor preživljavanja SALS bolesnika, i ovaj
objektivno merljiv i vremenski nepromenljiv parametar, zajedno sa kliničkim parametrima,
može unaprediti algoritme za predviđanje toka bolesti i omogućiti efikasniji terapeutske
pristupe kod SALS. | Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are motor
neurone diseases. SMA is the most frequent monogenic cause of infant mortality, whereas
ALS is an incurable late-onset disease with an inexorably progressive course. SMA is caused
by homozygous absence of the SMN1 gene located in the telomeric part of the 5q13.2
segmental duplication, while in its centromeric part resides a nearly identical copy named
SMN2 producing only ~10% of functional SMN protein. Clinical presentation of SMA is
extremely variable ranging from a severe infantile form (type I) to mild adult-onset form
(type IV). Homogeneity of the disease-causing mutation and extensive phenotypic variability
clearly indicate the existence of additional genetic, epigenetic and environmental factors
modulating SMA phenotype. High rate of unequal crossing over in 5q13.2 segmental
duplication results in copy number variations (CNVs) and complex structural rearrangements
of the two SMN genes and other genes residing in the same region, such as SERF1 and NAIP,
modulating disease progression and severity. Existing data suggest that, to some extent,
phenotypic discrepancies in SMA might be due to differences in methylation level of the
SMN2 promoter. Sporadic ALS (SALS) comprises ~90% of all ALS cases and has an
unknown aetiology. Even though aetiology, age at onset and progression may differ between
the diseases, due to similarities between ALS and SMA pathologies and the involvement of
SMN and some ALS-associated proteins in common molecular pathways, SMN1 and SMN2
genes were repeatedly studied as SALS susceptibility genes. The aim of this doctoral thesis
was to reconstruct genomic structure, the nature of rearrangements and epigenetic marks of
the 5q.13.2 segmental duplication in SMA patients from Serbia. Furthermore, the aim was to
investigate if copy numbers of the SMN1, SMN2, SERF1 and NAIP genes are associated with
SMA type, susceptibility to SALS and survival in SALS patients from Serbia.
In a group of 99 SMA patients and 122 patients’ parents 442 chromosomes were analysed by
multiplex ligation-dependent probe amplification (MLPA) and 20 different alleles of the
5q13.2 telomeric part were observed. Statistically significant inverse correlation was found
between SMN2, SERF1 and NAIP CNVs and SMA type (Spearman rank test, SMN2 p=2.2e-
16, SERF1 p=4.264e-10, NAIP p=2.722e-8). Generalised linear model and backward
selection, starting with a full model including the SMN2, SERF1 and NAIP copy number and
their two-way interactions, revealed that the best minimal model explaining phenotypic
variation in childhood-onset SMA with the smallest set of variables included the SMN2
(p<2e-16) and SERF1 (p<2e-16) copy number and their joint effect (p=0.02628). Analysis of
the methylation level of a part of the SMN2 gene promoter in patients with SMA type I, II
and III by high resolution melting analysis (HRM) showed its heterogeneous methylation
additionally confirmed by molecular cloning and Sanger sequencing of individual clones. No
statistically significant association was found between CNVs of SMN1 (p=0.650, Fisher's
exact test), SMN2 (p=0.369), SERF1 (p=0.257) or NAIP (p=0.908) genes and SALS
susceptibility in the examined group of 153 Serbian SALS patients. Survival curves based on
the Cox model obtained by the multivariable Cox regression analysis showed that three
SMN1 copies (HR=10.276, 95% CI 3.106-33.998), lower revised ALS functional rating scale
(ALSFRS-R) score at the time of diagnosis (HR=0.885, 95% CI 0.834-0.938), faster decline
of functionality over time (ΔALSFRS) (HR=2.193, 95% CI 1.618-2.972) and shorter
diagnostic delay (HR=0.939, 95% CI 0.888-0.994) result in shorter survival of Serbian SALS
patients.
Results of this study show extremely high inherent instability of the 5q13.2 region. Deletions
in the 5q13.2 telomeric part were mostly found in SMA type I patients, whereas SMN1-to-
SMN2 gene conversion was mainly a characteristic of type II and III patients. Copy numbers
of the SMN2, SERF1 and NAIP genes influence SMA clinical presentation and the best
minimal model explaining phenotypic variability of early-onset SMA included SMN2 and
SERF1 copy numbers and their joint ffect. In patients with SMA types I, II and III the
analysed part of the SMN2 gene promoter is characterised by highly heterogeneous
methylation for whose analysis HRM proved to be inappropriate. Higher SMN1 copy number
was a negative prognostic factor for the survival of SALS patients and this objectively
measurable time-invariant genetic factor, together with clinical variables, may help develop
better algorithms for disease prognosis and more targeted clinical trials in SALS. | true |
Adsorpciona i emulgujuća svojstva proteinskog izolata i hidrolizata semena tikve (Cucurbita pepo) | Adsorption and emulsifying properties of pumpkin (Cucurbita pepo) seed protein isolate and hydrolysate | Seme tikve (Cucurbita pepo) obiluje kako uljem
tako i proteinima. Nakon izdvajanja ulja, proteini se
koncentrišu u uljanoj pogači, sekundarnom proizvodu
procesa proizvodnje ulja, gde njihov sadrţaj dostiţe do
65%. Proteini semena tikve su primamljiv sastojak za
prehrambenu, farmaceutsku i kozmetičku industriju zbog
svoje
farmakološke
aktivnosti
i
visoke
biološke
vrednosti. Pored toga, budući da su mnogi proizvodi ovih
industrija po svojoj koloidnoj prirodi emulzije, proteini
semena tikve bi se u njima mogli naći i kao prirodne
površinski
aktivne
materije.
MeĎutim,
koloidna
funkcionalnost proteina semena tikve se još uvek
potcenjuje zbog globularne strukture za koju se vezuju
slabija funkcionalna svojstva u odnosu na proteine sa
fleksibilnijom
strukturom.
Prema
tome,
cilj
ove
disertacije
je
ispitivanje
funkcionalnih
osobina
proteinskog izolata semena tikve, pre svega njegovih
adsorpcionih i emulgujućih svojstava, kao i ispitivanje
uticaja promene proteinske strukture putem enzimske
hidrolize na ispitivana svojstva.
Pripremljeni su izolat proteina semena tikve
(IPST) i dva enzimska hidrolizata, H1 i H2. IPST, H1 i
H2 su okarakterisani odreĎivanjem sadrţaja vlage,
proteina
i
pepela,
zatim,
odreĎivanjem
prinosa,
molekulske mase i zeta potencijala. Ispitan je uticaj
koncentracije proteina/peptida (0,0001–1 g/100 cm3), pH
(3–8) i jonske jačine (0–1 mol/dm3 NaCl) na
rastvorljivost
i
adsorpciona
svojstva:
dinamički
meĎupovršinski pritisak (ulje/voda), statički površinski
(vazduh/voda) i meĎupovršinski (ulje/voda) pritisak,
kinetiku adsorpcije i dilatacionu reologiju proteinskih
adsorpcionih filmova. Nakon toga, ispitan je i uticaj
pomenutih parametara na emulziona svojstva IPST, H1 i
H2.
Emulgujuća
svojstva
IPST,
H1
i
H2
su
okarakterisana na osnovu prosečnog prečnika kapljica
emulzija, raspodele veličina kapljica i stabilnosti
emulzija.
UtvrĎeno je da je prinos IPST veći od prinosa oba
hidrolizata za oko 65 %. IPST ima najniţu rastvorljivost
na pH=5, što ujedno predstavlja i njegovu izoelektričnu
tačku. Enzimskom hidrolizom IPST značajno se
povećava rastvorljivost, posebno na pI=5. Povećanje
jonske jačine je izazvalo salting–in ili salting–out efekat
rastvorljivosti kod svih uzoraka u zavisnosti od pH.
IPST, H1 i H2 poseduju površinsku aktivnost pri čemu je
površinski/meĎupovršinski pritisak H1 i H2 manje
zavistan od promene pH i jonske jačine u poreĎenju sa
površinskim/meĎupovršinskim
pritiskom
IPST.
Adsorpcijom na granicu faza IPST i oba hidrolizata
obrazuju
adsorpcione
filmove
sa
dominantnom
elastičnom komponentom. Emulgujuća svojstva IPST,
H1 i H2 zavise od koncentracije uzorka, pH vrednosti i
jonske jačine kontinualne faze. Pri koncentraciji od 1
g/100 cm3 i Ic=0 mol/dm3 pripremljene emulzije su
stabilne na svim pH osim emulzije IPST na pH 5. Sve
emulzije podleţu gravitacionoj nestabilnosti. | Pumpkin (Cucurbita pepo) seed is rich source of
both, oil and proteins. Once the oil has been extracted,
proteins concentrate in oil cake, a by–product of the oil
extraction process, where their content can reach up to
65%. Pumpkin seed proteins are desirable ingredient in
food, pharmaceutical and cosmetic industry due to their
pharmacological activities and high biological value.
Moreover, since many of products of these industries are,
in colloidal terms, emulsions, pumpkin seed proteins
could serve as surface active materies. However,
colloidal functionality of pumpkin seed proteins is still
underestimated for their globular structure which entails
inferior functional properties to functional properties of
proteins with more flexible structure. Based on that, the
aim of this dissertation is to investigate functional
properties of pumpkin seed protein isolate, adsorption
and emulsifying properties, in the first place, and then to
investigate the influence of modification of the protein
structure, by means of enzymatic hydrolysis, on the
aforementioned properties.
Pumpkin seed protein isolate, IPST, and two
enzymatic hydrolysates, H1 and H2, were prepared.
IPST, H1 and H2 were characterized by determination of
moisture, ash and protein content, then, by determination
of protein recovery, molecular mass and zeta potential.
Influence of the protein/peptide concentration (0.0001–1
g/100 cm3), pH (3–8) i ionic strength (0–1 mol/dm3
NaCl) on the solubility and adsorption properties:
dynamic interfacial (oil/water) pressure, static surface
(air/water) and interfacial (oil/water) pressure, adsorption
kinetics and interfacial dilatational properties, was
investigated next. In the end, influence of the
aforementioned
pharameters
on
the
emulsifying
properties of IPST, H1 and H2 was investigated.
Emulsifying properties of IPST, H1 and H2 were
discussed in terms of mean droplet diameter, droplet size
distribution and emulsion stability.
Protein recovery of IPST was determined to be 65
% higher than recovery of H1 and H2. Solubility of IPST
was the lowest at pH 5, what presents the isoelectric
point. The enzymatic hydrolysis of IPST significantly
increased solubility, especialy at the isoelectric point.
Increase in the ionic strenght led to salting–in or salting–
out effect depending on pH of the sample. Three
investigated samples, IPST, H1 and H2 exhibited surface
activity, however, sufrace/interfacial pressure of H1 and
H2 were found to be less influenced by change in pH or
ionic strenght of the solution in comparison to the IPST.
Once adsorbed to the interface IPST and both
hydrolysates form interfacial film with dominant elastic
component. Emulsifying properties of IPST, H1 and H2
depend on the concentration, pH and ionic strength of the
continuous phase. Stabile emulsions were formed at
concentration of 1 g/100 cm3 and Ic=0 mol/dm3
regardless of pH, with the exception of the IPST at pH 5.
All
emulsions
were
susceptibile
to
gravitational
separation. | true |
Bioinformatička analiza proteina uključenih u patogenezu mijeloidnih maligniteta | Bioinformatics analysis of proteins involved in pathogenesis of myeloid malignancies | Mijeloidni maligniteti su klonalne bolesti ćelija mijeloidne krvne loze, koje su, pre
svega, posledica poremećaja samoobnavljanja i diferencijacije hematopoetskih matičnih
ćelija. Godišnja incidenca u Evropi je 7,5-8,6/100 000, a prosečna petogodišnja stopa
preživljavanja pacijenata, uz primenu standardne terapije, je 37%. S obzirom da maligniteti
najčešće nose više od jedne vodeće – ‘driver’ mutacije, pri čemu je velika intra- i
interkancerska genetička heterogenost, postoji potreba za individualizovanim terapeutskim
pristupom svakom pacijentu. Personalizovana medicina označava princip postizanja
maksimalne terapijske efikasnosti kroz upotrebu ciljanih terapeutika kod biološki
okarakterisanih pacijenata. Bioinformatika ima značajnu ulogu u identifikaciji ciljnih
molekula za razvoj terapije, racionalnom dizajnu lekova i identifikaciji informativnih
biomarkera za prateću dijagnostiku.
Predmet istraživanja u ovom radu bili su geni i njihovi proteinski produkti koji imaju
važnu ulogu u patogenezi mijeloidnih maligniteta. WT1 je transkripcioni faktor, koji
kontroliše ekspresiju gena uključenih u apoptozu, proliferaciju i diferencijaciju. Ovaj gen
ima povećanu ekspresiju u 70-90%, a mutiran je u ~10% akutnih mijeloidnih leukemija
(AML) i učestvuje u preko 40 protein-protein interakcija, koje su vremenski i kontekstno
zavisne. Mapiranje celokupne mreže WT1 kofaktora doprineće identifikaciji novih
molekula i interakcija, koje su potencijalni targeti za ciljanu terapiju AML.
Nedavno otkriven tumor supresor u karcinomu dojke, NISCH, ispoljava efekat preko
RAC signalnog puta, koji je ključan u regulaciji hematopoeze i uključen u patogenezu
mijeloidnih maligniteta, i smatra se atraktivnim targetom za ciljanu terapiju. Poznavanje
funkcionalnih domena NISCH-a će omogućiti dizajn i optimizaciju jedinjenja sa
antikancerskim delovanjem.
Poslednjih desetak godina je utvrđen značaj epigenetičkih regulatora, koji su, pored
transkripcionih faktora i signalnih molekula, neophodni za normalno funkcionisanje
hematopoetskog sistema. Ovi molekuli imaju tumor supresorski efekat, a mutacije u
ASXL1, DNMT3A, EZH2 i TET2 su prisutne u svim tipovima mijeloidnih maligniteta i
pretpostavlja se da dovode do nastanka ovih bolesti. Detekcija ovih štetnih mutacija je
važan prateći dijagnostik za terapeutike koji utiču na epigenom.
U ovom radu su primenjeni bioinformatički programi zasnovani na metodi
informacionog spektra (ISM) za: i) predviđanje protein-protein interakcija (PPI) WT1, ii)
identifikaciju i karakterizaciju funkcionalnih domena proteina NISCH i iii) predviđanje
funkcionalnih efekata aminokiselinskih supstitucija (AAS) u epigenetičkim regulatorima
ASXL1, DNMT3A, EZH2 i TET2. ISM je metoda za analizu sekevenci, koja se zasniva na
brojčanom kodiranju aminokiselina i Furijeovoj transformaciji. Za predviđanje PPI proteina
WT1 je korišćen integrativni algoritam, koji, pored ISM-a, obuhvata i enrichment analizu,
mrežnu analizu i analizu koekspresije gena. Za funkcionalnu anotaciju domena proteina
NISCH, identifikovanih ISM-om, korišćena je enrichment analiza i GOSlims. Razvijen je
novi ISM algoritam za predikciju funkcionalnih efekata AAS, koji je evaluiran na skupu
genskih varijacija koje se nalaze van konzerviranih funkcionalnih domena (engl. not
Conserved Functional Domains, nCFD) epigenetičkih regulatora i upoređen sa najšire
korišćenim programima, PolyPhen-2 i SIFT.
Filogenetski konzervirana frekvenca F(0,00195) je dominantna u informacionom spektru
sve četiri glavne izoforme proteina WT1. Odnos signal-šum u kros-spektru na ovoj
frekvenci veći od 10 je karakterističan za realne interakcije WT1. Skriningom UniProt
baze, identifikovano je 2576 proteina sa ovom karakteristikom. Ovaj skup je statistički
obogaćen proteinima uključenim u regulaciju transkripcije i obradu iRNK. U njemu se
nalazi 504 proteina anotiranih terminom: ‘DNK-zavisna regulacija transkripcije’. Presek
ovog skupa proteina i skupa od 500 proteina dobijenog mrežnom analizom čini 51 protein.
Od njih, 36 su koeksprimirani sa WT1 u K562 ćelijskoj liniji i predstavljaju kandidate za
njegove nove interaktore. Ovim analizama je humani proteom sveden na odgovarajući broj
hipoteza za eksperimentalno testiranje.
Bioinformatičkom analizom su identifikovana dva funkcionalna domena NISCH-a, koji
obuhvataju rezidue 680-739 i 966-1020. Računarska funkcionalna karakterizacija je
pokazala da je NISCH, preko prvog domena, asociran sa: i) procesima posttranslacionih
modifikacija proteina, specifično ubikvitinacijom, i ii) procesima sinaptogeneze. Preko
drugog domena, NISCH je uključen u: i) transkripciju, ii) apoptozu i iii) signalnu
transdukciju.
Tačnost predviđanja funkcionalnih efekata nCFD AAS u epigenetičkim regulatorima
ASXL1, DNMT3A, EZH2 i TET2 PolyPhen-2 je 52%, a SIFT 57%. Zapažena niska
efikasnost je najverovatnije posledica visokih korelacija predikcija ovih alata i pozicije
AAS u CFD/nCFD regionima (~0,50). Razvijeni ISM algoritam je na istim podacima imao
tačnost od 70%. Poređenje ova tri alata je pokazalo da je jedino ISM algoritam statistički
značajno diferencirao nCFD mutacije i SNP-ove u epigenetičkim regulatorima.
Ovo istraživanje je pokazalo da se primenom računarskih alata zasnovanih na ISM-u u
analizi proteina koji su uključeni u nastanak i razvoj mijeloidnih maligniteta efikasno mogu
ekstrahovati informacije koje su relevantne za mehanizme i terapiju ovih bolesti. | Myeloid malignancies are clonal diseases of myeloid cell line, which arise, primary, as a
consequence of aberrant self-renewal and differentiation of hematopoietic stem cells.
Annual incidence in Europe is 7.5-8.6/100 000 and the mean 5-year survival rate, with
standard therapy, is 37%. Since malignancies often carry more than one driver mutation,
with high intra- and inter-cancer genetic heterogeneity, there is a need for individualized
therapeutic approach to each patient. Personalized medicine is based on the principle of
achieving maximal therapeutic efficacy through the use of targeted drugs for biologically
characterized patients. Bioinformatics has an important role in identification of molecules
for targeted therapy, rational drug design and identification of informative biomarkers for
companion diagnostics.
The subject of this research included genes and their protein products that have
important roles in the pathogenesis of myeloid malignancies. WT1 is a transcription factor
that controls the expression of genes involved in apoptosis, proliferation and differentiation.
This gene is overexpressed in 70-90%, mutated in ~10% of acute myeloid leukemia (AML)
and it is engaged in more than 40 time- and context-dependant protein-protein interactions.
Characterizing entire network of WT1 cofactors can improve identification of new
molecules and their interactions that could be targets for therapy of AML.
Recently identified tumor suppressor in breast cancer, NISCH, affects RAC signaling
pathway, which is important in regulation of hematopoiesis and has a role in pathogenesis
of myeloid malignancies, so it is considered an attractive target for targeted therapy.
Characterization of functional domains of NISCH can enable design and optimization of
compounds with anticancer effect.
In the last decade, the importance of epigenetic regulators is determined, which are,
besides transcription factors and signaling molecules, necessary for normal functioning of
hematopoietic system. These molecules have tumor suppressing effect and mutations in
ASXL1, DNMT3A, EZH2 and TET2 are present in all types of myeloid malignancies and
probably lead to the genesis of these diseases. Detection of these deleterious mutations is
important companion diagnostic for epigenome-affecting therapeutics.
In this research, bioinformatics programs based on the Informational Spectrum Method
(ISM) were applied for: i) the prediction of protein-protein interactions (PPI) of WT1, ii)
the identification and characterization of NISCH functional domains and iii) the prediction
of functional effects of amino acid substitutions (AAS) in epigenetic regulators ASXL1,
DNMT3A, EZH2 and TET2. ISM is a method for sequence analysis, based on the
numerical coding of amino acids and Fourier transform. For the prediction of WT1 PPIs,
we used integrative algorithm, which contains, besides ISM analysis, enrichment, protein
network and gene co-expression analyses. For the functional annotation of NISCH protein
domains, identified by ISM, enrichment and GOSlims analyses were used. Finally, new
ISM algorithm for prediction of functional effects of AAS was evaluated on the set of
genetic variations positioned outside conserved functional domains (nCFD – not Conserved
Functional Domains) of epigenetic regulators and compared with the most commonly used
programs, PolyPhen-2 and SIFT.
Phylogenetically conserved frequency F(0.00195) is dominant in the informational
spectra of all four major WT1 protein isoforms. Signal-to-noise ratio on this frequency in
cross-spectrum higher than 10 is characteristic for the real WT1 interactions. Screening of
UniProt database identified 2 576 proteins with this characteristic. This set is enriched in
proteins involved in transcription regulation and mRNA processing. In this dataset, 504
proteins are annotated with the term: 'DNA-templated regulation of transcription'. Overlap
of this set of proteins and the set resulting from the protein network analysis contains 51
proteins. Of these, 36 are co-expressed with WT1 in the K562 cell line and represent
candidates for its interacting partners. With these analyses we constricted human proteome
to experimentaly testable number of hypothesis.
Bipinformatics analysis identified two functional NISCH domains encompassing
residues 680-739 and 966-1020. Computational functional analyses showed that NISCH
associates, through the first domain, with: i) protein posttranslational modification
processes, specifically ubiquitination, and ii) processes of sinaptogenesis. Through the
second domain, NISCH is involved in: i) transcription, ii) apoptosis and iii) signal
transduction.
Accuracy of prediction of the functional effects of nCFD AAS in epigenetic regulators
ASXL1, DNMT3A, EZH2 and TET2 is 52% for the PolyPhen-2 and 57% for the SIFT.
The observed low efficacy probably arises from high correlations of the predictions and the
AAS position in the CFD/nCFD regions (~0.50). Developed ISM algorithm has, on the
same data, accuracy of ~70%. Comparison of the eficacy of these three tools showed that
only ISM algorithm statistically significantly differentiated between nCFD mutations and
SNPs in epigenetic regulators.
This research showed that trough the application of computational ISM-based tools for the
analyses of proteins involved in occurrence and progression of myeloid malignancies one
can efficiently extract information relevant for the mechanisms and therapy of these
diseases. | true |
Diverzitet vaskularne flore nizijskih šuma ravnog Srema | Diversity of vascular flora of lowland forests of ravni Srem | Kako su plavne šume u Evropi vrlo fragilni ekosistemi i povećava se njihova ugroženost prevashodno antopopresijom, postoji rastuća potreba za povećanjem obima stavljanja sastojina ovih šuma pod specijalni režim zaštite. Upravo sprovdena, floristička i ekološka istraživanja u ovoj disertaciji obezbeđuju jednu širu ekološku osnovu za zaštititu prirodnih vrednosti ovih šuma i održivi menadžment nizijskih šumskih ekosistema, distribuiranih u plavnom i izvanplavnom području Ravnog Srema, ali i šre u svakom drugom šumskom području koje pripada kompleksu aluvijalnih higrofilnih šuma. Da bi se detektovala i analizirala bazična floristička diferencijacija tipološki izdvojenih jedinca šumske vegetacije, primenjeno je shodno odgavarujućim pravilima za različite setove vegetacijskih podataka niz ordinacionih metoda (CA, DCA, PCA i nMDS) a od klasifikcionih - UPGMA, kao hijerarhijski, aglomerativni metod. Da bi detektovali nivo florističkog ili ekološkog diskontinuiteta i njegovu statističku signifikantnost primenjena je ANOSIM analiza. Multivarijantnom SIMPER procedurom je dodatno kvantifikovan doprinos svakog pojedinačnog taksona ili faktora postojećem obrazcu ukupnog florističkog odnosno ekološkog diskontinuiteta između analiziranih setova vegetacijskih odnosno ekoloških podataka. Korespodentna analza (CA) i klasifikacioni UPGMA metod su pokazali da se u odnosu na recentno stanje florističkog sastava i sastojinske strukture nizijske šume plavnog i izvanplavnog područja Ravnog Srema diferenciraju na četiri velike grupe sastojina, koje u potpunosti korespondiraju sa cenoekološkim grupama tipova šuma: močvarne šume crne jove, šume bele vrbe i topola, šume lužnjaka i jove i šume lužmjaka i graba. Shodno multivarijantnoj ANOSIM analizi, utvrđeno je da najveći stepen florističkog diskontinuiteta postoji upravo između cenogrupe močvarne šume crne jove i cenogrupe lužnjaka i graba. Po osnovu SIMPER procedure najznačajnijim vrstama, koje doprinose (na ≈15% kumultativnog doprinisa) prethodno utvrđenom obrazcu florističkog diskontinuiteta sastojinske strukture, na nivou istraženih cenotipova, samtraju se sledećim redosledom: Amorpha fruticosa 2, Populus euroamericana cl. I -214 (1), Quercus robur 1, Amorpha fruticosa 3, Fraxinus angustifolia 1, Carpinus betulus 1 i Fraxinus angustifolia 3... | As floodplains in Europe are very fragile ecosystems, increasingly threatened by anthropopression, there is a growing need for increase in number of forest stands of this type placed under a special protection regime. The most recent floristic and ecological studies presented in this dissertation provide a wider ecological foundation for protection of natural values of these forests, as well as for sustainable management of lowland forest ecosystems distributed in the floodplain and out-of-floodplain areas, both in Ravni Srem and in any other forest area belonging to the complex of alluvial hygrophilous forests. In order to detect and analyze the basic floristic differentiation of typologically separated units of forest vegetation, a number of ordination methods (CA, DCA, PCA and nMDS) and the classification method UPGMA (as a hierarchical agglomerative method) were applied according to the scientifically recognized rules for various sets of vegetation data. The ANOSIM method was used in order to detect the level of floristic or ecological discontinuity and its statistical significance. The multivariate SIMPER procedure was used for additional quantification of contribution of each individual taxon or factor to the existing pattern of overall floristic or ecological discontinuity between the analyzed sets of vegetation or ecological data. The correspondent analysis (CA) and classification UPGMA method have shown that,regarding the recent condition of floristic composition and stand structure, the lowland forests of floodplain and out-of-floodplain areas of Ravni Srem are differentiated into four large groups of stands, completely corresponding with the coenoecological groups of forest types: Swamp forest of Black Alder, forests of White Willow and Poplar, forests of Pedunculate Oak and Alderand forests of Pedunculate Oak and Hornbeam. The multivariate ANOSIM analysis has shown the greatest degree of floristic discontinuity between the coeno-group of Swamp forest of Black Alder and the coeno-group of Pedunculate Oak and Hornbeam. According to the SIMPER procedure, the most important species (contributing with ≈ 15% of cumulative contribution) to the previously determined pattern of floristic discontinuity of stand structure at the level of studied coeno-types, are in following order: Amorphafruticosa 2, Populuseuroamericana cl. I -214 (1), Quercus robur 1, Amorphafruticosa 3, Fraxinusangustifolia 1, Carpinus betulus 1 andFraxinusangustifolia 3... | false |
Određivanje profila ekspresije i unutarćelijske lokalizacije proteina ANKRD2 u srcu i skeletnim mišićima čoveka | Determination of expression profile and localization of ANKRD2 protein in human cardiac and skeletal muscle | Protein Ankrd2 pripada familiji mišićnih proteina sa ankirinskim ponovcima (eng. Muscle Ankyrin Repeat Proteins – MARP). U sarkomeri ima ulogu strukturnog elementa i signalnog molekula u procesu mehanotransdukcije. Pod dejstvom fiziĉkih i hemijskih stresora migrira u jedro, gde ostvaruje ulogu regulatora transkripcije. Ekspresija Ankrd2 je indukovana mehaniĉkim stresom i zavisna od stepena diferencijacije mišićne ćelije i faze razvića organizma. Osim u skeletnim mišićima, protein ANKRD2 je u manjoj meri eksprimiran i u srcu ĉoveka. U cilju otkrivanja novih funkcija koje ANKRD2 ima u ovom organu odreĊeni su njegov profil ekspresije i lokalizacija u srcu ĉoveka i uporeĊeni sa karakteristikama homolognog ANKRD1, koji je eksprimiran samo u srcu i detaljno okarakterisan. Utvrdili smo da je nivo ekspresije ANKRD2 dosta niţi u poreĊenju sa ANKRD1 i da je preteţno lokalizovan u sarkomeri kardiomiocita. Interkalirajući diskovi su identifikovani kao novo mesto lokalizacije oba proteina. S obzirom da su ĉesto i uspešno korišćen model sistem za prouĉavanje funkcije srĉano-specifiĉnih gena, kardiomiociti neonatalnih pacova (KMNP) su okarakterisani po pitanju ekspresije i lokalizacije endogenog Ankrd2 i egzogenog ANKRD2. Ankrd2 je lokalizovan i u jedru i u citoplazmi KMNP, a sliĉnu dualnu lokalizaciju je pokazivao i egzogeno eksprimirani ANKRD2. Za razliku od inhibitornog dejstva na ekspresiju Ankrd1, kardiotoksiĉni agens doksorubicin nije doveo do promene nivoa ekspresije proteina Ankrd2 u KMNP. U ovoj tezi su okarakterisane tri izoforme ANKRD2 od 446 (L-ANKRD2), 360 (M-ANKRD2) i 333 (S-ANKRD2) aminokiselina. Diskriminacija izoformi u popreĉno prugastim mišićima ĉoveka je postignuta korišćenjem specifiĉno dizajniranih antitela i graniĉnika. Utvrdili smo brojne sliĉnosti izmeĊu izoformi S- i M-ANKRD2, kao i znaĉajne razlike koje mogu da se odraze na njihove specifiĉne funkcije. Izoforma LANKRD2 nije detektovana ni u srcu, ni u skeletnim mišićima... | Ankrd2 is a member MARP (Muscle Ankyrin Repeat Proteins) protein family. In sarcomere, it has a role of a structural element and signaling molecule in mechanotransduction. Ankrd2 is highly responsive to physical and chemical stress, which results in its translocation to the nucleus where it acts as a transcriptional regulator. Expression of Ankrd2 is upregulated in response to mechanical stresses such as exercise and stretch, as well as during muscle development and myoblast differentiation. Apart from skeletal muscles, ANKRD2 has been also detected in adult human heart, but at lower levels. In order to explore the cardiac-specific function of ANKRD2, we determined its expression and localization in human heart and compared them with the profiles of homologous cardiac protein ANKRD1. It was found that ANKRD2 is preferentially localized in sarcomere of cardiomyocytes and that level of its expression was lower when compared to ANKRD1. Intercalated disks were identified as a novel site of localization of both proteins. Neonatal rat cardiomyocytes (NRCM) are commonly used as a model system for functional analysis of cardiac-specific genes. One of the goals of this thesis was to characterize this model regarding expression and intracellular localization of endogenous Ankrd2 and exogenous ANKRD2. Nuclear and cytoplasmic localization of Ankrd2 was observed in NRCM, while similar dual localization was established for exogenous ANKRD2 as well. In the contrast to inhibitory effect on Ankrd1 expression, cardio-toxic drug doxorubicin did not change the protein level of Ankrd2 in NRCM. In this thesis we also characterized three ANKRD2 isoforms, containing 446 (LANKRD2), 360 (M-ANKRD2) i 333 (S-ANKRD2) amino acids. Their discrimination in human striated muscles was achieved by specifically designed antibodies and PCR primers. Various similarities between S- and M-ANKRD2 isoforms were discovered, as well as several significant differences, which could reflect on their specific functions... | false |
Biološka aktivnost etarskih ulja odabranih aromatičnih biljaka na vrste rodova Staphylococcus, Streptococcus, Lactobacillus, Pseudomonas, Entercoccus i Candida izlovane iz usne duplje čoveka | Biological activity of essential oils of selected aromatic plants on species of the genera Staphylococcus, Streptococcus, Lactobacillus, Pseudomonas, Enterococcus and Candida isolated from the human oral cavity | Oralno zdravlje smatra se važnim delom opšteg zdravlja ljudi, ali je često zanemarena oblast,
a razlozi tome su raznovrsni i kompleksni. Visoka učestalost oralnih oboljenja ima veliki
socijalni i ekonomski uticaj. Najčešća oboljenja usne duplje su karijes, parodontalna oboljenja
i oralne infekcije, dok su glavni uzročnici ovih oboljenja vrste rodova Streptococcus mutans-
grupa, Staphylococcus, Lactobacillus, Pseudomonas, Enterococcus i Candida. Efikasan
tretman i razvoj novih antiseptika koji poseduju antimikrobnu aktivnost je od velikog značaja
za medicinu. Najčešće se koriste preparati koji sadrže hlorheksidin, povidon jod i
cetilpiridinijum hlorid, zatim sintetička sredstva kao što su penicilin, cefalosporini, tetraciklini
i njihovi derivati, nistatin, amfoterecin B, flukonazol itd. Ovi agensi poseduju antimikrobno
dejstvo i kontrolišu upalu desni, ali imaju ograničenu efikasnost i veliki broj negativnih
posledica kao što su bojenja zuba, iritacije sluzokože usne duplje, pojave alergije,
nefrotoksičnost i endokrinu toksičnost, a povezuju se i sa kancerom i drugim sekundarnim
oboljenjima. Problem rezistencije prema Svetskoj zdravstvenoj organizaciji je najveća pretnja
globalnom zdravlju čoveka. Jedna od alternativa koja je poslednjih decenija u žiži naučnih
interesovanja jesu etarska ulja kao izvor biološki aktivnih jedinjenja.
U cilju dobijanja novih biološkli aktivnih jedinjenja, koja neće biti toksična za humane i
animalne ćelije, u ovoj disertaciji analiziran je hemijski sastav i biološka aktivnost in vitro 60
komercijalnih uzoraka etarskih ulja ekstrahovano iz 47 biljnih vrsta: Cananga odorata,
Foeniculum vulgare, Pimpinella anisum, Pastinaca sativa, Anethum graveolens, Matricaria
recutita, Achillea millefolium, Helichrysum italicum, Boswellia carterii, Canarium luzonicum,
Commiphora myrrha, Juniperus communis, J. virginiana, Gaultheria procumbens, G.
fragrantissima, Pelargonium graveolens, Hyssopus officinalis, Mentha piperita, M. pulegium,
Lavandula angustifolia, Thymus algeriensis, T. vulgaris, T. serpyllum, Rosmarinus officinalis,
Salvia lavanudilifolia, S. officinalis, S. sclarea, S. triloba, Satureja montana, Laurus nobilis,
Leptospermum petersonii, L. scoparium, Eucalyptus citriodora, E. globulus, Melaleuca
alternifolia, M. quinquenervia, Syzygium aromaticum, Abies procera, A. grandis, A. alba,
Pseudotsuga douglasii, Pinus silvestris, Piper nigrum, Cymbopogon martinii, Rosa centifolia,
Citrus aurantium spp. aurantium i C.limon. Cilj rada je ispitivanje biološke aktivnosti i uticaja
etarskih ulja i komponenti odabranih aromatičnih biljaka na mikroorganizme izolovane iz usne
duplje čoveka i primena u prevenciji i lečenju oralnih bolesti i infekcija. Kvalitativna i
kvantitativna analiza uzoraka etarskih ulja vršena je gasnohromatografski korišćenjem GC/MS
i GC/FID procedura. Ukupno je identifikovano 342 komponente iz grupa jedinjenja
oksidovanih monoterpena, monoterpenskih ugljovodonika, seskviterpenskih ugljovodonika,
oksidovanih seskviterpena, fenilpropanoida, spiroetra, alifatičnih ketona, viših alkana,
oksidovanih diterpena, alifatičnih ugljovodonika i oksidovana alifatična jedinjenja.
U periodu 2010-2011. godine izvršena je izolacija i identifikacija mikroorganizama iz usne
duplje pacijena sa Klinike za dečju i preventivnu stomatologiju Stomatološkog Fakulteta,
Univerziteta u Beogradu. Antibakterijska aktivnost testirana je na 8 kliničkih izolata
(Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, Lactobacillus
acidophilus, Streptococcus salivarius, Streptococcus sanguinis, Pseudomonas aeruginosa i
Enterococcus feacalis). Antifungalna aktivnost ispitana je na 58 kliničkih izolata (Candida
albicans, C. glabrata i C. krusei) i dva referentna soja (C. albicans i C. tropicalis).
Antimikrobna aktivnost etarskih ulja određivana je pomoću mikrodilucione i bioautografske
metode na TLC pločama. Određivane su minimalne inhibitorne (MIK) i minimalne
baktericidne i fungicidne (MBK/MFK) koncentracije. Kao pozitivne kontrole korišćeni su
komercijalni antibiotici i mikotici (Ampicilin, Streptomicin i Flukonazol) i preparati za
održavanje oralne higijene (Hexoral® i Curasept®), kao i koloidni rastvor srebrne vode i
ozona.
Rezultati mikrodilucione metode su pokazali da etarsko ulje T. serpyllum poseduje najbolji
antibakterijski potencijal, dok su ulja M. recutita1 i J. virginiana pokazala najslabije dejstvo na
ispitivane bakterije. Najbolji antifungalni potencijal pokazalo je etarsko ulje S. montana, dok
je najslabiju aktivnost imalo etarsko ulje H. italicum2. Prema rezultatima korišćenja
bioautografske metode na TLC pločama sva etarska ulja i izolovana jedinjenja pokazala su
antimikrobnu aktivnost. Za potvrđivanje antimikrobne aktivnosti i ispitivanje mehanizma
dejstva etarskih ulja i komponenti korišćena je skening elektronska mikroskopija (SEM),
kojom je potvrđena njihova aktivnost na destabilizacije ćelijske membrane ćelija C. albicans.
Korišćenjem kriterijuma jačine antimikrobne aktivnosti etarska ulja odabrana su za ispitivanje
međusobne interakcije, antiquorum sensing, antibiofilm i citotoksičnog efekta/potencijala su
bila C. limon, P. nigrumZ, M. alternifolia2, T. algeriensis, T. serpyllum, T. vulgaris, G.
fragrantissima, P. graveolens, C. limon, R. centifolia, L. petersonii, S. montana, C. martinii i
C. myrrha. Međusobne interakcije etarskih ulja i njihovih komponenti testirane su na
reprezentativne kliničke izolate S. mutans i C. albicans. Rezultati ispitivanja pokazuju
sinergističko, aditivno i antagonističko dejstvo. U okviru antiqourum-sensing aktivnosti,
ispitan je uticaj ulja na proces kretanja ćelija P. aeruginosa PAO1 (twiching i flagella test),
produkciju piocijanina kao i sposobnost formiranja biofilma. Odabrana etarska ulja su se
pokazala vrlo efikasnim u inhibiciji quorum-sensing zavisnih procesa i pokazala jak
antiqourum sensing potencijal. Testirane minimalne inhibitorne i sub-inhibitorne koncentracije
etarskih ulja inhibirale su proces formiranja biofilma kod S. mutans i C. albicans, u odnosu na
kontrolnu grupu. Citotoksičnost etarskih ulja ispitivana je pomoću sulforodamin B testa (Sr-B
test) i test redukcije tetrazola (MTT test) na ćelijske linije MCF-7, NCI-H 46, HCT15, HeLa,
HepG2, PLP2, LS-174, K562, A549, Fem-X i MRC-5. Kvantifikacijom stepena citotoksičnosti
odabranih etarskih ulja, utvrđeno je da pokazuju dejstvo na tumorske ćelijske linije. Prikazana
biološka aktivnost odabranih etarskih ulja na mikroorganizme iz usne duplje čoveka ukazuje
na mogućnost izrade preparata na bazi etarskih ulja i njegove upotrebe u terapijske i
profilaktičke svrhe.
Naučna oblast: Biologija
Uža naučna oblast: Mikologija
UDK broj: [582.28:616.31-083]:581.135.5:616-022.7(043.3) | Oral health is considered an important part of general health, but is often neglected area, and
the reasons for this are diverse and complex. The high incidence of oral disease has a huge
social and economic impact. The most common diseases of the oral cavity are dental caries,
periodontal disease, and oral infections, while the main causal agents of these diseases are
species of genus Streptococcus mutans-group, Staphylococcus, Lactobacillus, Pseudomonas,
Enterococcus and Candida. Effective treatment and developing new drugs that have
antimicrobial activity is of great significance for medicine. The most commonly used
preparations containing chlorhexidine, povidone iodine and cetylpyridinium chloride, followed
by synthetic drugs such as penicillin, cephalosporins, tetracyclines and their derivatives,
nystatin, amphotericin B, fluconazole, etc. These agents possess antimicrobial activity and
control gum inflammation, but have limited efficacy and a large number of negative effects
such as tooth staining, mucosal irritation, allergy, nephrotoxicity and endocrine toxicity, and
are also associated with cancer and other secondary illnesses. The problem of resistance to the
World Health Organization is the biggest threat to global human health. One of the alternatives
that in recent decades are in focus of scientific interest are essential oils as a source of
biologically active compounds.
In order to obtain new biologically active compounds, which will not be toxic to human and
animal cells, in this dissertation analysis of the chemical composition and biological activity in
vitro of 60 commercial samples of essential oils extracted from 47 plant species were used:
Cananga odorata, Foeniculum vulgare, Pimpinella anisum, Pastinaca sativa, Anethum
graveolens, Matricaria recutita, Achillea millefolium, Helichrysum italicum, Boswellia
carterii, Canarium luzonicum, Commiphora myrrha, Juniperus communis, J. virginiana, G .
fragrantissima, Pelargonium graveolens, Hyssopus officinalis, Mentha piperita, M. pulegium,
Lavandula angustifolia, Thymus algeriensis, T. vulgaris, T. serpyllum, Rosmarinus officinalis,
Salvia lavanudilifolia, S. officinalis, S. sclarea, S. triloba, Satureja montana, Laurus nobilis,
Leptospermum petersonii, L. scoparium, Eucalyptus citriodora, E. globulus, Melaleuca
alternifolia, M. quinquenervia, Syzygium aromaticum, Abies procera, A. grandis, A. alba,
Pseudotsuga douglasii, Pinus silvestris, Piper nigrum, Cymbopogon martinii, Rosa centifolia,
Citrus aurantium spp. aurantium and C. limon. The aim was to test their biological activity and
the impact of essential oils and components from selected aromatic plants to microorganisms
isolated from human oral cavity for the application in the prevention and treatment of oral
diseases and infections. Qualitative and quantitative analysis of essential oils Gas
chromatographic was performed using GC / MS and GC / FID procedures. A total of 342
compounds were identified belonging to different groups such as oxidized monoterpenes,
monoterpenskih hydrocarbons, sesquiterpene hydrocarbons, oxidized sesquiterpenes
phenylpropanoids, spiro ethers, aliphatic ketones, higher alkanes, oxidized diterpenes, aliphatic
hydrocarbons and oxidized aliphatic compounds.
In the period 2010-2011 the isolation and identification of microorganisms from the oral cavity
were collected of patients who attend the Clinic for Pediatric and Preventive Dentistry, Faculty
of Dentistry, University of Belgrade. The antibacterial activity was tested on 7 clinical isolates
of bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans,
Lactobacillus acidophilus, Streptococcus salivarius, Streptococcus sanguinis, Pseudomonas
aeruginosa and Enterococcus faecalis). Antifungal activity was investigated on 58 clinical
isolates (Candida albicans, C. glabrata and C. krusei) and two reference strains of fungi (C.
albicans and C. tropicalis). The antimicrobial activity of essential oils was determined by
microdilution and bioautography method on TLC plates. Determination of minimal inhibitory
concentrations (MIC) and minimum bactericidal and fungicidal (MBC / MFC) concentration
were done. As a positive control we used a commercial antibiotics and antimycotics
(Ampicillin, Streptomycin and Fluconazole), and preparations for oral hygiene (Hexoral® and
Curasept®), as well as colloidal silver, and a ozone gas.
Results from the microdilution method showed that the essential oil of T. serpyllum has the
best antibacterial potential, while oil M. recutita1 and J. virginiana showed the lowest effect
on the examined bacteria. Best antifungal potential was achived by the essential oil of S.
montana, while the lowest activity had essential oil of H. italicum2. According to the results of
bioautography method used on TLC plates, all essential oils and isolated compounds showed
antimicrobial activity. To confirm the antimicrobial activity and the potential mechanism of
action of essential oils and components scanning electron microscope was used, which
confirmed their activity on the cell membrane destabilisation to C. albicans. The essential oils
with the best antimicrobial activity, as main criteria, were selected for further study as testing
interactions, antiquorum sensing, antibiofilm and cytotoxic effect / potential C. limon, P.
nigrumZ M. alternifolia2, T.algeriensis, T. serpyllum, T. vulgaris, G. fragrantissima, P .
graveolens, R. centifolia, L. petersonii, S. montana, C. martinii and C. myrrha. Mutual
interactions of essential oils and their components are tested on a representative clinical isolates
of S. mutans and C. albicans. The test results demonstrate the synergistic, additive and
antagonistic effects. Within antiqourum-sensing activities, the effects of the oils on the process
of moving of P. aeruginosa PAO1 (twiching and flagella test), piocyanin production, as well
as the ability to form biofilms were investigated. Selected essential oils have proven very
effective in the inhibition of quorum sensing dependent processes and showed strong
antiqourum sensing potential. Tested minimum inhibitory and sub-inhibitory concentrations of
essential oils were found to inhibit the process of biofilm formation of S. mutans and C.
albicans in comparison to the control group. The cytotoxicity of the essential oils was tested
using a sulforhodamine B assay (Sr-B test) and the test of the tetrazole salt reduction (MTT
assay) in the cell lines MCF-7, NCI-H 46, HCT15, HeLa, HepG2, PLP2, LS-174, K562, A549,
Fem-X and MRC-5. The results of the quantification of cytotoxicity of the essential oils
selected, it has been found to exhibit the effect on all tumor cell lines. Displayed biological
activity of selected essential oils on microorganisms from the oral cavity as man point show
the possibility of making preparations based on essential oils and their use in therapeutic and
prophylactic purposes.
Scientific field: Biology
Specific scientific field: Plant Physiology
UDC number: [582.28:616.31-083]:581.135.5:616-022.7(043.3) | true |
Karakterizacija i in vitro biološke aktivnosti fenolnih jedinjenja graška (Pisum sativum L) | Characterization and in vitro biological activities of phenolic compounds from pea (Pisum sativum L.) | Porodica mahunarki (Fabaceae) obiluje ekonomski i poljoprivredno značajnim vrstama
među kojima se po zastupljenosti gajenja u Srbiji izdvaja grašak (Pisum sativum L.) sa udelom
od 4,5% u ukupnoj povrtarskoj proizvodnji. Semenjače graška, posebno genotipova sa izrazito
pigmentisanim tamnim semenima predstavljaju značajan izvor fenolnih jedinjenja čiji su sastav i
biološke aktivnosti nedovoljno ispitani, te stoga ni potencijal ovog materijala nije iskorišćen na
odgovarajući način. Semenjače graška čine i do 10% suve mase semena i uglavnom predstavljaju
otpadni materijal koji nastaje u procesu industrijske izolacije proteina semena koji se koriste u
humanoj i animalnoj ishrani. U ovom radu je ispitivan sadržaj fenolnih jedinjenja prisutnih u
semenjačama deset evropskih genotipova graška kao i njihove biološke aktivnosti. Pored toga je
ispitivan i uticaj različitih rastvarača, termičke obrade i in vitro digestije semena graška na prinos
fenolnih jedinjenja i antioksidativne aktivnosti dobijenih ekstrakata.
Dobijeni rezultati su pokazali da termički tretman i in vitro digestija dovode do statistički
značajnog povećanja sadržaja fenolnih jedinjenja, kako u vodenim ekstraktima semena graška
tako i u ekstraktima pripremljenim pomoću organskog rastvarača. Utvrđeno je da peptidi koji
nastaju u toku in vitro digestije proteina semena graška mogu značajno interferirati sa testovima
za određivanje antioksidativne aktivnosti, pogotovo u vodenim rastvorima, pošto i sami poseduju
detektabilne antioksidativne aktivnosti. Pokazano je da kombinacija rastvarača koja sadrži 70%
acetona, 29% destilovane vode i 1% sirćetne kiseline najpodesnije sredstvo za ekstrakciju
ukupnih fenola iz semenjače graška, koje daje najveći prinos kako kod genotipova sa bledom,
tako i kod genotipova sa izrazito tamno pigmentisanom semenjačom. Sadržaj ukupnih fenola u
ekstraktima dobijenim primenom pomenutog rastvarača se kod pojedinih genotipova (Assas,
MBK 168 i Aslaug) kretao u opsegu 41,8-46,6 mg GAE/ g s.m., što su najviše do sada
zabeležene vrednosti kod graška. Isti genotipovi su ujedno sadržali najviše taninskih materija i
antocijanina i pokazivali su najizraženije sposobnosti uklanjanja DPPH radikala i redukcije Fe3+
jona. Analizom ekstrakata semenjača pomoću UHPLC–LTQ OrbiTrap MS tehnike
identifikovano je 25 najzastupljenijih fenolnih jedinjenja i tom prilokom je detektovano 9
jedinjenja čije prisustvo do sad nije bilo zabeleženo u semanjačama graška. Prvi put je
detektovano prisustvo flavanona hrizina, hesperetina, naringenina i naringina kao i flavanola
katehin galata i galokatehin galata. Takođe su detektovane i manje količine morina, galangina i
ruzmarinske kiseline kod većine genotipova. Analiza glavnih komponenata (PCA) je pokazala da
se na osnovu sadržaja pojedinačnih fenolnih jedinjenja kao i antioksidativnih aktivnosti
ekstrakata ispitivani genotipovi mogu svrstati u dve glavne grupe. Prvu grupu čine genotipovi sa
bledo pigmentisanim semenjačama Golf, Törsz i MBK 88, u drugoj grupi se nalaze genotipovi sa
tamnom semenjačom Aslaug, Assas, Dora, Poneka, MBK 168 i MBK 173, dok je genotip MBK
90 bio van pomenutih grupa. Ono što je značajno istaći je da raspodela genotipova na osnovu
PCA analize odgovara raspodeli genotipova u grupe na osnovu boje semenjače. Ekstrakti osam
od deset ispitivanih genotipova, u kojima su koncentracije najzastupljenijih komponenti bile
ispod 1µM, su ispoljavali protektivni efekat na ćelije NT2-D1 linije humanog teratokarcinoma
umanjujući stvaranje reaktivnih vrsta kiseonika koje je bilo izazvano egzogenom primenom
vodonik peroksida. Ekstrakti semenjača genotipova: Aslaug, Assas, Dora, Poneka, Golf, MBK
168 i MBK 173 su pokazivali citotoksične efekte zavisne od koncentracije na svim ispitivanim
ćelijskim linijama (humanog karcinoma dojke MDA-MB-453, adenokarcinoma kolona LS174,
hronične mijeloidne leukemije K562 i karcinoma pluća A549). Iako nije primećena značajna
selektivnost u antitumorskom dejstvu, ekstrakti su pokazivali slabiji intenzitet dejstva prema
normalnoj MRC-5 ćelijskoj liniji. U eksperimentima na LS174 i K562 linijama zabeleženo je
značajno povećanje broja ćelija koje se nalaze u subG1 fazi ćelijskog ciklusa nakon tretmana
pomenutim ekstraktima. Eksperimenti u kojima su LS174 ćelije tretirane inhibitorom kaspaze 3,
pre tretmana ekstraktima su pokazali da vodeni ekstrakti semenjača graška mogu pokrenuti i neki
drugi put aktivacije ćelijske smrti, različit od apoptotskog posredovanog kaspazama. | The family of legumes (Fabaceae) is rich in economicaly and agriculturally important
species among which the most abundant in Serbia stands peas (Pisum sativum L.) with a share of
4.5% of the total vegetable production. Pea seed coats, especially in genotypes with highly
pigmented dark seeds are an important source of phenolic compounds, whose composition and
biological activity have not been tested in detail, thus this valuable material is still underutilized.
Pea seed coat makes up to 10% of the seed dry weight and largely represents waste material
generated in the process of industrial isolation of seed proteins, which are used in human and
animal nutrition. In this study the content of phenolic compounds in seeds of ten European pea
genotypes, as well as their biological activities has been examined. In addition, the effect of
different solvents, heat treatment and in vitro digestion of pea seeds on yield of phenolic
compounds and antioxidant activity in extracts was also studied.
The obtained results showed that the thermal treatment and in vitro digestion led to a
statistically significant increase of the total phenolic content, both in aqueous extracts of pea
seeds and in the extracts prepared by using an organic solvent. It was shown that peptides which
were formed during the in vitro digestion of the pea seed proteins can significantly interfere with
the assays for determination of antioxidant activity, especially in aqueous solutions, since they
possess a detectable antioxidant activity. It has been shown that the combination of solvents
containing 70% acetone, 29% distilled water and 1% acetic acid was the most suitable means for
the extraction of total phenols from pea seed coats, which gave the highest yield in both bright
colored and dark colored seed genotypes. Total phenolic content of extracts obtained by
mentioned solvent in certain genotypes (Assas, MBK 168 and Aslaug) ranged from 41.8 to 46.6
mg GAE / g s.m., which are the highest recorded values in pea. The same genotypes also
possessed the highest tannin and anthocyanin content and showed the most pronounced ability
for DPPH radical scavenging as well as for the reduction of Fe 3+ ions. The analysis of seed coat
extracts, using the LTQ Orbitrap UHPLC-MS technique, identified 25 most abundant phenolic
compounds, from which 9 compounds have not been recorded in pea seed coats of previously
studied genotypes. It was the first report of flavanones: chrysin, hesperetin, naringenin and
naringin. Certain flavanols: catechin gallate and gallocatechin gallate were also detected. Small
amounts of morin, galangin and rosmarinic acid were detected in most genotypes. The principal
components analysis (PCA) based on the content of individual phenolic compounds and
antioxidant activity of extracts grouped genotypes into two main groups. The first group consists
of genotypes with pale seed coats Golf, Törsz and MBK 88, in the second group were genotypes
with dark seed coat Aslaug, Assas, Dora, Poneka, MBK 168 and MBK 173, while MBK 90
genotype was outside the groups. What is important to emphasize is that the distribution of
genotypes in groups based on PCA analysis corresponds to the distribution according to seed
coat color. Extracts of eight out of ten investigated genotypes, in which the concentration of the
most abundant compounds were below 1 μM, were displaying a protective effect on NT2-D1
human teratocarcinoma cell line by reducing the formation of reactive oxygen species, which
was caused by exogenous application of hydrogen peroxide. Seed coat extracts obtained from
genotypes: Aslaug, Assas, Dora, Poneka, Golf, MBK 168 and MBK 173 showed cytotoxic
effects dependent on the concentration in all tested cell lines (human breast cancer MDA-MB-
453, colon adenocarcinoma LS174, chronic myeloid leukemia K562 and A549 lung carcinoma).
Although the significant selectivity in the antitumor activity of extracts has not been recorded
they showed weaker cytotoxic activities against normal MRC-5 cell line. In experiments on
LS174 and K562 lines a significant increase in the number of cells located in subG1 phase of the
cell cycle was observed after treatment of the above mentioned extracts. Experiments in which
the LS174 cells were treated with an inhibitor of caspase-3 before treatment with extracts
showed that aqueous extracts of pea seed coats can induce some other pathway of the cell death,
other than apoptotic caspase-mediated pathway. | true |
Populaciona dinamika i ekotoksikologija crnog američkog patuljastog soma (Ameiurus melas Rafinesque, 1820) u Savskom jezeru | Population dynamic and and ecotoxicology of the black bullhead (Ameiurus melas Rafinesque, 1820) in Sava Lake | Crni američki patuljasti som (Ameiurus melas Rafinesque, 1820) je
najzastupljenija i najuspešnija alohtona vrsta ribe koja naseljava kopnene vode Evrope.
Istraživanja koja su sprovedena u proteklih nekoliko godina ukazuju na to da je ovo
dominantna invazivna vrsta i u mnogim kopnenim vodama u Srbiji.
Cilj istraživanja se odnosio na ispitivanje populacionih atributa patuljastog soma
u Savskom jezeru, kao i na bioakumulaciju teških metala i mikroelemenata u različitim
tkivima. Terenska istraživanja vršena su u periodu od 2009. do 2012. godine. Jedinke su
uzorkovane jednom mesečno, četiri dana za redom. Laboratorijska obrada prikupljenih
uzoraka obuhvatila je merenje totalne dužine tela (TL) i ukupne težine tela ulovljenih
primeraka. Nakon disekcije jedinki merena je težina tela bez utrobe, kao i masa jetre i
gonada.
Struktura populacije utvrđena je kombinovanjem dve metode – analizom starosti
jedinki i analizom dužinskih frekvencija. Starost jedinki određena je takođe uz pomoć
dve metode – očitavanjem naraštajnih zona otolita i Batačarija metodom. Analiza
uzrasne strukture populacije pokazala je prisustvo pet uzrasnih kategorija: od 0+ do 4+.
Klasa 1+ bila je najdominatnija uzrasna klasa, kako po godinama, tako i po sezonama
uzorkovanja. Prosečna totalna dužina jedinki iznosila je 13,3 cm, a prosečna težina
38,26 g. Prosečna relativna težina jedinki u Savskom jezeru, u celokupnom uzorku,
iznosila je 84,78 ± 1,09 g. Utvrđeno je prisustvo ukupno 8 kohorti.
Na osnovu analize dužinsko-težinskih odnosa izračunate su i vrednosti faktora
alometrije b koje su se kretale od 2,93 do 3,16, dok je vrednost ovog faktora u celom
poduzorku iznosila 3,00. Analizom krive dužinsko-težinskog odnosa utvrđeno je da
tačka infleksije ne postoji.
Ribolovni napor (CPUE) je korišćen za analizu distribucije i relativne gustine
jedinki, kako po staništima, tako i po godinama uzorkovanja i sezonama. Tokom 2009,
2010. i 2012. godine CPUE vrednosti varirale su od 0,4 ± 0,5 do 41,5 ± 22,1 jedinki po
vrši po danu. Ove vrednosti su 2011. godine bile niske i prilično ujednačene, a kretale
Rezime
vii
su se u opsegu od 0,2 ± 0,3 do 3,4 ± 4,5 jedinki po vrši po danu. CPUE vrednosti bile su
najveće u zoni makrofitske vegetacije, kao i tokom jesenjeg i letnjeg perioda godine.
Teoretski rast dužine procenjen je korišćenjem fon Bertalanfijevog modela.
Paremetri fon Bertalanfijeve krive rasta imali su sledeće vrednosti: L∞ = 26,8 cm, K =
0,43 i t0 = - 0,13, bez razlike između polova. Vrednost koeficijenta φ’ iznosila je 2,49.
Rezultati su pokazali da je srednja vrednost totalne dužine tela tokom prve godine
života (0+) 9,70 cm TL; druge godine (1+) 12,78 cm TL; treće godine (2+) 16,29 cm
TL; četvrte godine (3+) 18,61 cm TL i pete godine (4+) 21,68 cm TL. Rast jedinki je
najbrži između prve (0+) i druge (1+) godine života.
Za analizu ishrane korišćeni su sledeći indeksi: indeks vakuiteta, procentualna
učestalost, procentualni udeo i indeks značajnosti. Širina niše za korišćenje resursa
hrane po sezonama izračunata je prema Šenonovom indeksu diverziteta. Vrednosti
indeksa vakuiteta kretale su se od 72,86% do 100%. Spektar ishrane sačinjavalo je 14
različitih kategorija plena iz pet gupa: Mollusca, Crustacea, Insecta, Teleostei i biljke.
Ribe su bile glavni plen u svim sezonama, a zatim vodeni beskičmenjaci. Jedinke su se
biljnim materijalom i terestričnim insektima hranile uglavnom u manjoj meri.
Zabeležena je i ishrana ikrom. Na osnovu istraživanja potvrđeno je da je ova vrsta
generalista i oportunista, čije jedinke konzumiraju hranu koja je najdostupnija i koje ima
u najvećoj meri, kao i da se ishrana menja tokom ontogenetskog razvića. Najmanja
vrednost trofičkog diverziteta zabeležena je kod najvećih jedinki, a najveća kod jedinki
srednje veličine. Najveća širina niše zabeležena je na proleće 2011. godine.
Gonadosomatski (GSI) i hepatosomatski (HSI) indeksi i faktor kondicije (K)
iskorišćeni su kako bi se dobila slika o alokaciji resursa i opštem stanju jedinki u odnosu
na različite sezone uzorkovanja. Vrednosti GSI bile su najveće u maju, da bi tokom juna
opale, a u julu ponovo porasle. Niske vrednosti GSI mogu se primetiti u jesenjem i
zimskom periodu, kada nema mresta. Zabeležene su visoke vrednosti HSI tokom jeseni
i zime. Vrednosti K su najveće tokom leta.
Od osobina životne istorije, analizom su obuhvaćene veličina jedinki pri
dostizanju polne zrelosti i fekunditet. Rezultati su pokazali da se veličina jedinki koje
prvi put dostižu polnu zrelost kreće u intervalu od 10 cm do 12 cm TL (1+). Od 15 cm
TL (2+), sve jedinke se smatraju polno zrelim. Vrednosti apsolutnog i relativnog
Rezime
viii
fekunditeta u aprilu (pred sam početak mresta) bile su izuzetno visoke. Krajem maja i
početkom juna opadaju, da bi tokom jula bile još niže.
Masovni pomor jedinki crnog američkog patuljastog soma dogodio se tokom
maja 2011. godine. Uzrok masovnog uginuća nije utvrđen sa sigurnošću, ali je
najverovatnije usledio kao posledica virusne infekcije (virus američkog somića) koja je
izazvala oboljenje epizootsku hematopoeznu nekrozu (EHN). Pokazatelj obnove
populacije nakon masovnog pomora bio je odnos juvenilnih i adultnih jedinki, odn.
regrutacija. Obnova populacije usledila je već tokom naredne godine, kada je udeo
juvenilnih jedinki (65,8%) bio dvostruko veći od udela adultnih jedinki (34,2%).
Zdravstveno stanje populacije utvrđeno je na osnovu analize ekstenziteta i
intenziteta parazitiranosti analiziranih jedinki. Osim kod jedne jedinke, svi endoparaziti
pripadali su razdelu Nematoda. Najverovatnije se radilo o vrsti Philometra richta.
Zdravstveno stanje populacije je tokom 2012. godine bilo znatno bolje u odnosu na
2011. godinu. Jedinke starosti 3+ bile su najzaraženije endoparazitima.
Selektivnost vrša procenjena je na osnovu odnosa uhvaćenih jedinki crnog
američkog patuljastog soma i drugih vrsta riba, za svaki mesec pojedinačno. Pored
jedinki crnog američkog patuljastog soma, uzorkovane su i jedinke grgeča, sunčice,
glavoča i bodorke. Ulov šarana, smuđa, štuke, babuške i deverike bio je na nivou
statističke greške. Zaključak je da se vrše mogu primenjivati u kontroli brojnosti jedinki
crnog američkog patuljastog soma, jer osim što su efikasne i selektivne, njima se veoma
lako rukuje, a pri tom se ne oštećuju druge vrste ribe.
Za analizu dužina i težina jedinki urađena je i trofaktorska MANOVA, nakon
koje je korišćen Dankanov test. Za testiranje hipoteze o distribuciji jedinki po nizovima,
sezonama i dubinama, korišćeni su neparametarski testovi: Kruskal-Valis za k
nezavisnih uzoraka i post-hok Man-Vitni test za dva nezavisna uzorka. Multivarijaciona
analiza je pokazala da jedino sezona značajno utiče kako na prosečne dužine i težine
izlovljenih jedinki (osim 2011. godine), tako i na brojnosti.
Demografska analiza je potvrdila veliku fleksibilnost osobina životne istorije.
Preživljavanje je najvažniji/najuticajniji parametar životne istorije. Starost pri kojoj
ženke dostižu polnu zrelost jeste parametar čija se fleksibilnost posebno povećava kada
populacija raste, dok fleksibilnost maksimalne starosti i preživljavanja opada sa rastom
Rezime
ix
populacije. Maksimalna starost je osobina koja je pokazala najmanju fleksibilnost.
Fekunditet i preživljavanje juvenilih jedinki imaju isti stepen fleksibilnosti.
Uzorci tkiva bili su liofilizovani i digestovani pre očitavanja vrednosti
koncentracije teških metala i mikroelemenata primenom metode induktivno spregnute
plazme-optičko emisione spektrometrije. Man-Vitni test je korišćen za statističku
analizu, dok je za procenu razlika između analiziranih tkiva korišćena analiza glavnih
komponenti (PCA). Koncentracija teških metala bila je najmanja u mišićima, a najveća
u
jetri
i
škrgama.
Koncentracija
elemenata
imala
je
sledeći
trend:
Fe>Al>Zn>Sr>As>Mn>Mo
u
škrgama,
Zn>Sr>As>Mo
u
mišićima
i
Fe>Zn>Sr>Cu>As>Mo>Mn u jetri. Svi analizirani elementi u mišićima bili su ispod
granica MDK propisanih nacionalnom legislativom i stoga jedinke ove vrste iz Savskog
jezera ne predstavljaju rizik za čoveka, ukoliko se koriste u ishrani. | abundant and successful non-native fish species in European freshwaters. Recent studies
indicate that the black bullhead is one of the most dominant invasive fish species in
Serbian waters as well.
The aim of this research was to determine the population characteristics of this
species in Sava Lake, as well as bioaccumulation of heavy metals and trace elements in
different tissues. Fieldwork was conducted from 2009 to 2012. Black bullhead samples
were collected monthly, for four days in a row. Once in the laboratory, specimens were
measured for their total length (TL) and body weight (W). After dissection, the
eviscerated body weight was measured, as well as liver and gonad weights.
Population structure was determined by combining two methods – analyzing age
and length-frequency distributions. Age was determined by combining otolith
examination and Bhattacharya’s method. Age structure analysis has revealed the
presence of five age classes: from 0+ to 4+. The class 1+ was the most dominant, by
both year and season of sampling. The average total length was 13.3 cm and the average
weight was 38.26 g. The mean value of the relative weight, across the entire sample,
was 84.78 ± 1.09. The research revealed the presence of eight cohorts, in total.
The length-weight allometric factor b ranged from 2.93 to 3.16, while the value
of this factor, across the entire sample, was 3.00.The analysis of the length-weight
relationship curve showed an absence of inflection points.
Catch-per-unit-effort (CPUE) was used as an indicator of the black bullhead
distribution and abundance in different habitats, years of sampling, and seasons. In
2009, 2010, and 2012, CPUE values fluctuated and ranged from 0.4 ± 0.5 to 41.5 ± 22.1
ind./net/night, while in 2011 they were low and rather uniform, ranging from 0.2 ± 0.3
to 3.4 ± 4.5 ind./net/night. CPUE values were highest in the macrophyte vegetation
zone, as well as during autumn and spring.
Abstract
xi
Theoretical length growth was assessed using the von Bertalanffy model. The
calculated parameters of the von Bertalanffy growth function were: L∞ = 26.8 cm, K =
0.43, t0 = - 0.13, without distinction between sexes. The value of φ’ was 2.49. Our
results indicate that the black bullhead in Sava Lake reached a mean length of 9.70 cm
TL during the first year of life (0+), 12.78 cm TL in the second year (1+), 16.29 cm TL
in the third year (2+), 18.61 cm TL in the fourth year (3+), and 21.68 cm TL in the fifth
year (4+). The growth of black bullhead specimens was fastest between the ages of 0+
and 1+.
For the diet analysis, we used the following indices: the vacuity index, frequency
of occurrence, numerical abundance, and prominence value. In addition, the seasonal
trophic niche breadth was calculated according to the Shannon index of diversity.
Values of the vacuity index ranged between 72.86% and 100%.The diet spectrum
consisted of 14 different prey categories from five groups: Mollusca, Crustacea, Insecta,
Teleostei, and plants. Fish were the main prey in all seasons, followed by aquatic
invertebrates. Plant material and terrestrial insects were used as food in relatively small
quantities. Egg predation was also detected. Our research confirmed that this species is
an opportunistic generalist, foraging on the most abundant and available prey.
Ontogenetic shift was also detected. The lowest value of trophic diversity was found in
the largest black bullheads, while the highest was found in the medium sized
individuals. The widest niche breadth was recorded in the spring of 2011.
Gonadosomatic (GSI) and hepatosomatic (HSI) indices and Fulton's condition
factor (K) were used to gain insight into allocation of resources and the general
condition of individuals in relation to different sampling seasons. GSI values were
highest in May. They declined during June and then went up again in July. Low GSI
values can be noticed during autumn and winter, when there is no spawning. HSI values
were highest during autumn and winter and K values were highest during summer.
Regarding the life history traits, the analysis included the size at first maturity
and fecundity. The results showed that length at sexual maturity ranged from 10 cm to
12 cm TL (1+). From 15 cm TL (2+), all individuals are considered sexually mature.
Absolute and relative fecundities were extremely high in April. In late May and early
June they declined, and in July these values were even lower.
Abstract
xii
Mass mortality of black bullhead in Sava Lake occurred in May 2011. Although
the cause of the mass mortality was not determined with certainty, it was probably the
result of a viral infection (European catfish virus) that causes epizootic haematopoietic
necrosis (EHN). The indicator of the recovery potential of the black bullhead population
was the relationship between the juvenile and the adult specimens, i.e. the recruitment.
The studied black bullhead population recovered within the timeframe of a single year
when the proportion of juvenile specimens (65.8%) was twice as high as the proportion
of the adult specimens (34.2%).
The health status of the population was determined based on the analysis of
extensiveness and intensity of parasitism in analyzed specimens. Except for one
individual, all endoparasites belonged to the phylum Nematoda, most likely the species
Philometra richta. The health status of the population in 2012 was much better
compared to 2011. Individuals aged 3+ were most infected by endoparasites.
The selectivity of fyke nets was estimated based on the ratio of the captured
individuals of black bullhead and other fish species, for each month. Besides specimens
of black bullhead, individuals of European perch, pumpkinseed, gobies, and roach were
also sampled. The catch of carp, pikeperch, pike, Prussian carp, and bream was at the
level of statistical error. The conclusion is that fyke nets can be applied to control the
abundance of black bullhead because, in addition to being effective and selective, they
are easy to handle and cause no damage to other fish species.
Three-way MANOVA was used for the analysis of individual length and weight.
Following that, the Duncan test was used. To test the hypotheses about the distribution
of specimens per rows, seasons, and depths, the following nonparametric tests were
used: the Kruskal-Wallis for k independent samples and post-hoc Mann-Whitney test
for two independent samples. Multivariate analysis showed that only the season
significantly affected both the average length and weight of sampled specimens (except
for 2011), as well as abundance.
Demographic analysis confirmed a high flexibility of life history traits. Survival
was the most influential life history parameter, followed by age at which females reach
maturity. The age at which females reach maturity is the parameter whose flexibility
especially increases when the population grows, while flexibility of longevity and
survival decreases with increasing population size. Maximum longevity was the
Abstract
xiii
characteristic that showed the least flexibility. Fecundity and survival of juvenile
specimens have the same degree of flexibility.
Tissue samples were prepared using a lyophilizer and microwave digestion prior
to reading values by using inductively coupled plasma – optical emission spectrometry
(ICP-OES). For statistical analysis, the Mann-Whitney U test was used, while principal
components analysis (PCA) was used to assess the difference between the analyzed
tissues. Elemental concentrations were lowest in muscles and highest in liver and gills.
The concentration of elements showed the following trend: Fe>Al>Zn>Sr>As>Mn>Mo
in gills, Zn>Sr>As>Mo in muscles, and Fe>Zn>Sr>Cu>As>Mo>Mn in liver. All
analyzed elements in muscle samples were below MAC prescribed by the national
regulation of the Republic of Serbia; therefore, the consumption of these fish from Sava
Lake does not pose a risk to human health. | true |
Citokininska homeostaza i ekspresija gena uključenih u kontrolu ćelijskog ciklusa tokom procesa regeneracije kelerabe (Brassica oleracea L. var. gongylodes) in vitro | Cytokinin homeostasis and expression of the genes involved in cell cycle control during kohlrabi (Brassica oleracea L. var. gongylodes) regeneration in vitro | Regeneracija in vitro gajenog biljnog tkiva kontrolisana je regulatorima rastenja,
naročito citokininima i auksinima. Egzogeno primenjeni biljni regulatori rastenja mogu
interagovati sa endogenim biljnim hormonima i uticati na njihovu biosintezu ili
distribuciju, odnosno menjati hormonalnu homeostazu koja utiče na razviće in vitro.
Proces regeneracije biljaka in vitro podrazumeva intenzivne ćelijske deobe, a centralnu
ulogu u kontroli ćelijskog ciklusa i razvića organa imaju kompleksi ciklina i kinaza
zavisnih od ciklina na čiju sintezu, odnosno aktivnost, utiču pomenuti biljni hormoni. U
tom smislu, ciljevi ovog rada bili su uspostavljanje efikasnih protokola za regeneraciju
biljaka kelerabe (Brassica oleracea L. var. gongylodes) in vitro, sa posebnim osvrtom
na identifikaciju i kvantifikaciju različitih grupa citokinina i indol-3-sirćetne kiseline
(IAA) u eksplantatskim tkivima sa regenerisanim izdancima, kao i analiziranje obrazaca
i intenziteta ekspresije gena koji kodiraju cikline i kinaze zavisne od ciklina tokom de
novo organogeneze izdanaka. Efikasan sistem za repetitivnu somatsku embriogenezu
(SE) kod kelerabe, sorta Bečka Plava (BP), je uspostavljen iz nezrelih zigotskih
embriona. Najveća frekvencija SE detektovana je kod nezrelih zigotskih embriona u
ranom kotiledonarnom stadijumu razvića gajenih na hranljivoj podlozi bez regulatora
rastenja.
Uspešna indukcija de novo organogeneze izdanaka kelerabe, sorte BP i Bečka Bela
(BB), postignuta je kod odsečaka hipokotila i intaktnih klijanaca, na podlogama sa
pojedinačnim citokininima (CK): 6-benzilaminopurin (BAP), tidiazuron (TDZ), trans-
zeatin (transZ) ili cis-zeatin (cisZ). Frekvencija regeneracije kao i vijabilnost
regenerisanih izdanaka obe sorte je bila veća kod intaktnih klijanaca, dok su među
primenjenim citokininima BAP i TDZ bili najefikasniji u indukciji de novo
organogeneze. Kvantifikacija endogenog sadržaja citokinina i IAA u odsečcima
hipokotila sa regenerisanim izdancima (HRI) kao i u intaktnim klijancima sa
regenerisanim izdancima (KRI) je ukazala na to da uočene razlike u organogenom
odgovoru ova dva tipa početnog biljnog materijala mogu biti povezane sa njihovim
hormonskim statusom. Nivo ukupnih endogenih citokinina je bio viši kod HRI, dok su
se KRI odlikovali višim odnosom IAA i bioaktivnih citokinina. Izraziti porast nivoa
endogenih citokinina u odnosu na kontrolu koja je gajena na podlozi bez regulatora
rastenja zabeležen je na tretmanima sa zeatinom, dok je prisustvo BAP i TDZ imalo
mali efekat ili je efekat potpuno izostao. U poređenju sa kontrolom, egzogena primena
citokinina dovela je do porasta endogenog nivoa IAA i u HRI i u KRI kod obe sorte
kelerabe, čime se verovatno može objasniti odsustvo potrebe za auksinima u hranljivim
podlogama tokom indukcije de novo organogeneze izdanaka.
Dinamiku pojave regenerisanih izdanaka kelerabe na različitim citokininskim
tretmanima je pratio i nivo aktivnosti tri analizirana gena koji kodiraju kinaze zavisne
od ciklina (CDK) i cikline (CYC): CDKB2;1, CYCB2;4 i CYCH;1 gen. Obrasci
ekspresije sva tri gena u odnosu na vreme i različite tretmane su bili slični, sa najvišim
vrednostima zabeleženim na podlozi sa TDZ u skoro svim vremenskim tačkama.
Dobijeni rezultati ukazuju na to da su primenjeni citokinini imali ulogu u kontroli
ćelijskog ciklusa tako što su uticali na ekspresiju pomenutih gena, kao i da je promena
aktivnosti tih gena bila u korelaciji sa određenim fazama de novo organogeneze
izdanaka kelerabe. Iako razjašnjenje uloge citokinina u procesu regeneracije izdanaka
kelerabe zahteva dodatna istraživanja, dobijeni rezultati predstavljaju polaznu osnovu za
dalje rasvetljavanje mehanizama de novo organogeneze kod biljaka koje nisu uobičajeni
model-sistemi za izučavanje procesa regeneracije in vitro. | vitro plant regeneration is under control of plant growth regulators (PGRs), especially
cytokinins and auxins. Exogenously applied PGRs can affect the homeostasis of
endogenous plant hormones by influencing their biosynthesis and distribution,
subsequently altering in vitro development. Intensive cell divisions represent the basis
of in vitro plant regeneration, and they are controlled by complexes of cyclins and
cyclin-dependent kinases. The activity of these complexes during cell cycle and organ
development is affected by plant hormones. Respecting this, the aims of this study were
developing efficient protocols for in vitro regeneration of kohlrabi (Brassica oleracea
L. var. gongylodes) with an emphasis on identification and quantification of different
groups of endogenous cytokinins and indole-3-acetic acid (IAA) in explant tissue with
respective regenerated shoots, as well as analyzing expression of genes encoding cyclins
and and cyclin-dependent kinases during de novo shoot organogenesis. Efficient system
for repetitive somatic embryogenesis (SE) was developed for kohlrabi cv. Vienna
Purple (VP) using immature zygotic embryos. The highest frequency of SE was
obtained from immature zygotic embryos at the early cotyledonary stage grown on
PGR-free medium.
Successful induction of de novo shoot organogenesis in kohlrabi cv VP and cv. Vienna
White (VW) was achived using hypocotyl explants and intact seedlings, cultivated on
media supplemented with single cytokinin (CK): 6-benzylaminopurine (BAP),
thidiazuron (TDZ), trans-zeatin (transZ) or cis-zeatin (cisZ). Higher frequency of shoot
regeneration as well as viability of regenerated shoots was detected for intact seedlings,
with BAP and TDZ being the most efficient in de novo organogenesis induction.
Quantification of endogenous cytokinin and IAA contents in both hypocotyl explants
and seedlings with regenerated shoots (HRSs and SRSs, respectively) suggested that the
observed differences in organogenic response between these two types of starting plant
material were related to their cytokinin and IAA contents. The level of total CKs was
higher in HRSs, while SRSs displayed a higher IAA/bioactive CKs ratio. Significant
increase in the endogenous CK levels compared to control grown on PGR-free medium
was recorded only when cis- and transZ were applied, while presence of BAP and TDZ
in the shoot regeneration media contributed to little or no increase. Compared to control,
exogenous addition of CKs to the regeneration media contributed to an increase in
endogenous levels of IAA in both HRSs and SRSs of both cultivars indicating that
elevated IAA levels could be sufficient to induce de novo organogenesis without
exogenously applied auxins.
The dinamics of regenerated shoots development were accompanied by expression of
the three analyzed genes encoding cyclin-dependent kinases (CDKs) and cyclins
(CYC): CDKB2;1, CYCB2;4 and CYCH;1 gene. Expression patterns of all three genes
in terms of time and different treatments were similar, with the highest values recoded
on TDZ treatment in almost all time points. Our results suggested the role of applied
CKs in cell cycle control by influencing the expression of analyzed genes, and that the
changes in activity of these genes was correlated with distinct phases of de novo shoot
organogenesis in kohlrabi. Although clarification of cytokinin role in regeneration of
kohlrabi shoots requires additional research, these results present useful background for
further investigation of mechanisms involved in de novo organogenesis in plants that do
not represent common model systems for in vitro regeneration study. | true |
Karakterizacija molekulskih vrsta specifičnog antigena prostate čoveka primenom imunoafinitetne hromatografije na čipu | Characterization of molecular species of human prostate-specific antigen by on-chip immunoaffinity chromatography | KARAKTERIZACIJA MOLEKULSKIH VRSTA SPECIFIČNOG ANTIGENA
PROSTATE ČOVEKA PRIMENOM IMUNOAFINITETNE HROMATOGRAFIJE
NA ČIPU
REZIME
Specifičan antigen prostate (prostate-specific antigen; PSA) ili kalikrein 3 (hK3),
pripada grupi ekstracelularnih serin-zavisnih proteaza iz porodice kalikreina. PSA u
najvećoj meri eksprimiraju epitelne ćelije acinusa i duktusa prostate i periuretralne
žlezde, a u manjoj meri i neka druga tkiva. PSA je, u normalnim fiziološkim uslovima,
prisutan u semenoj tečnosti u izrazito visokoj koncentraciji za razliku od seruma gde je
ona veoma niska. Usled narušene strukture prostate, do koje može doći pri različitim
poremećajima i oboljenjima, nivo PSA se u sistemskoj cirkulaciji znatno povećati pa se
on koristi kao serumski biomarker. Brojne studije su pokazale da PSA nije, u striktnom
smislu, specifičan samo za prostatu, već da ga eksprimiraju i neka hormon-zavisna tkiva
žene, pre svega dojka, endometrijum i ovarijum, kako u normalnom, tako i u patološkim
stanjima. Ekspresija gena za PSA je, u tkivu prostate, regulisana androgenima, dok je, u
ćelijskim linijama kancera dojke ona posredovana aktivacijom receptora za progesteron,
androgene, mineralokortikoide i glukokortikoide, ali ne i receptora za estrogene.
PSA je glikoprotein molekulske mase 28,43 kDa, koji sadrži jedan biantenaran lanac N-
acetil-laktozaminskog tipa sa sijalinskom kiselinom na krajevima obe grane. Ovi podaci
se odnose na kanonsku sekvencu/strukturu enzimski aktivnog molekula PSA. On se u
cirkulaciji nalazi u kompleksima sa inhibitorima proteaza kao što je α1-antihimotripsin
(ACT) ili α2-makroglobulin, ali pored njega postoje i molekuli PSA koji nemaju
enzimsku aktivnost ili je ona znatno smanjena. Njihova zastupljenost u kompleksima je
niska i oni predstavljaju formu slobodnog PSA (fPSA).
Za razliku od ispitivanja PSA formi, njegove pojedinačne molekulske vrste koje
pripadaju ovim formama do sada nisu bile predmet sistematske analize. Proteinska vrsta
je najmanja strukturna i funkcionalna jedinica proteoma i označava jedan individualan
protein iz familije proteina koji kodira pojedinačan gen. Ispitivanje specijacije proteoma
i kinetike njegovih proteinskih vrsta se nalazi u fokusu savremenih proteomskih
istraživanja. Cilj ove doktorske disertacije je da se ustanovi profil molekulskih vrsta
Rezime
PSA u subproteomima klinički relevantnih bioloških tečnosti: serum, urin i semena
plazma.
U ovom radu su korišćeni uzorci seruma osoba sa benignom hiperplazijom prostate,
kancerom prostate, zdravih ženskih osoba i ženskih osoba sa kancerom dojke. Pored
toga, ispitani su urin i semena plazma (SP) zdravih muških osoba, kao i prvi
internacionalni standard fPSA, koji predstavlja prečišćeni preparat PSA poreklom iz
semene plazme. Analizirani su nefrakcionisani uzorci ili uzorci seruma razdvojeni gel
filtracijom u frakcije koje sadrže PSA u imunskom kompleksu sa imunoglobulinom M
(PSA-IgM), PSA u kompleksu sa ACT (PSA-ACT) ili slobodan PSA (fPSA).
Imunoafinitetna hromatografija odgovarajućih frakcija je vršena na biohemijskoj
površini koja je dobijena imobilizacijom monoklonskog antitela prema epitopu I
molekula PSA, na preaktiviranom proteinskom čipu, PS20. Jonoizmenjivačka
hromatografija je vršena na čipu sa inkorporiranim amonijumovim jonima, Q10 ili
karboksimetil grupom, CM 10. Detekcija proteina na čipu je bila bazirana na SELDI-
TOF (Surface enhanced laser desorption / ionization – time of the flight) masenoj
spektrometriji, a dobijeni spektri su analizirani primenom softvera Ciphergen Express
Software 3,0. Za bioinformatičko predviđanje sastava i strukture glikana detektovnih
fPSA-imunoreaktivnih vrsta su korišćeni softveri: PeptideMass, GlycanMass i
GlycoWorkBench. Alatka PeptideMass je bila korišćena za izračunavanje mase
peptidnih fragmenata ili celog polipeptidnog lanca PSA na osnovu poznate
amnokiselinske sekvence. Alatka GlycanMass je korišćena za izračunavanje teorijske
mase oligosaharidne strukture koja je pridružena molekulskim vrstama PSA uočenim u
odgovarajućim masenim spektrima. Program GlycoWorkBench je upotrebljen za crtanje
oligosaharidnih struktura PSA glikoformi.
Dobijeni rezultati su pokazali da se fPSA-imunoreaktivne molekulske vrste u serumu,
na osnovu molekulske mase i pretpostavljene glikozilacije, mogu grupisati u četiri
glikopeptidne grupe: gp28 (26,6-29,6 kDa), gp22 (20,9-23,1 kDa), gp18 (17,1-18,6
kDa) i gp12 (12,2-12,7 kDa). Pored toga, uočeno je prisustvo brojnih molekulskih vrsta
na 24-25,7 kDa, 19,8-20,0 kDa i 13,5-16,3 kDa koje bi mogle predstavljati produkte
fragmentacije glikozilovanog ili neglikozilovanog zrelog ili „nicked“ PSA.
Rezime
Procena glikozilacije odgovarajućih molekulskih vrsta, izvršena na osnovu izračunate
mase njihovog glikana, ukazala je na postojanje mono-, bi- i triantenarnih
oligosaharidnih lanaca u okviru grupe gp28, kao i oligomanoznih struktura u grupi
gp18. U grupi gp28, mikroheterogenost se mogla dovesti u vezu sa razlikama u
sijalinizaciji, fukozilaciji i prisustvu bisektnog GlcNAc, a u okviru grupe gp18 sa
razlikama u broju manoza u oligomanoznom lancu.
Profili fPSA-imunoreaktivnih vrsta iz fPSA frakcija BPH- i PCa-seruma, su pokazali
visok stepen sličnosti, posebno u grupi gp28. Najdominantnija razlika je uočena u
slučaju vrste na 27595,81 Da koja je bila prisutna samo kod BPH-seruma i kojoj je
pridružen monoantenaran N-glikan. Pored ovoga, prisustvo molekulskih vrsta malih
masa, pre svega onih u grupi gp22, bilo je izraženije kod BPH. Najveći broj ovih vrsta
je poticao od “nicked” PSA koji ima prekid između Lys145/Lys146 (gp18) ili
Lys182/Ser183 (gp22).
Pokazano je da u sastav kompleksa sa IgM kod BPH i PCa, predominantno ulaze
molekulske vrste “nicked” PSA koji ima prekid između Lys145/Lys146, a za koje je
procenjeno da sadrže oligomanozni N-glikan. Uočene razlike između BPH i PCa su se
odnosile na prisustvo molekulske vrste na 26889,27 Da u PCa PSA-IgM kompleksu,
kojoj je pridružena paucimanozna struktura.
Nije dobijen detaljan uvid u molekulske vrste PSA koji se oslobađa iz kompleksa sa
ACT, s obzirom na to da su one detektovane kao autoproteolitički fragmenti. Isto važi i
za urinarni PSA čiji se profil znatno razlikovao od profila serumskog PSA, a koji su
karakterisale dominantne molekulske vrste malih masa koje bi mogle biti rezultat
proteolize ili autoproteolize.
Za ekstraprostatičan PSA, u serumu zdravih ženskih osoba kao i ženskih osoba sa
kancerom dojke, bilo je karakteristično prisustvo glikopeptidnih i peptidnih grupa u koje
su svrstane i molekulske vrste prostatičnog PSA. Najizrazitija razlika se odnosila na
gp18, koja je bila prisutna u PCa serumima, a koja nije detektovana u BCa serumima ili
je bila na granici detekcije u serumima zdravih ženskih osoba. Procena glikozilacije
PSA kod BCa je ukazala na prisustvo tri- i tetraantenarnih lanaca kod molekulskih vrsta
na 12,6 kDa, 23,1 kDa i 29,6 kDa koje su bile karakteristične za BCa-serume, a nisu
detektovane kod PCa.
Rezime
Sumarni rezultati ovoga rada dobijeni primenom imunoafinitetne hromatografije na čipu
su upotpunili i proširili saznanja o strukturnoj heterogenosti PSA i mogućem
biomarkerskom potencijalu njegovih pojedinih molekulskih vrsta. PSA je zajednička
biohemijska karakteristika kancera prostate i kancera dojke. Za potpuno razumevanje
biološkog značaja njegove heterogenosti i organ-ograničene distribucije, nephodno je da
se precizno opiše identitet i vremenska distribucija svih molekulskih vrsta PSA u
različitim subproteomima. Pored dopune bazičnih znanja o strukturi, ispitivanje
kompozicije proteinskih vrsta i validacija modifikacija njegovog proteinskog i
glikanskog dela su od opšteg interesa i u laboratorijskoj dijagnostici u smislu
standardizacije imunometrijskih testova koji se upotrebljavaju za određivanje
koncentracije PSA kao serumskog tumorskog markera. Dobijeni rezultati usmeravaju
dalji rad ka produkciji antitela koja bi specifično prepoznavala pojedinačne molekulske
vrste PSA koje se isključivo/predominantno javljaju u određenim fiziološkim i
patološkim stanjima, i koja bi pored primene u dijagnostici mogla imati i poseban
značaj u imunoterapiji kancera prostate i dojke. | CHARACTERIZATION OF MOLECULAR SPECIES OF HUMAN
PROSTATE-SPECIFIC ANTIGEN BY ON-CHIP IMMUNOAFFINITY
CHROMATOGRAPHY
ABSTRACT
Prostate-specific antigen (PSA), also known as kallikrein-3 (hK3), is an extracellular
serine protease belonging to the human kallikrein family. PSA is primarily produced by
the epithelial cells of the acini and ducts of the prostate gland and periurethral glands,
and to a lesser extent in other tissues. PSA is normally present at high concentration in
seminal plasma and very low concentration in serum.
Disrupted normal prostate glandular structure which occurs due to a variety of disorders
and diseases may result in elevated level of PSA in circulation, so that PSA can be used
as a serum biomarker. A number of previous studies have shown that PSA is also
expressed in non-prostatic tissues, especially in some hormon-dependent tissues of
women (breast, endometrium and ovary) in both normal and pathological conditions.
PSA gene expression is androgen regulated in prostate tissues, while PSA production in
breast cancer cell lines is mediated through the action of progesterone, androgen,
mineralocorticoid and glucocorticoid receptors, but not the estrogen receptor. PSA is a
28.4 kDa glycoprotein having a biantennary N-linked oligosaccharide chain of the N-
acetyllactosamine type with a sialic acid group at the end of the two branches. These
data refer to the canonical sequence / structure of enzymatically active PSA. In
circulation, it is complexed with extracellular protease inhibitors, such as α1-
antichymotrypsin (ACT) or α2-macroglobulin. In addition to active PSA, there are
molecules of PSA with considerably reduced or no existing enzymatic activity in
circulation. They essentially do not form complexes and represent free PSA form
(fPSA).
In comparison with these forms, individual molecular species of PSA forms have not
been yet systematically investigated. Protein/molecular species is the smallest structural
and functional unit of proteome and represents one individual protein from protein
family encoded by one, individual gene. In the focus of contemporary proteomic
research is speciation of proteome and kinetic of its protein species. The aim of this
Abstract
doctoral thesis was established the profile of PSA molecular/protein species in
subproteomes of clinical relevant biological fluids: serum, urine and seminal plasma.
In this study we have used serum samples of patients with benign prostatic hyperplasia
(BPH) and prostate cancer (PCa), as well as, sera of healthy females (HF) and women
with breast cancer (BCa). In addition, we have analyzed urine and seminal plasma of
healthy males and first international reference material for fPSA which represents
purified PSA preparation originating from the seminal plasma. Unfractionated and
fractionated serum samples were analyzed. Fractions separated by gel filtration
contained PSA complexed with IgM (PSA-IgM), PSA complexed with ACT (PSA-
ACT) and free PSA (fPSA). Immunoaffinity chromatography was carried out on
biochemical chip surface with immobilized monoclonal antibody for epitope I of PSA,
PS20, while ion-exchange chromatography was performed on chip with incorporated
quaternized ammonium groups, Q10 or carboxymethyl groups, CM10. On-chip protein
detection was based on surface enhanced laser desorption/ionization–time of the flight
mass spectrometry (SELDI-TOF/MS), and obtained spectra were analyzed using
softwere Ciphergen Express Software 3,0. Software tools, PeptideMass, GlycanMass
and GlycoWorkBench were used for the bioinformatic prediction of glycan composition
and structure of detected fPSA-imunoreactive species. PeptideMass was utilized for
mass calculation of polypeptide fragments or mature peptide chain of PSA on the basis
to known aminoacid sequence. GlycanMass was used to calculate masses of
oligosaccharide structures attached to PSA species detected in appropriate mass spectra.
GlycoWorkBench was used to draw the oligosaccharide structures of PSA glycoforms.
Obtained results have shown that fPSA-immunoreactive species in sera can be grouped
according to molecular masses and assumed glycosylation into four glycopeptide
groups: gp28 (26.6-29.6 kDa), gp22 (20.9-23.1 kDa), gp18 (17.1-18.6 kDa) and gp12
(12.2-12.7 kDa). In addition, there were numerous fPSA-immunoreactive species at 24-
25.7 kDa, 19.8-20.0 kDa and 13.5-16.3 kDa which might represent degradation
products of glycosylated/non-glycosylated mature or “nicked” PSA.
Glycosylation assessment of selected molecular species, based on their calculated
glycan masses, indicated the differences in the presence of mono-, bi- or triantennary
oligosaccharide chain in the group gp28, as well as oligomannose structures in the
group gp18. Within the group gp28 i.e. gp18, microheterogeneity could be related to the
Abstract
differences in sialylation, fucosylation and the presence of bisected GlcNAc i.e. in the
number of mannoses in the oligomannose chain, respectively.
fPSA-immunoreactive species of BPH and PCa fPSA fractions showed a high degree of
similarity, especially within the group gp28. Molecular species at 27595.81 Da with
associated monoantennary N-glycan was only present in BPH sera. It was the most
dominant difference between BPH and PCa. In addition, the presence of low molecular
mass species within the group gp22 was more pronounced in BPH. The large number of
these species originated from “nicked” PSA internally cleaved between Lys145/Lys146
(gp18) or Lys182/Ser183 (gp22).
Molecular species of “nicked” PSA internally cleaved between Lys145/Lys146 were
predominantly complexed with IgM, in both, BPH and PCa. It was estimated that these
species contained oligomannose N-glycan. The difference between BPH and PCa was
reflected in the presence of molecular species at 26889.27 Da in PCa PSA-IgM
complex. This species had associated pauci-mannose structure.
The detailed insight into PSA molecular species released from the complex with ACT
was not obtained, given that they were considered autoproteolytic fragments. The same
was for urinary PSA which profile was notably different in comparison to serum PSA
profile. The urinary PSA was characterized by low molecular mass species that could be
the result of proteolysis or autoproteolysis.
Extraprostatic PSA in sera of healthy female and women with breast cancer contained
the same peptide and glycopeptide groups of molecular species as prostatic PSA. The
main difference was found in the case of gp18 group present in PCa sera, but not
detected in BCa, or hardly detectable in HF sera. The glycosylation assessment
indicated that molecular species at 12.6 kDa, 23.1 kDa and 29.6 kDa, characteristically
presented in BCa sera, contained tri- and tetraantennary N-glycan chains. These species
did not detect in PCa.
Summarized results of this study were indicated that the knowledge of the structural
heterogeneity of PSA and possible biomarker potential of its individual molecular
species were complemented and extended by application of on-chip immunoaffinity
chromatography. PSA is a common biochemical characteristic of prostate and breast
cancer. For a complete understanding of the biological importance of its heterogeneity
and organ-limited distribution, it was necessary to accurately describe the identity and
Abstract
temporal distribution of molecular species in different PSA subproteomes. In addition to
the amendments to the basic knowledge about the structure, testing of protein species
composition and validation of its protein and glycan modification are of general interest
in laboratory diagnosis in terms of standardization immunometric tests used to
determine the serum concentration of PSA as a tumor marker. The results obtained in
this study directed further research towards the production of antibodies that specifically
recognize individual molecular species of PSA that are exclusively/predominantly
present in certain physiological and pathological conditions, and that, in addition to
applications in diagnosis, could have special significance for immunotherapy of prostate
and breast cancer. | true |
Mutacije u genima koji doprinose riziku za oboljevanje od karcinoma dojke i/ili jajnika kod BRCA1 i BRCA2 negativnih visokorizičnih porodica u Srbiji | Mutations in breast cancer susceptibility genes in BRCA1 and BRCA2 negative hereditary breast and ovarian cancer families in Serbia | Uvod: Nasledni karcinom čini 5-10% od svih slučajeva karcinoma dojke i prvenstveno je
povezan sa štetnim mutacijama u visoko penetrabilnim genima BRCA1 i BRCA2. I pored
intenzivnog istraživanja ostatka nasledne komponente, treći visoko penetrabilni gen u
karcinomu dojke (BRCA3) još uvek nije pronadjen. Pod okriljem poligenog modela
otkrivene su tri grupe alela koji doprinose riziku za karcinom dojke: visoko penetrabilni,
koji povećavaju rizik na 40-85% (BRCA1, BRCA2, TP53), srednje penetrabilni (povećavaju
rizik na 20-40%) kao što su CHEK2, RAD51, RAD51C i XRCC1, i učestale genske
varijante koje su nisko penetrabilne. Pored štetnih mutacija u ovim genima, odredjene
polimorfne varijante mogu uticati na efikasnost popravke grešaka u DNK i na nastanak
karcinoma.
Cilj: Obzirom da prisustvo mutacija u BRCA1/2 genima ne može da opiše celu genetičku
osnovu naslednog karcinoma dojke, cilj ovog rada je istraživanje prisustva populaciono
specifičnih mutacija u drugim genima (TP53, CHEK2, NBS1, RAD51, RAD51C i XRCC1)
kod BRCA1/2 negativnog, naslednog karcinoma dojke u Srbiji. Utvrdjivanje asocijacije
polimorfnih varijanti u ovim genima sa rizikom za nastanak karcinoma dojke, kao i njihove
korelacije sa parametrima tumora i parametrima domaćina tumora, predstavlja dodatni cilj
ovog rada.
Materijal i metode: 57 ispitanika poreklom iz visokorizičnih porodica kod kojih je
sekvenciranje BRCA1/2 gena dalo negativan rezultat (nasledni uzorak) i 107 pacijentkinja
sa karcinomom dojke koji nije odredjen u odnosu na porodičnu istoriju bolesti (sistematski
uzorak), uključeno je u ovu studiju. Kontrolnu grupu je činilo 114 briseva grlića materice
zdravih žena sa normalnim Papa nalazom i bez prethodne istorije pre-kancerskih i
kancerskih lezija. Veliki genski rearanžmani u BRCA1/2 genima su ispitivani MLPA
metodom, a utvrdjivanje prisustva mutacija u CHEK2 genu je radjeno alelski specifičnim
PCR-om. RAD51C gen je ispitivan DHPLC tehnikom, a detekcija štetnih mutacija u TP53 i
NBS1 genima je radjena uz pomoć HRM metode. Sve detektovane mutacije i profili koji su
odstupali od kontrolnih su sekvencirani. Polimorfne varijante u TP53, RAD51 i XRCC1 su
analizirane RFLP metodom. Za statističku obradu podataka korišćeni su Fišerov egzaktni,
χ2 i odds-ratio testovi.
Rezultati: MLPA tehnikom je pokazano odsustvo velikih genskih rearanžmana u BRCA1/2
genima u naslednom karcinomu dojke. U istoj grupi nisu pronadjene 1100delC u CHEK2 i
657del5 u NBS1 genu. Kod obolelih mladjih od 40 godina iz grupe naslednog karcinoma,
nisu pronadjene promene u 8. egzonu TP53 gena. Detektovana je jedna Del5395 mutacija u
CHEK2 genu (1/57) sa učestalošću od 1,7% u ispitivanoj grupi.
Analiza celog kodirajućeg regiona i egzon-intron granica RAD51C gena u naslednom
karcinomu dojke pokazala je 5 promena: 2 missense (c.790G>A i c.859A>G), jednu
intronsku (c.904+34T>C), jednu u 5’UTR (c.1-26C>T) i jednu u promotorskom regionu
gena (c.1-118G>A).
Ispitivanje Arg72Pro polimorfizma TP53 gena pokazalo je njegovu statistički značajnu
vezu sa naslednim oblikom karcinoma dojke i to kod nosilaca Pro alela (Pro/Pro+Arg/Pro
vs. Arg/Arg: OR=2,34; 95%CI=1,17-4,70).
Nosioci C alela RAD51 135G>C polimorfne varijante u homozigotnom stanju, imaju
značajno povećan rizik za oboljevanje od karcinoma dojke ukoliko u porodici imaju više
obolelih (C/C vs. C/G+G/G: OR=10,28; 95%CI=1,12-94,5). Ispitivanje asocijacije RAD51
135G>C polimorfizma sa karakteristikama biološkog ponašanja tumora pokazalo je razliku
u distribuciji C/C+G/C vs. G/G genotipova izmedju ER- i ER+ karcinoma (OR= 2,65; 95%
CI=0,96-7,34). Pokazana je i asocijacija ovog polimorfizma sa prognostički najlošijim
oblicima karcinoma dojke (triple-negative karcinomi).
Ispitivanje XRCC1 Arg399Gln polimorfizma pokazalo je povećan rizik za oboljevanje od
naslednog karcinoma dojke za nosioce Gln alela (OR=4,47; 95% CI=1,64-12,2). Značajna
veza Gln alela sa rizikom za oboljevanje pokazana je i kod mladjih od 40 godina
(OR=3,36; 95%CI=1,30-8,70). Poredjenje naslednog i sistematskog karcinoma dojke
pokazalo je značajnu vezu Gln alela sa naslednim oblikom bolesti (OR=3,05; 95%
CI=1,09-8,47). Ispitivanje asocijacije XRCC1 Arg399Gln polimorfizma sa histološkim
karakteristikama tumora pokazalo je razliku u distribuciji genotipova Gln/Gln+Arg/Gln vs.
Arg/Arg izmedju invazivnih duktalnih i lobularnih karcinoma (χ2=4,29; OR=2,92; 95%
CI=1,03- 8,28).
Zaključak: Nedostatak velikih genskih rearanžmana u BRCA1/2 genima u ispitivanoj grupi,
kao i štetnih mutacija u TP53, RAD51C i NBS1 genima, ukazuje na potrebu analize
prisustva mutacija u drugim srednje penetrabilnim genima, koji bi eventualno objasnili
naslednu predispoziciju i povećan rizik za oboljevanje u uzorku visokorizičnih, BRCA1/2
negativnih porodica. Prisustvo CHEK2 Del5395 mutacije ohrabruje dalja istraživanja ove
promene i njenog mogućeg osnivačkog efekta u Srbiji. Ispitivane polimorfne varijante
TP53, RAD51 i XRCC1 gena su asocirane sa povećanim rizikom za oboljevanje od
karcinoma dojke, naročito naslednog oblika ove bolesti. RAD51 135G>C se pokazao i kao
faktor koji je povezan sa prognostički najlošijim oblikom karcinoma dojke (triple-
negative). | Background: 5-10% of all breast cancer cases are hereditary, and are often caused by
deleterious mutations in high penetrance BRCA1 and BRCA2 genes. Despite significant
efforts in unraveling the rest of the familial component, BRCA3 could not be identified.
Under the polygenic model of inheritance, three groups of genetic predisposition alleles
have been identified so far: high-risk genes, conferring 40–85% lifetime risk (BRCA1,
BRCA2, TP53), moderate-risk genes (20–40% lifetime risk) including CHEK2, RAD51,
RAD51C and XRCC1, and low-risk common alleles. Besides the deleterious mutations in
these genes, certain polymorphic variants have functional significance in DNA repair
pathways, and may be responsible for cancer development.
Aim: Considering the fact that mutations in BRCA1/2 can’t explain the whole range of
hereditary predisposition to breast cancer, we aimed to investigate the presence and
frequency of population-specific mutations in other susceptibility genes (TP53, CHEK2,
NBS1, RAD51, RAD51C and XRCC1) in BRCA1/2 negative hereditary breast cancer cases
in Serbia. Determining association between polymorphic variants in these genes with breast
cancer risk, as well as their correlation with patient’s characteristics and histological and
pathological characteristics of breast cancer, were also the aims of this study.
Material and methods: 57 subjects from high-risk families negative for small alterations in
BRCA1/2 genes (hereditary group) and 107 breast cancer patients non-selected according to
family history (consecutive group) were included in the study. The control group was
consisted of 114 cervical smears of gynecological healthy women with normal Papa test
results and without previous history of pre-cancer and cancer lesions. Large genomic
rearrangements in BRCA1/2 genes were investigated by MLPA technique, while
genotyping for specific CHEK2 mutations was done by allele specific PCR. The coding
region of RAD51C was assessed through DHPLC method, and detection of TP53 and NBS1
deleterious mutations was done by HRM. All suspected mutations, or profiles that differed
from the control ones, were confirmed by direct sequencing. Polymorphic variants in TP53,
RAD51 and XRCC1 were analyzed by RFLP method. Fisher exact, χ2 and odds-ratio tests
were employed for statistical analysis.
Results: MLPA method revealed the absence of large genomic rearangements in BRCA1/2
genes in the high-risk, hereditary group. This group was also negative for 1100delC
mutation in CHEK2, and 657del5 in NBS1 genes. Young breast cancer patients from this
group (≤40 years) were screened for deleterious mutations in 8. exon of TP53, but they
showed no alterations in this genomic region. One Del5395 in CHEK2 was detected in the
same group (1/57) which makes its frequency of 1,7% in the tested group.
Screening the whole coding region and exon-intron boundaries of RAD51C gene in the
hereditary group revealed 5 DNA alterations including two missense (c.790G>A and
c.859A>G), one intronic (c.904+34T>C), one variant in the 5’UTR (c.1-26C>T) and one
variant in the promoter region of the gene (c.1-118G>A).
Regarding the Arg72Pro TP53 polymorphism, we found statistically important correlation
of Pro allele with cancer development among subject from hereditary group
(Pro/Pro+Arg/Pro vs. Arg/Arg: OR=2,34; 95%CI=1,17-4,70).
Considering RAD51 135G>C polymorphism, 135C allele carriers in homozygous state have
significantly higher risk for developing breast cancer if they also had family history of the
disease (C/C vs. C/G+G/G: OR=10,28; 95% CI=1,12-94,5). Investigation of association of
RAD51 135G>C polymorphism with histological characteristics of breast cancer showed
difference in distribution of C/C+G/C vs. G/G genotypes between ER- and ER+ carcinomas
(χ2=3,67; OR= 2,65; 95% CI=0,96-7,34). We also showed association of RAD51 135G>C
with triple negative breast carcinomas which have the worst prognostic outcome.
XRCC1 Arg399Gln polymorphism showed high risk for hereditary form of breast cancer
for Gln allele carriers (OR=4,47; 95% CI=1,64-12,2). Gln allele importance was shown
also for younger than 40 years (OR=3,36; 95% CI=1,30-8,70). Comparing hereditary and
consecutive groups we showed significant association of Gln allele with hereditary disease
(OR=3,05; 95%CI=1,09-8,47). Investigation of Arg399Gln association with histological
characteristics of breast cancer showed difference in distribution of Gln/Gln+Arg/Gln vs.
Arg/Arg genotypes between invasive ductal and invasive lobular carcinoma (χ2=4,29;
OR=2,92; 95% CI=1,03- 8,28).
Conclusion: The absence of large genomic rearrangements in BRCA1/2 genes as well as
truncating mutations in TP53, RAD51C and NBS1 genes indicates that deleterious
mutations in other breast cancer susceptibility genes may account for the increased
susceptibility in our cohort of high-risk BRCA1/2 negative families. The presence of
CHEK2 Del5395 mutation encourages its further examination as possible founder mutation
in Serbian population. RAD51, TP53 and XRCC1 polymorphic variants are associated with
higher risk for breast cancer development, especially in hereditary form of the disease.
Polymorphic variant RAD51 135G>C can be predictor for the triple-negative disease, the
type of breast cancer that has the worst prognostic outcome. | true |
Analiza mikrometastatske limfogene propagacije karcinomskih ćelija jetre kolorektalnog porekla | Analysis of micrometastatic lymphogenic propagation of colorectal carcinoma metastases | Cilj: Ispitati učestalost i distribuciju mikrometastatskih limfnih nodusa hepatičnog sliva i
prognostički značaj rutinske regionalne limfadenektomije s posebnim osvrtom na pojavu recidiva
maligne bolesti.
Metod: Ukupno 50 bolesnika sa metastatskom bolesti jetre podvrgnuto je resekciji jetre sa
rutinskom limfadenektomijom.
Rezultati: Pozitivni limfni nodusi hepatičnog sliva prisutni su kod 14 bolesnika (28%) bolesnika.
Standardna H&E patohistološka analiza verifikuje 6 bolesnika (12%) sa pozitivnim limfnim
nodusima hepatičnog sliva. Imunohistohemijska analiza dodatno verifikuje još 8 bolesnika
(16%) pozitivnih limfnih nodusa hepatičnog sliva preostalih limfnih nodusa. Limfni nodusi duž
zajedničke hepatične arterije (grupa 8a i 8p) najčešće sadrže mikrometastaze kolorektalnog
karcinoma (53,84%, i 19,23%). MSCT trbuha je od koristi za preoperativnu dijagnostiku
limfadenopatije (18,42%). Slobodni interval do pojeve metastaza je u proseku 15 meseci.
Recidiv maligne bolesti javio se kod 38,0% bolesnika. Volumen metastatske bolesti jetre
značajan faktor recidiva maligne bolesti (p<0,05)
Zaključak: Rutinska limfadenektomija hepatičnog sliva je bezbedna i preporučljiva procedura u
prevenciji širenja kolorektalne metastatske bolesti, koja neznatno povećava vreme trajanja
operacije u celini (prosečno 32 minuta) i ne povećava učestalost postoperativnih komplikacija. | Objective: To investigate the frequency and distribution of micrometastatic lymph nodus of the
hepatic basin and the prognostic significance of routine regional lymphadenectomy with
particular reference to the occurrence of relapse of malignant disease.
Methods: A total of 50 patients with metastatic liver disease underwent resection of the liver
with routine lymphadenectomy.
Results: Positive lymph nodes of the hepatic basin are present in 14 patients (28%) of patients.
The standard H & E pathohistological analysis verifies 6 patients (12%) with positive lymph
nodes of the hepatic basin. Immunohistochemical analysis further verified another 8 patients
(16%) of positive lymph nodes of the hepatic basin of the remaining lymph nodes. Lymph nodes
along the common hepatic artery (group 8a and 8p) most commonly contain colorectal
carcinoma micrometastases (53.84%, and 19.23%). MDCT abdomen is beneficial for
preoperative diagnosis of lymphadenopathy (18.42%). The free interval to metastasis rates is on
average 15 months. The recurrence of malignancy occurred in 38.0% of patients. The volume of
metastatic liver disease is an important factor of relapse of malignant disease (p <0.05).
Conclusion: Routine lymphadenectomy of the hepatic basin is a safe and recommended
procedure in the prevention of the spread of colorectal metastatic disease, which slightly
increases the duration of the operation as a whole (an average of 32 minutes) and does not
increase the incidence of postoperative complications. | true |
Uticaj različitih vrsta hrane na parametre oksidativnog stresa i masnokiselinski profil fosfolipida jetre pacova | Effects of different types of diets on the parameters of oxidative stress and the fatty acid profile of rat liver phospholipids | Sastav masnih kiselina lipida, posebno fosfolipida (PL) glavnih konstituenata ćelijskih
membrana zavisi od brojnih faktora. U poslednje vreme povećano je interesovanje za
istraživanja novih antioksidanasa u ishrani, uključujući omega-3 masne kiseline i
bioaktivne proteine prisutne u mleku. Uticaj različitih vrsta hrane, odnosno njihovog
masnokiselinskog sastava na profil masnih kiselina fosfolipida tkiva nije dovoljno
ispitivan. Stoga, cilj ove doktorske disertacije je bio ispitivanje efekta unosa tri različite
vrste hrane (standardne hrane, hrane obogaćene ribljim brašnom i hrane obogaćene
mlekom u prahu) na biohemijske parametre u krvi, masnokiselinske profile fosfolipida
jetre i na parametre oksidativnog stresa u jetri pacova Wistar soja, starosne dobi četiri
meseca u trajanju od četiri nedelje. U okviru ove studije, poređen je manokiselinski
profil u hranama različitog sastava, a takođe i u fosfolipidima jetri životinja nakon
tretmana. Preciznije, cilj je bio izdvajanje pojedinačnih masnih kiselina, koje su najviše
doprinele razlici u sastavu fosfolipida jetre, kao i ispitivanje polno specifičnog odgovora
tkiva na primenjene vrste hrane.
Rezultati ove disertacije su pokazali da hrana obogaćena ribljim brašnom i hrana
obogaćena mlekom u prahu menja sastav masnih kiselina fosfolipida i njihov odnos u
jetri pacova, na polno specifičan način. Primenjene hrane su promenile odnos ukupnih
zasićenih (SFA), mononezasićenih (MUFA) i polinezasićenih masnih kiselina (PUFA),
ali su efekti polno specifični. Hrana obogaćena mlekom u prahu je smanjila sadržaj SFA
i povećala udeo MUFA kod mužjaka, i PUFA kod ženki, u odnosu na standardnu hranu.
Ista vrsta hrane je smanjila procenat ukupnih n-3, povećala procenat ukupnih n-6 i
odnos n-6/n-3 kod mužjaka u odnosu na druge vrste hrana. S druge strane, kod ženki,
hrana obogaćena ribljim brašnom povećala je procenat ukupnih n-3, smanjila procenat
ukupnih n-6 i odnos n-6/n-3 u poređenju sa standardnom hranom i hranom obogaćenom
mlekom u prahu. Hrana je na polno specifičan način uticala i na odnos pojedinačnih
masnih kiselina u PL jetre. Dok je kod ženki hrana obogaćena ribljim brašnom smanjila
sadržaj arahidonske (AA) i povećala udeo eikosapentaenske (EPA), dokosapentaenske
(DPA) i dosaheksaenske kiseline (DHA), ista vrsta hrane je povećala samo udeo DHA
kod mužjaka. Grupa pacova koja je hranjena hranom obogaćenom ribljim brašnom
imala je veće aktivnosti GPx i CAT u odnosu na kontrolu i grupu koja je hranjena
hranom obogaćenom mlekom u prahu. Uprkos nešto nižim vrednostima lipidne
peroksidacije u grupi koja je hranjena hranom obogaćenom mlekom u prahu, nisu
utvrđene značajne razlike u poređenju sa drugim grupama. Inicijalne polne razlike u
sastavu masnih kiselina PL jetre pacova mogu biti modulisane tipom ishrane. Pored
toga, modulatorni efekti hrane obogaćene mlekom u prahu i hrane obogaćene ribljim
brašnom na profil masnih kiselina PL jetre su polno specifični. Ishrana bogata ribom bi
mogla delovati povoljno na oksidativni status preko poboljšanja aktivnosti
antioksidativnih enzima jetre.
Ovim je otvoreno značajno polje istraživanja uticaja potencijalno funkcionalne hrane, u
eksperimentalnim uslovima, na profil lipida u različitim tkivima. Takođe, veliki značaj
bi imala i istraživanja polno-specifičnog odgovora tkiva na promenu hrane. | The composition of fatty acids especially phospholipids (PL) as the main constituents of
cell membranes depends on many factors. Recently, there has been an increased interest
in novel dietary antioxidants, including omega-3 fatty acids and bioactive proteins
present in milk. There is limited data regarding the effects of fatty acid composition of
different types of diets on tissue phospholipids fatty acid profile. The aim of this
doctoral dissertation was to examine the effects of different types of diets (standard diet,
fish based and milk based diets) on plasma biochemical parameters, FA profiles of liver
phospholipids, and parameters of oxidative stress in liver, of four months old Wistar
rats, during four-weeks. Within this study, fatty acid profile was compared in diets of
different composition, as well as in liver phospholipids of animals after treatment. The
aim was to detect individual fatty acids mostly contributed to the difference in
composition of liver phospholipids, and to explore the association of tissue response
with gender.
The results of this dissertation have shown that both, milk- and fish-based diet, changed
the composition and ratio of rat liver phospholipids FA, in gender-specific manner.
Although, applied diets changed the ratio of total saturated fatty acids (SFA),
monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), but the
effects were sex specific. Milk-based diet lowered the amount of SFA and elevated the
ratio of MUFA in males, and PUFA, in females comparing to standard diet. The same
diet decreased n-3, increased n-6 and n-6/n-3 ratio in males comparing to the other type
of diets. On the other hand, in females, fish-based diet increased n-3, decreased n-6 and
n-6/n-3 ratio comparing to standard and milk-based diet. However, the ratio of
individual FA in liver PL was also dietary-influenced, but with gender specific manner.
While in females fish-based diet decreased AA (arachidonic acid) increased level of
EPA (eicosapentaenoic acid), DPA
(docosapentaenoic acid) and DHA (docosahexaenoic acid), the same diet elevated only
DHA levels in males. Statistically significant higher activities of GPx and CAT were
detected in rats fed with fish-based meal, in comparison to both the control and the
group fed with milk-based diet. Despite somewhat lower values of lipid peroxidation in
the milk-treated group, no significant differences were detected in comparison to the
other groups. Initially present sex differences in FA composition of rat liver PL appear
to be dietary modulated. Furthermore, the modulatory effects of milk- and fish-based
diets on liver phospholipids FA profiles appeared to be sex-specific. Diet enriched with
fish could improve one’s oxidative status by enhancing the activities of antioxidant
enzymes in liver tissue.
This has opened an important field of research of the potentially functional foods
influence, in experimental conditions, on the profile of lipids in various tissues. The
research related to sex-specific differences in response of tissues to different types of
diets would also be of great importance. | true |
Mehanizmi tolerancije slobodnoživećih bakterija i bakterija u biofilmovima na teške metale | Mechanisms of heavy metal tolerance in free living bacteria and bacterial biofilms | Teški metali, čija se koncentracija u sredini sve više povećava kao rezultat povećane
industrijske aktivnosti i tehnološkog razvoja, ubrajaju se u najčešće zagađivače životne
sredine. Kada su prisutni u većim koncentracijama toksični su i predstavljaju ozbiljnu
pretnju kako za životnu sredinu tako i za zdravlje ljudi, jer se ne mogu razgraditi i jer se
nagomilavaju u lancima ishrane. Sa druge strane, ovakve sredine predstavljaju dobar izvor
mikroorganizama tolerantnih na teške metale, sa potencijalnom primenom u biološkom
uklanjanju (bioremedijaciji) i oslobađanju (bioluženju) teških metala. Cilj ovog rada je bila
izolacija, identifikacija, analiza diverziteta i aplikativnog potencijala novih bakterijskih
sojeva sa sposobnošću tolerancije visokih koncentracija teških metala iz površinskog i
podzemnog sedimenta iz rudnika bakra Bor. Metagenomskom analizom je detektovan veći
mikrobiološki diverzitet u uzorku površinskog sedimenta u poređenju sa podzemnim
sedimentom. Iz oba uzorka je izolovano 6 bakterija tolerantnih na visoke koncentracije
teških metala, primenom direktne kultivacije. Ovi izolati su taksonomski identifikovani
kao predstavnici rodova Arthrobacter i Staphylococcus. Pored ovih izolata, u ovu studiju je
bilo uključeno još 8 sojeva za koje je tolerancija na metale prethodno opisana u literaturi,
tako da je aplikativni potencijal slobodnoživećih bakterija i definisanih bakterijskih
zajednica, kao i njihovih biofilmova, ispitan kod ukupno 14 sojeva. Među 14 testiranih
sojeva, 6 sojeva je imalo sposobnost rasta u prisustvu nikla, 6 u prisustvu kadmijuma, 4 u
prisustvu bakra, 3 u prisustvu hroma i 1 je imao sposobnost rasta u prisustvu žive, dok
nijedan od sojeva nije imao sposobnost rasta u prisustvu gvožđa i cinka. Izdvojio se izolat
Staphylococcus sp. MSI08, koji je jedini imao sposobnost rasta u prisustvu tri teška metala
(nikl, kadmijum, hrom). Analizom genetičkih determinanti mehanizama tolerancije na
teške metale, kod 2 soja je detektovano prisustvo gena za determinantu tolerancije na
kadmijum, kod 2 soja prisustvo gena za determinantu tolerancije na hrom i kod 2 soja je
detektovano prisustvo gena za determinantu tolerancije na bakar. Svi testirani sojevi su
imali sposobnost formiranja biofilmova, s tim što su najveći potencijal za formiranje
biofilmova pokazali Staphylococcus sp. MUI10, Arthrobacter sp. MSI31, Pseudomonas
aeruginosa PAO1 i Cupriavidus metallidurans CH34, dok su Staphylococcus sp. MSI08,
Rhodococcus sp. TN113, Pseudomonas putida KT2440 i Cupriavidus necator H16
pokazali nešto slabiji potencijal za formiranje biofilmova. U uslovima višednevnog
izlaganja bakterijskih biofilmova dejstvu teških metala pokazano je da biofilmovi sojeva
Rhodococcus sp. TN113, Cupriavidus metallidurans CH34 i Pseudomonas aeruginosa
PAO1 imaju povećanu toleranciju na teške metale u poređenju sa slobodnoživećim
bakterijama. Pokazano je takođe da se na problem kompleksnog zagađenja sredine teškim
metalima može odgovoriti upotrebom soja Pseudomonas aeruginosa PAO1 kao i
upotrebom četiri definisane bakterijske zajednice, koji su imali sposobnost rasta u prisustvu
smeše bakra, kadmijuma i hroma. U ovom radu je prvi put testiran uticaj tri odabrana
biofilma sojeva koji ne učestvuju aktivno u procesu bioluženja na proces bioluženja
pomoću Acidithiobacillus ferrooxidans i pokazano je da ti biofilmovi mogu povećati
efikasnost bioluženja pojedinih metala i da se ovaj pristup može dalje optimizovati u
pravcu poboljšanja procesa bioluženja. | Heavy metals are one of the most common environmental pollutants due to increased
industrial activities and technological development. When present in excessive
concentrations, heavy metals are highly toxic and they pose a serious threat both to
environment and human health, due to their persistence in nature and accumulation in the
food chain. On the other hand, polluted sites have proven to be valuable sources of bacteria
capable to tolerate increased concentration of heavy metals that could be potentially applied
in biological removal (bioremediation) and release (bioleaching) of heavy metals. The aim
of this study was to obtain, identify, analyze diversity and application potential of novel
bacterial strains with the ability to tolerate high heavy metal concentrations from surface
and underground sediments from the copper mine Bor. Metagenomic analysis revealed a
higher microbial diversity in surface sediment compared to underground sediment. From
both sediments, 6 bacterial strains able to tolerate high heavy metal concentrations were
isolated using direct cultivation approach. These isolates were taxonomically identified as
members of Arthrobacter and Staphylococcus genus. In addition, this study involved 8
more strains with heavy metal tolerance previously described in the literature. Thus, 14
bacterial strains were included in analysis and evaluation of heavy metal tolerance and
application potential of free living bacteria and defined bacterial consortia, as well as their
biofilms. Among 14 tested strains, 6 strains had the ability to grow in the presence of
nickel, 6 in the presence of cadmium, 4 in the presence of copper, 3 in the presence of
chromium and 1 had the ability to grow in the presence of mercury, while none of the
strains had the ability to grow in the presence of iron and zinc. Staphylococcus sp. MSI08
singled out as the only strain with the ability to grow in the presence of three heavy metals
(nickel, cadmium, chromium). Analysis of genetic determinants conferring heavy metal
tolerance revealed presence of cadmium tolerance genes in 2 strains, chromium tolerance
genes in 2 strains, and copper tolerance genes were also detected in 2 strains. All strains
showed ability to form biofilms, but Staphylococcus sp. MUI10, Arthrobacter sp. MSI31,
Pseudomonas aeruginosa PAO1 and Cupriavidus metallidurans CH34 demonstrated the
highest potential for the biofilm formation, while Staphylococcus sp. MSI08, Rhodococcus
sp. TN113, Pseudomonas putida KT2440 and Cupriavidus necator H16 were less
successful in biofilm formation. When exposed to heavy metals for several days,
Rhodococcus sp. TN113, Cupriavidus metallidurans CH34 and Pseudomonas aeruginosa
PAO1 biofilms exhibited enhanced tolerance to heavy metals compared to free living
bacteria. It has been demonstrated that Pseudomonas aeruginosa PAO1 and four defined
bacterial consortia could be employed in solving problem of complex heavy metals
pollution, since they had the ability to grow in the simultaneous presence of copper,
cadmium and chromium. In this study the influence of 3 selected biofilms, consisting of
non-bioleaching bacterial strains, on Acidithiobacillus ferrooxidans bioleaching was
examined for the first time. It has been shown that these biofilms could increase the
bioleaching efficiency for certain metals and this approach can be further optimized in
order to improve the bioleaching process. | true |
Ispitivanje mehanizma antitumorskog delovanja derivata antrahinona in vitro | An in vitro investigation of the mechanism of antitumor activity of anthraquinone derivatives | Prirodni antrahinoni, važna klasa hinonskih jedinjenja, se mogu naći u velikom
broju viših biljaka, mahovinama, lišajevima kao i drugim organizmima. Ova hinolna
jedinjenja poseduju laksativno, antibiotičko, antiinflamatorno i antipiretičko kao i
antiviralno dejstvo, a njihov potencijal u tretmanu kancera, i inhibiciji proliferacije i
metastaziranja malignih ćelija ih čini jednom od najzanimljivijih grupa jedinjenja u razvoju
novih terapeutika. Njihov mehanizam dejstva podrazumeva učešće u metaboličkim
procesima, oksidativnoj fosforilaciji, interkalalaciju u DNK i RNK molekule, inhibiciju
telomeraze i topoizomeraze II. Najpoznatija podgrupa ove klase jedinjenja su antraciklini,
antrahinonski antibiotici. Antraciklini kao što su doksorubicin i daunorubicin imaju
izuzetno široku primenu u terapiji kancera, mada su izuzetno kardiotoksični.
Tiosemikarbazoni predstavljaju grupu iminskih derivata koji sadrže atom sumpora u
svom molekulu, i odlikuje ih izuzetna sposobnost helacije jona metala. Odlikuje ih
antiviralno, antibakterijsko, antifungalno, kao i citotoksično i antiproliferativno dejstvo.
Halkoni su prekursori flavonoida i izoflavonoida, grupa jedinjenja sa velikim
terapeutskim potencijalom, i prisutni su u prirodnim izvorima kao što su voće, začinsko
bilje i čajevi. Za njih je utvrđeno da poseduju antioksidativni, antibakterijski, antimalarijski,
antiviralni, antihiperglikemijski i antitumorski efekat.
Cilj ovog projekta je ispitivanje grupe od trinaest novosintetisanih derivata
antrahinona, i analiza njihovih molekularnih mehanizama. Ova jedinjenja su dizajnirana sa
ciljem da im se poboljša aktivnost i specifičnost u odnosu na prirodne derivate koji su
trenutno
u
upotrebi.
Devet
jedinjenja
ove
grupe
predstavljaju
antrahinonske
tiosemikarbazone, dok su četiri halkonski antrahinoni.
Prvi korak u istraživanju je predstavljalo određivanje potencijalnog antitumorskog
dejstva derivata antrahinona prema sedam malignih ćelijskih linija poreklom iz različitih
tipova kancera. Selektivnost u nivou toksičnog dejstva određena je uz pomoć
eksperimenata na uspostavljenoj liniji normalnih humanih ćelija.
Dalje istraživanje se fokusiralo na rasvetljavanje molekularnih mehanizama
delovanja ispitivanih supstanci. Morfološke promene na tretiranim ćelijama su posmatrane
primenom fluorescentnog mikroskopa, uz pomoć akridin oranž/etidijum bromid smeše, i na
ovaj način se određivao i tip ćelijske smrti (apoptoza i/ili nekroza) koju indukuju ova
jedinjenja. Analizirana je distribucija malignih i normalnih ćelija po fazama ćelijskog
ciklusa nakon tretmana ispitivanim supstancama uz pomoć protočne citometrije, određivane
su ciljne kaspaze preko kojih ispitivana jedinjenja ostvaruju apoptotski efekat, a utvrđen je i
antimetastatski potencijal ovih jedinjenja, odnosno da li antrahinonski derivati inhibiraju
matriksne metaloproteinaze, korišćenjem želatinske zimografije. Anti-angiogenetski
potencijal i anti-invazivni potencijal ispitivanih jedinjenja je bio određen uz pomoć Tube
formation assay i Wound-healing assay.
Ispitivana grupa novosintetisanih derivata antrahinona pokazuje izuzetno dobro
citotoksično, kao i antiproliferativno dejstvo prema malignim ćelijskim linijama, a ova
jedinjenja su se pokazala i kao selektivna u odnosu na normalne transformisane ćelijske
linije. Najveću citotoksičnost ova grupa jedinjenja ispoljava prema malignoj ćelijskoj liniji
poreklom od mijeloidne leukemije, a najrezistentnije su ćelije izolovane iz metastatskih
mesta poreklom od kancera dojke. Samo jedinjenje sa heksil grupom kao bočnim lancem
pokazuje izuzetno dobro dejstvo prema ovoj ćelijskoj liniji. Mehanizam dejstva
antrahinonskih halkona podrazumeva indukciju G2/M zastoja u ćelijskom ciklusu tretiranih
ćelija, a pokazano je da sva ispitivana jedinjenja izazivaju apoptozu u malignim ćelijama in
vitro.
Takođe, ispitivana jedinjenja su pokazala izuzetan potencijal sprečavanja metastaze,
neoangiogeneze, kao i inhibitorno dejstvo na potencijal migracije tretiranih malignih ćelija.
Ova istraživanja su pokazala da članovi grupe antrahinonskih halkona predstavljaju
veoma dobre kandidate za dalji razvoj i in vivo ispitivanja ovih jedinjenja u terapeutske
svrhe, dok se iz grupe antrahinonskih tiosemikarbazona posebno ističu jedinjenja 1 i 3, sa
fenil i heksil suptituentima.
Dobijeni rezultati pružaju osnovu za dalja istraživanja ovih grupa jedinjenja kao
potencijalnih hemoterapeutika sa veoma izraženim citotoksičnim, antimetastatskim kao i
antineoangiogenetskim svojstvima. | Naturally occuring anthraquinones, an important class of quinone compounds, are
numerous in plants, moss, lichen and other organizms. These quinol compounds posses
laxative, antibiotic, antiinflamatory, antipyretic and antiviral properties, and their potential
for cancer treatment and inhibition of proliferation and metastasis of malignant cells is what
makes them one of the most interesting classes of compounds in cancer drug development
field. Their mechanism of action includes involvement in the metabolic processes,
oxidativve phosporylation, DNA and RNA intercalation and inhibition of telomerase and
topoisomerase II. The most well-known compounds belonging to this class are
anthracyclines, anthraquinone antibiotics. Anthracyclines, such as doxorubicin and
daunorubicin, are widely used in treatment of cancer, even though they are highly
cardiotoxic.
Thiosemicarbazones are a class of imine derivatives which posses in their molecule
an atom of sulfur. They are characterized by an exceptional ability to chelate metal ions.
They have antiviral, antibacterial, antifungal and cytotoxic and antiproliferative activities.
Chalcones are flavonoid and isoflavonoid precursors, a class of compounds with a
significant therapeutic potential, and they can be found in natural sources like fruit, herbs
and teas. It was previously determined that they can have anti-oxidative, antibacterial,
antimalarial, antiviral, antihyperglycemic and antitumor effects.
The aim of this research is to investigate a group of thirteen newly synthesized
anthraquinone derivatives, and to analyze their mechanisms of action. These compounds
were designed in a way that aims for increased activity and specificity compared to the
currently used natural derivatives. Nine of the thirteen compounds are anthraquinone-
thiosemicarbazone derivatives, and another four belong to the subgroup of anthraquinone-
chalcones.
First step in this research was to determine potential for antitumor activity of the
tested anthraquinone derivatives on seven malignant cell lines, originated from different
cancer types. Selectivity in levels of cytotoxicity was determined using an established line
of normal human cells.
Further research was focused on determination of the molecular mechanisms of
action of the investigated compounds. Changes in cell morphology after treatment with the
compounds were followed using fluorescence microscopy and ethidium bromide double
staining , and this helped to determine the cell death type (apoptosis and/or necrosis), that is
induced by these compounds. The effects of the investigated compounds on cell cycle
distribution of malignant and normal cells were determined using flow cytometry, which
was also used to determine the activity of caspase-3, -8 and -9 in the treated cells.
Antimetastatic potential, i.e. the potential to inhibit matrix metalloproteinases, of the
investigated compounds was investigated using gelatin zymography. Antiangiogenic and
anti/invasive potential of anhtraquinone derivatives was determined using Tube formation
assay and Wound-healing assay.
The investigated group of novel anhtraquinone derivatives shows very good
cytotoxic and antiproliferative activity toward malignant cell lines, but these compounds
also show significant selectivity compared to normal transformed cell lines. The most
sensitive cell line is malignant cell line originated from erythroleukemia, and the most
resistant cells were isolated from brain metastasis of breast cancer. The only compound that
had a significant effect on this cell line is a compound with hexyl group as a side chain in
the molecule. Mechanism of action of anthraquinones with chalcone substituents includes
inducement of G2/M arrest in the treated cells, and it was shown that all tested compounds
induce apoptosis in the malignant cells in vitro.
Moreover, the investigated compounds demonstrated a significant effect on
inhibition of metastasis and neoangiogenesis, and also inhibitory effects on migratory
potential of the treated malignant cells.
This research showed that compounds that belong to the group of chalcone
substituted anhtraquinones represent very good candidates for further development and in
vivo investigations of these compounds for use in therapy, while in the anthraquinone-
thiosemicarbazone group, compounds 1 and 3, containing phenyl and hexyl moieties,
respectively, are particularly noteworthy.
The obtained results provide the basis for further investigations of these classes of
compounds as potential therapeutics with a pronounced cytotoxic, antimetastatic and
antineoangiogenetic properties. | true |
Uticaj dugotrajne restrikcije hrane na ekspresiju glukokortikoidnog receptora u prednjem mozgu pacova tokom starenja | The effect of long-term food restriction on te expression of glucocorticoid receptor in the rat forebrain during aging | Dugotrajna restrikcija hrane produžava životni vek i odlaže pojavu mnogih bolesti koje se
javljaju sa starenjem. Brojni literaturni podaci ukazuju da kod životinja kojima je unos hrane
smanjen izostaje karakterističan starosno-zavistan pad u kognitivnim funkcijama, međutim,
mehanizam ovakvog neuroprotektivnog dejstva restrikcije hrane nije u potpunosti razjašnjen. U
isto vreme, kod životinja na dugotrajnoj restrikciji hrane se povećava nivo kortikosterona u
plazmi što ukazuje da ovaj tretman deluje kao blagi stresor. Do sada nije ispitivano do kakvih
promena u signalnom putu glukokortikoida dovodi smanjeni unos hrane tokom starenja u mozgu.
Centralno polje istraživanja procesa starenja neizbežno podrazumeva ispitivanje promena do
kojih dolazi u korteksu i hipokampusu, strukturama mozga koje su ključne za kognitivne
funkcije. Sa druge strane, iste strukture učestvuju u regulaciji HHA ose kao važni regioni
delovanja negativne povratne sprege.
Cilj doktorske disertacije je da se ispita uloga signalnog puta glukokortikoida u korteksu i
hipokampusu pacova tokom starenja i pod uticajem dugotrajne restrikcije hrane.
Eksperimentalne životinje (mužjaci pacova soja Wistar) starosti 6 meseci su podeljene u dve
grupe. Prva grupa, označena kao ad libitum (AL), je imala neograničen pristup hrani, dok je
druga grupa (označena kao DR) podvrgnuta režimu redukovane ishrane koji je podrazumevao
dobijanje 100% dnevnog unosa hrane AL životinja svakog drugog dana. Životinje su analizirane
kada su dostigle starost od 18 i 24 meseca. Životinje stare 6 meseci su predstavljale kontrolnu
grupu. Glukokortikoidna signalizacija u ispitivanim strukturama je praćena na prereceptorskom
nivou, kao i na nivou ekspresije i aktivacije glukokortikoidnog receptora primenom imunoesej,
imunoblot i PCR metoda, kao i imunohistohemijske analize.
Rezultati doktorske disertacije pokazuju da tokom starenja dolazi do povećanja
koncentracije kortikosterona u korteksu pacova, dok dugotrajna restrikcija hrane povećava nivo
kortikosterona u obe ispitivane strukture. Utvrđeno je da nivo ključnog enzima koji u moždanom
tkivu reguliše dostupnost kortikosterona za receptore, 11β-HSD1, raste u hipokampusu pacova
koji imaju neograničen pristup hrani. Tretman dugotrajnom restrikcijom hrane dovodi do
povećanja nivoa 11β-HSD1 u korteksu, dok u hipokampusu izostaje efekat na nivo ovog enzima.
Najizraženije promene na nivou glukokortikoidnog receptora tokom starenja su uočene u
hipokampusu životinja koje su imale slobodan pristup hrani. Detektovan je značajan pad nivoa
glukokortikoidnog receptora, praćen smanjenim nivoom fosforilisanog oblika receptora.
Pokazano je da primena dugotrajne restrikcije hrane povećava nivo glukokortikoidnog receptora
i u korteksu i u hipokampusu, dok se uticaj na fosforilacioni status receptora ostvaruje samo u
hipokampusu najstarije ispitivane grupe, gde se značajno povećava nivo fosforilisanog oblika
receptora, kao i njegova jedarna lokalizacija u piramidalnim neuronima CA1 regiona i
granularnim ćelijama dentatnog girusa. Funkcionalna posledica aktivacije glukokortikoidnog
receptora u hipokampusu životinja podvrgnutih dugotrajnoj restrikciji hrane se ogleda u
povećanju ekspresije sgk-1 gena. Takođe, kod životinja kojima je unos hrane smanjen dolazi do
porasta nivoa mineralokortikoidnog receptora u hipokampusu. Sa druge strane, dugotrajna
restrikcija hrane u korteksu sprečava povećanje nivoa transkripcionog regulatora NFκB u
jedarnoj frakciji do kojeg dolazi sa starenjem i dalje deluje u pravcu održavanja reaktivnosti
kortikalnih neurona tokom starenja, na šta ukazuje povećana ekspresija c-fos gena.
Utvrđeno je da dugotrajna restrikcija hrane ostvaruje uticaj na signalni put glukokortikoida
na strukturno-specifičan način. Promene koje su detektovane u korteksu su uočljivije na
prereceptorskom nivou, i ogledaju se u povećanoj koncentraciji kortikosterona i povećanom
nivou enzima 11β-HSD1. U hipokampusu je efekat restikcije hrane najizraženiji na nivou samog
receptora, i ostvaruje se preko povećanja nivoa ukupnog receptora i njegove fosforilisane forme.
Rezultati dobijeni u okviru disertacije ukazuju na značaj signalnog puta glukokortikoida u
ostvarivanju efekata dugotrajne restrikcije hrane, čime proširuju dosadašnje uvide u mehanizme
neuroprotektivnog dejstva smanjenog unosa hrane. Imajući u vidu važnu ulogu signalnog puta
glukokortikoida u procesima učenja i pamćenja, rezultati su značajni i sa aspekta potencijalnog
korišćenja redukovane ishrane u očuvanju kognitivnih kapaciteta tokom starenja kod ljudi. | Moderate restriction in food intake (dietary restriction, DR) extends the life-span and
delays the onset of many age-related diseases. In addition, food-restricted animals exhibit
attenuated cognitive deficits during aging as they perform better in learning and memory tasks
than their ad libitum fed counterparts. At the same time, the animals on chronic food restriction
have elevated levels of plasma corticosterone, which implies that this treatment could be
considered as a mild stressor. So far, there are no literature data regarding the effects of food
restriction on glucocorticoid signaling in the brain during aging and the mechanisms by which
DR exhibits its neuroprotective effects are poorly understood. Two brain regions of interest are
cortex and hippocampus, regions particularly prone to age-related changes. The same regions are
also involved in cognition and regulation of the hypothalamic–pituitary–adrenal (HPA) axis
activity.
The aim of the present study was to evaluate the effect of long-term food restriction on
glucocorticoid signaling pathway in the cortex and hippocampus of rats during aging.
Experimental animals (male Wistar rats, 6 months old) were divided into two groups. The AL
group was fed ad libitum, whereas the food restricted group (DR) received 100% of the mean
daily intake of the AL animals every other day. The animals were examined at the age of 18 and
24 months. Six-month-old animals were used as a control. Glucocorticoid signaling in specific
brain regions was examined at the pre-receptor level, as well as regarding the level of expression
and glucocorticoid receptor activity by using immunoassays, Western blot, PCR and
immunohistochemistry.
The results of this study demonstrate an age-related increase in cortical corticosterone
concentration. The increase in the hormone level was further detected in DR animals, in both
brain regions examined. However, 11β-HSD1, a key enzyme in brain tissue that regulates the
availability of corticosterone to its receptors, was increased only in the hippocampi of AL rats
and in the cortex of rats subjected to the long-term food restriction. The most pronounced
changes in the level of glucocorticoid receptor during aging were observed in the hippocampi of
AL animals. A significant decrease in the level of glucocorticoid receptor protein was
accompanied by the decrease in the level of phosphorylated receptor form (pGR). Conversely,
following long-term food restriction, an increase in the level of glucocorticoid receptor both in
the cortex and hippocampus was observed. The effect of food restriction on pGR was observed
only in the hippocampi of old animals, where increased level of pGR together with its prominent
nuclear staining in CA1 pyramidal and DG granule neurons of aged DR rats was observed. These
changes were accompanied by increased sgk-1 gene expression, pointing to upregulated
transcriptional activity of GR. In the hippocampus, DR also induced an increase in the
expression of the mineralocorticoid receptor. Furthermore, reduced food intake in the cortex
prevented an age-related increase in the level of nuclear NFκB and induced increase in the
expression of c-fos, indicating that DR during aging tends to maintain the reactivity of cortical
neurons.
The results presented herein reveal that long-term food restriction affects the
glucocorticoid signaling pathway in a region-specific manner. In the cortex, more prominent
changes were observed at the pre-receptor level, where increased corticosterone concentration
and elevated 11β-HSD1 level were detected. In the hippocampus, the most pronounced effects
were on the receptor itself, as long-term food restriction induced an increase in the total GR and
pGR levels.
Overall, this study demonstrates how long-term food restriction affects the
glucocorticoid signaling pathway in the brain and thus provides new insights into the
mechanisms by which reduced food intake exerts its neuroprotective effects. Considering the
importance of glucocorticoid signaling pathway in learning and memory, these results also
provide important information in relation to the potential use of reduced food intake in
preserving cognitive capacity during aging in humans. | true |
Rasprostranjenje, horološka struktura i centri diverziteta balkanske endemične flore u Crnoj Gori | Distribution, chorological structure and centers od diversity of the balkan endemic flora in Montenegro | Glavni cilj ovog rada je utvrđivanje ukupnog broja balkanskih endemičnih
taksona ranga vrste i podvrste na teritoriji Crne Gore. Terenskim istraživanjima, i
pregledom obimne literaturne građe i herbarskih zbirki utvrđeno je da u Crnoj Gori rastu
372 balkanska endemična taksona. Podaci koji obuhvataju rasprostranjenje, ekološke
karakteristike kao i literaturne i herbarske izvore za svaki pojedinačan takson
inkorporirani su u bazu podataka Microsoft Excel koja broji 7215 zapisa.
Popisani su svi lokaliteti na kojima je svaki pojedinačan takson bio konstatovan.
Urađene su areal karte sa UTM koordinatama 10x10 km na kojima je prikazano
rasprostranjenje svakog pojedinačnog taksona na teritoriji Crne Gore. Analizom opšteg
rasprostranjenja tj. rasprostranjenja na Balkanskom poluostrvu, za svaki takson je
određen florni elemenat, areal podgrupa i areal grupa. Prikazane su i ekološke
karakteristike endemičnih taksona koje su podrazumijevale utvrđivanje pripadnosti
odgovarajućoj životnoj formi, tipova staništa koje naseljava dati takson, dijapazona
nadmorskih visina i tipova geološke podloge. U slučajevima preuzimanja literaturnih
podataka iz fitocenoloških radova unošene su i informacije o ekspoziciji i nagibu terena
staništa, te stepenu prisutnosti endemičnih taksona u određenoj asocijaciji.
Taksonomska analiza balkanske endemične flore Crne Gore pokazala je da su
najbogatije familije Asteraceae, Caryophyllaceae, Campanulaceae, Fabaceae i
Lamiaceae, dok čak 13 familija ima predstavnika u jednom endemičnom taksonu. U
okviru najbrojnije familije Asteraceae je i najbrojniji rod Hieracium sa 71 taksonom,
slijede rodovi Dianthus, Edraianthus i Campanula. Rodovi Stachys, Cerastium i
Alchemilla su zastupljeni sa istim brojem endemičnih vrsta i podvrsta. Zanimljivo je da
čak 80 rodova ima po jednog endemičnog predstavnika.
Horološka struktura balkanskih biljnih endemita Crne Gore ukazuje da gotovo
dvije trećine endemičnih taksona čine orofite koje pripadaju dvjema horološkim
grupama: južno evropsko planinskoj (146 taksona ili 39,2%) i srednje evropskoj
planinskoj (99 taksona ili 26,6%). Shodno tome može se govoriti o apsolutnoj dominaciji
planinskog i visokoplaninskog endemizma u flori Crne Gore. Srazmjerno velikim brojem
taksona (81 ili 21,8%) zastupljena je i mediteransko-submediteranska horološka grupa,
što ukazuje na snažne uticaje mediteranske i submediteranske flore koji dopiru duž
kanjonskih dolina rijeka jadranskog sliva duboko u unutrašnjost zemlje. Srednje evropska
horološka grupa, karakteristična za šumska i livadska staništa unutrašnjeg niza Dinarida u
Crnoj Gori, predstavljena je sa znatno manje endemičnih biljaka (45 ili 12,3%) u
ukupnom horološkom spektru balkanske endemične flore Crne Gore, dok je pontska areal
grupa predstavljena samo jednim endemičnim taksonom.
Analiza biološkog spektra balkanske endemične flore Crne Gore ukazuje da
dominiraju životne forme hemikriptofita (H) i hamefita koje zajedno čine 87% biološkog
spektra. Učešće hemikriptofita je 63%, a hamefita 24%. Većina biljaka Holarktika ili
umjerenog pojasa, pa i Balkanskog poluostrva pripada životnoj formi hemikriptofita.
Osim toga, hemikriptofite i hamefite naseljavaju brdsko-planinska područja, a najveći dio
teritorije Crne Gore je brdsko-planinskog karaktera.
Detaljnije analize ekoloških karakteristika balkanske endemične flore u Crnoj
Gori podrazumjevale su utvrđivanje preferencije svakog pojedinačnog taksona u odnosu
na dijapazone nadmorskih visina, kao i tip geološke podloge i koju svaka od njih
naseljava. Definisane su tri grupe geoloških podloga (karbonati, silikati, ostalo) i urađena
je zastupljenost broja endemičnih taksona u odnosu na ove tipove substrata. Ispostavilo se
da se najviše endemita javlja na karbonatnoj geološkoj podlozi (342 taksona), a znatno
manje na silikatnoj (svega 40 vrsta i podvrsta). U odnosu na dijapazone nadmorskih
visina u kojima se javljaju endemične vrste i podvrste na teritoriji Crne Gore, izdvojeno je
pet klasa i utvrđen je broj endemičnih taksona koji se javljaju u tako definisanim
dijapazonima nadmorskih visina. Najviše endemičnih taksona javlja se u visinskom
dijapazonu 1500-2000 m (156 taksona), kao i u dijapazonu 1000-1500 m (132 vrste i
podvrste), dok se najmanje endemičnih biljaka javlja u visinskom opsegu od 2500-2600
m (samo 7 taksona).
Urađena je i analiza prisustva balkanskih endemičnih taksona po regionima i
podregionima Crne Gore. Teritorija Crne Gore je podijeljena u dva osnovna regiona
Primorski i Planinsko-kotlinski, pri čemu je Primorski podijeljen na 2 podregiona
mediteranski (M) i submediteranski (SM), a Planinsko-kotlinski na 4 podregiona:
centralni (C), istočni (I), sjeverni (S) i zapadni (Z). Utvrđeno je da se najveći broj
endemičnih biljaka javlja u istočnom podregionu (223 vrste i podvrste) i sjevernom
podregionu (205 taksona), dok je najmanje endemičnih biljaka zabeleženo u
mediteranskom podregionu (samo 56 biljaka).
Sumarne karte rasprostranjenja endemičnih biljaka koje pripadaju određenom
areal tipu, kao i osnovnoj životnoj formi, a konačno i sumarna karta rasprostranjenja svih
balkanskih endemičnih taksona na teritoriji Crne Gore, urađene su u cilju izdvajanja
centara diverziteta ove specifične fitogeografske kategorije biljaka u Crnoj Gori.
Konačnim analizama distribucije i stepena zastupljenosti endemičnih taksona na UTM
areal kartama 10x10 km, visokoplaninski region Durmitora sa kanjonskim dolinama
rijeka Pive i Tare izdvojio se kao apsolutni centar diverziteta balkanske endemične flore
na teritoriji Crne Gore. Izuzetnim bogatstvom endemične flore odlikuje se i planinski
masivi Komova i primorskog Lovćena, dok planine Orjen i Rumija u submediteranskom
podregionu i Moračke planine u centralnoj Crnoj Gori takođe spadaju u područja sa
visokim stepenom diverziteta balkanske endemične flore u Crnoj Gori. | The main objective of this study is to determine the total number of the Balkan
endemic taxa of species and subspecies rank on the territory of Montenegro. Field
research and extensive literature and herbarium collection review has found that there are
372 Balkan endemic taxa in Montenegro. The data which include distribution, ecological
characteristics, as well as literature and herbarium sources for each individual taxon have
been incorporated in a Microsoft Excel database which includes 7215 records.
The study lists all sites in which individual taxa were identified. Areal maps with
UTM coordinates 10x10 km have been made, representing the distribution of each
individual taxon on the territory of Montenegro. Analysis of the general distribution, i.e.
the distribution on the Balkan Peninsula has defined the floral element, areal subgroup
and areal group for each taxon. Ecological characteristics of endemic taxa have also been
stated, which implied determining the affiliation to appropriate life form, habitat types
inhabited by the given taxon, range of altitudes and geological substrate types. When
citing literature data from phytocoenological papers, information have been entered on
habitat exposure and slope, as well as on endemic taxa presence in a certain association.
Taxonomic analysis of the Balkan endemic flora in Montenegro has shown that
Asteraceae, Caryophyllaceae, Campanulaceae, Fabaceae and Lamiaceae are the richest
families, while as many as 13 families have a representative in one endemic taxon. The
most numerous Asteraceae family includes the largest Hieracium genus with 71 taxa,
followed by Dianthus, Edraianthus and Campanula genera. Stachys, Cerastium and
Alchemilla genera are represented with an equal number of endemic species and
subspecies. It is interesting to note that as many as 80 genera have one endemic
representative.
Chorological structure of the Balkan endemic plants in Montenegro shows that
almost two thirds of endemic taxa are orophytes that belong to two chorological groups:
south European mountain (146 taxa or 39.2%) and central European mountain group (99
taxa or 26.6%). Consequently, it can be said that there is absolute domination of mountain
and high-mountain endemism in the flora of Montenegro. The Mediterranean and
sub-Mediterranean chorological group is also represented with a proportionally large
number of taxa (81 or 21.8%), which indicates a strong influence of the Mediterranean
and sub-Mediterranean flora reaching along the canyon valleys of the Adriatic basin
rivers deep into the continent. Central European chorological group, typical of forest and
meadow habitats of the inner Dinarides in Montenegro, is represented by far less endemic
plants (45 or 12.3%) in the total Balkan endemic flora chorological spectrum in
Montenegro, while the Pontic areal group is represented by only one endemic taxon.
Biological spectrum analysis of the Balkan endemic flora in Montenegro
indicates that life forms of hemicryptophytes (H) and chamaephytes dominate,
amounting together to 87% of the biological spectrum. Individually, emicryptophytes
account for 63% and chamaephytes for 24%. Most of the plants from Holarctic or
temperate zone and from the Balkan Peninsula belong to hemicryptophyte life form. In
addition to this, hemicryptophytes and chamaephytes populate highland areas, and a
major part of the territory of Montenegro is of highland character.
More detailed analysis of ecological characteristics of the Balkan endemic flora in
Montenegro involved determining the preferences of each individual taxon in relation to
the altitude ranges and type of geological substrate inhabited by each of them. Three
groups of geological substrate (carbonates, silicates, others) have been defined, as well as
the presence of the number of endemic taxa in relation to these types of substrates. It
turned out that the majority of endemic species occur on carbonate geological substrate
(342 taxa), and far less on silicate substrate (only 40 species and subspecies). In relation
to altitude ranges at which endemic species and subspecies occur on the territory of
Montenegro, five classes have been defined and the number of endemic taxa occurring at
altitude ranges defined in such a manner has been determined. The majority of endemic
taxa occur at the height range of 1500–2000 m (156 taxa), as well as at the range of
1000-1500 m (132 species and subspecies), while the least number of endemic plants
occur at the height range of 2500–2700 m (only 7 taxa).
Analysis of the Balkan endemic taxa presence across regions and subregions of
Montenegro has also been made. The territory of Montenegro has been divided into two
major regions – Coastal and Mountain Basin ones, with the Coastal region further divided
into two subregions: Mediterranean (M) and sub-Mediterranean (SM), and Mountain
Basin region into 4 subregions: central (C), eastern (I), northern (S) and western (Z). It
has been found that the largest number of endemic plants occur in the eastern subregion
(223 species and subspecies) and northern subregion (205 taxa), while the least number of
endemic plants has been recorded in the Mediterranean subregion (only 56 plants).
Summary maps of endemic plant distribution which belong to a particular areal
type as well as basic life form, and finally a summary distribution map of all Balkan
endemic taxa on the territory of Montenegro have been made with a view to single out the
centres of diversity of this specific phytogeographic plant category in Montenegro. The
final analysis of distribution and degree of presence of endemic taxa on UTM areal maps
10x10km singled out high mountain region of Durmitor together with canyon valleys of
Piva and Tara rivers as the absolute centre of diversity of the Balkan endemic flora on the
territory of Montenegro. The mountain ranges of Komovi and Lovćen are also
characterized by an exceptional wealth of endemic flora, while the coastal mountains
Orjen and Rumija, and more continental Moračke Planine mountain are also areas with
high degree of the Balkan endemic flora diversity in Montenegro. | true |
Spavanje i elektroencefalografski ritmovi kao indikatori poremećaja funkcionalno različitih holinergičkih inervacija velikog mozga pacova | Sleep and electroencephalographic rhythms as an indicators of the functionally distinct cholinergic innervation disorders in rat brain | Alchajmerova bolest (AB) i Parkinsonova bolest (PB) su najčešće neurodegenerativne bolesti starenja... | Alzheimer's disease (AD)and Parkinson's disease are the most common neurodegenerative diseases in elderly... | false |
Molekulski mehanizmi citotoksičnog delovanja trans-kompleksa platine(II) i platine(IV) sa piridinskim derivatima kao ligandima | Molecular mechanisms of the cytotoxicity of trans-platinum(II) and trans-platinum(IV) complexes with pyridine ligands | Uvod: Hemioterapija koja se zasniva na platinskim agensima je ključna u lečenju
solidnih tumora, a jedan od vodeći itostatika koji se koristi je isplatina. Međutim, i
pored visoke efikasnosti cisplatine, toksičnost i razvoj rezistentnosti ograničili su njenu
upotrebu. Stoga su pokrenuta brojna istraživanja sa iljem pronalaska novi kompleksa
koji bi bili kancer-selektivniji, koji bi posedovali drugačije mete delovanja od isplatine,
a ujedno bi bili manje toksični i ne bi dovodili do pojave rezistentnosti. Mnogi
kompleksi metala, među kojima su brojni trans-platinski kompleksi, za koje se dugo
smatralo da su neaktivni, pokazali su se kao dobri kandidati, što utiče na stalan razvoj
novih jedinjenja kao potencijalnih antikancerskih agenasa.
Cilj: Ispitivanje itotoksičnog poten ijala novosintetisani trans-kompleksa platine(II) i
platine(IV) sa piridinskim derivatima kao ligandima na nekoliko ćelijskih linija
(kompleks 1 - trans-[PtCl2(3-acpy)2]; kompleks 2 - trans-[PtCl2(4-acpy)2]; kompleks 3 -
trans-[Pt(3-acpy)2Cl4]; kompleks 4 - trans-[Pt(4-acpy)2Cl4]). In vitro ispitivanje
molekulskih mehanizama antikancerske aktivnosti trans-platinskih kompleksa na
odabranim ćelijskim linijama na kojima su ispoljili značajno itotoksično delovanje.
Ispitivanje korelacije strukture i aktivnosti kompleksa: uticaj (cis-/trans-) geometrije
kompleksa na itotoksičnu aktivnost i mehanizam delovanja – poređenje trans-
platinskih kompleksa sa cisplatinom; korelacija u odnosu na oksidaciono stanje platine
– poređenje trans-platina(II) i trans-platina(IV) kompleksa; uticaj strukture liganada na
itotoksičnu aktivnost i me anizam delovanja.
Materijal i metode: Antiproliferativna aktivnost ispitivanih kompleksa nakon 48 h
delovanja ispitana je MTT i SRB testovima na šest tumorski ćelijskih linija (HeLa,
MDA-MB-453, B16, FemX, A549, LS-174), dve transformisane endotelijalne linije
humanog (EA. y 926) i mišjeg (MS1) porekla, i jednoj normalnoj, zdravoj ćelijskoj
liniji fetalni umani fibroblasta pluća (MRC-5). Potencijal ispitivanih kompleksa da
indukuju promene na nivou ćelijskog ciklusa određen je protočnom citometrijom nakon
bojenja tretirani HeLa i EA. y 926 ćelija propidijum-jodidom. Dvokolornim bojenjem
ćelija aneksinom i propidijum-jodidom i analizom na protočnom citometru ispitan je
potencijal kompleksa da indukuju apoptozu. Unutarćelijska akumula ija i distribucija
platine(II) u proteinskoj i DNK frakciji HeLa i EA. y 926 ćelija tretiranih ispitivanim
kompleksima utvrđena je korišćenjem metoda ICP-OES i ICP-MS. Kako bismo utvrdili
da li ispitivani kompleksi indukuju odgovor ćelija zavisan od DNK reparacije kao
rezultat interakcija sa DNK, ispitivali smo iRNK i proteinski nivo ekspresije ERCC1
metodama qRT-PCR i Western blot. In vitro antimetastatski potencijal ispitivanih
kompleksa na HeLa i EA. y 926 linijama ispitan je praćenjem iRNK i proteinskog
nivoa ekspresije matriksnih metaloproteinaza (MMP-2 i MMP-9) metodama qRT-PCR i
želatinske zimografije. In vitro antiangiogeni potencijal kompleksa ispitan je testom in
vitro formiranja tubula (“tube formation assay”) na MS1 i EA. y 926 ćelijskim
linijama. Uticaj kompleksa na morfološke karakteristike ćelijske smrti EA. y 926 ćelija
i ekspresiju markera apoptoze (bax i kaspaza 3 proteina) ispitan je fluorescentnom
mikroskopijom, odnosno Western blot metodom. Cisplatina je korišćena u svim
analizama radi poređenja.
Rezultati: Ispitivani trans-platinski kompleksi su pokazali značajan antiproliferativni
potencijal na svim tumorskim ćelijskim linijama, naročito na HeLa i EA. y 926
ćelijama, koji u nekim slučajevima prevazilazi aktivnost isplatine, a jači je od onoga
koji kompleksi imaju na normalnim MRC-5 ćelijama. Pokazano je da postoje razlike u
ćelijskom odgovoru na delovanje kompleksa u HeLa, odnosno EA.hy 926 ćelijama,
kako u mehanizmima indukcije ćelijske smrti tako i u procesima angiogeneze i
metastaziranja in vitro. Utvrđeno je da trans-platina(II) kompleksi poseduju značajniju
itotoksičnu i antitumorsku aktivnost in vitro, pokazuju bolju unutarćelijsku
akumula iju i reaktivnost sa ćelijskim proteinima i DNK, u poređenju sa trans-
platina(IV) kompleksima. Kompleks 2 poseduje najveću itotoksičnu aktivnost i
ispoljava značajne efekte u smislu antitumorskog dejstva in vitro: indukuje značajnu
unutarćelijsku akumula iju platine i značajnu platina iju DNK i proteina; poseduje
antiangiogeni potencijal; indukuje smanjenje genske i proteinske ekspresije ERCC1;
indukuje apoptozu.
Zaključak: Upoređivanjem aktivnosti ispitivani trans-platinskih kompleksa sa
cisplatinom utvrđeno je da geometrija kompleksa (cis-/trans-) utiče na aktivnost,
odnosno itotoksični poten ijal i generalno me anizam delovanja – biološku aktivnost
in vitro. Pokazano je da male razlike u strukturi ispitivani kompleksa, kao što je
pozicija acetil-supstituenta u piridinskom ligandu, mogu značajno uti ati na razlike u
molekulskom
mehanizmu
itotoksičnog
delovanja
kompleksa.
Trans-platinski
kompleksi sa piridinskim derivatima kao ligandima su dobri potencijalni antikancerski
agensi, pri čemu se kompleks 2 (trans-[PtCl2(4-acpy)2]) pokazao kao najbolji kandidat
za dalja ispitivanja. | Introduction: Platinum-based anticancer chemotherapy constitutes a cornerstone for the
treatment of various solid tumors. Cisplatin was one of the first chemotherapeutic
agents to exhibit broad efficacy and it remains among the most widely used agents in
the treatment of cancer. Its introduction inspired great efforts to design similarly
effective platinum agents with different molecular targets and with the ability to
overcome main limitations of cisplatin - toxicity and tumor resistance. This has
provided the motivation for developing novel metal complexes as anticancer agents
with different mechanisms of action, including numerous trans-platinum complexes that
were considered inactive for a long time.
Aim: Investigation of the cytotoxic potential of novel trans-platinum(II) and trans-
platinum(IV) complexes with pyridine ligands in several cell lines (complex 1 - trans-
[PtCl2(3-acpy)2]; complex 2 - trans-[PtCl2(4-acpy)2]; complex 3 - trans-[Pt(3-
acpy)2Cl4]; complex 4 - trans-[Pt(4-acpy)2Cl4]). In vitro investigation of molecular
mechanisms of their anticancer action. Analyzing the structure-activity relationship of
the complexes: investigating if cis-/trans- geometry affects the cytotoxicity and
mechanism of action – comparison of trans-platinum complexes to cisplatin; correlation
in terms of oxidation states of platinum – comparison of trans-platinum(II) with trans-
platinum(IV) complexes; investigating if structure of the ligands affects the anticancer
activity of the complexes in vitro.
Material and methods: The cytotoxic activity of the complexes was investigated by SRB
and MTT assays for 48 h of continual action on six tumor cell lines (HeLa, MDA-MB-
453, B16, FemX, A549, LS-174), two transformed endothelial cell lines (EA.hy 926
and MS1), and one normal lung fibroblast cell line (MRC-5). Potential of the
investigated complexes to induce cell cycle perturbations was determined after staining
of treated HeLa and EA.hy 926 cells with propidium iodide (PI) and analysis on flow
cytometer. Potential of the complexes to induce apoptotic changes was analyzed using
flow cytometry after two-color staining with Annexin V-FITC and PI. Total
intracellular platinum accumulation and platinum(II) distribution among the DNA and
protein fractions in HeLa and EA.hy 926 cells were determined by inductively coupled
plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass
spectrometry (ICP-MS). Affinity for the interaction with DNA and expression of
ERCC1 mRNA and protein levels in HeLa cells were also analyzed by qRT-PCR and
Western blot and compared to cisplatin. The inhibitory activity of the complexes on
matrix metalloproteinases (MMP-2, MMP-9) was investigated by gelatin zymography
and qRT-PCR in HeLa and EA.hy 926 cell lines. In vitro antimetastatic potential of the
trans-platinum complexes at subtoxic concentrations was examined in MS1 and EA.hy
926 cells using tube-formation assay. Morphological changes and expression of bax and
caspase 3 proteins in EA.hy 926 treated cells were analyzed by fluorescent microscopy
and Western blot. All the analyses were performed with cisplatin as a reference
compound.
Results: The tested complexes showed significant cytotoxic activity in all used tumor
cell lines, with the greatest effects in HeLa and EA.hy 926 cells. Their antiproliferative
activity in vitro overcame the activity of cisplatin in some cell lines, and also the
activity in MRC-5 normal cell line. Cytotoxicity differences in HeLa and EA.hy 926
cells may be attributed to the ability of the complexes to produce different cellular
effects related to damage-processing and signal activation pathways, including cell
death mechanisms and angiogenesis and metastasis in vitro, and to induce multiple
cellular responses. In comparison to trans-platinum(IV) complexes, trans-platinum(II)
complexes showed significant cytotoxic and antitumor activity in vitro, better
intracellular platinum accumulation and binding to cellular DNA and proteins. Complex
2 showed the highest cytotoxic potential, as well as significant antitumor activity in
vitro: it induced significant intracellular platinum accumulation and DNA and protein
platination; showed antiangiogenic potential; inhibited mRNK and protein expression of
ERCC1; induced apoptosis.
Conclusion: The obtained results suggest that structural settings play a fundamental
role, since they are responsible for the interaction between trans-platinum drugs and
cellular DNA and proteins that led to the observed biological effects. Biological activity
in vitro and molecular mechanism of action depend on cis-/trans- geometry of the
complexes, and different positioning of the acetyl substituent on the pyridine ring.
Altogether, properties of substituted trans-platinum pyridines, especially complex 2,
encourage further investigations in search for compounds with different modalities of
action toward cancer cells in comparison to CDDP. | true |
Utvrđivanje varijabilnosti i diferencijacije prirodnih populacija Pinus nigra Arnold u Srbiji korišćenjem fitohemijskih i molekularnih markera | Determination of variability and diferentiation of native populations of Pinus nigra Arnold in Serbia using phytochemical and molecular markers | U ovom radu analizirana je fitohemijska i genetiţka varijabilnost i diferencijacija sedam autohtonih populacija Pinus nigra u Srbiji koje pripadaju razliţitim infraspecijskim taksonima (ssp. nigra, var. gocensis, ssp. pallasiana i var. banatica). U dvogodišnjim ţetinama 195 jedinki iz sedam populacija odreŤen je sadrţaj i varijabilnost n-alkana i terpena. Kutikularne voskove ţetina istraţivanih populacija crnog bora odlikuje serija nalkana od C16 do C33, osim u populacijama opisanim kao ssp. nigra u kojima je detektovana serija od C18 do C33. Najzastupljeniji su n-alkani sa neparnim brojem C atoma: C23, C25, C27 i C29 (proseţni sadrţaj 10.3, 16.5, 15.1 i 10.7%, respektivno). U etarskim uljima ţetina utvrŤeno je ukupno 75 komponenti, od koji je 58 identifikovano. Dominantne komponente su α-pinen (43.6%) i germakren D (29.8%), koje zajedno ţine 73.4% mase ulja. Multivariacione statistiţke analize (PCA, CDA i HCA) koriššenih fitohemijskih markera su, generalno, pokazale tendenciju formiranja tri grupe populacija (nigra, pallasiana i banatica). Na osnovu varijabilnosti terpena, populacije Priboj i Goţ (determinisane kao var. gocensis) pokazale su umerenu tendenciju odvajanja od prve grupe (ssp. nigra), dok je prema sastavu n-alkana populacija Priboj pokazala još vešu tendenciju odvajanja. Pretpostavljeno je da takson gocensis predstavlja tranzicionu formu izmeŤu taksona nigra na zapadu i pallasiana na jugoistoku Srbije. U cilju odreŤivanja genetiţkog diverziteta i diferencijacije sedam istraţivanih populacija P. nigra iz Srbije, testirano je 114 jedinki pomošu jedarnih (nuDNK), hloroplastnih (cpDNK) i mitohondrijalnih (mtDNK) markera koji imaju bi-parentalno, paternalno i maternalno nasleŤivanje, respektivno, kao i razliţitu brzinu evolucije. Zbog nedostatka jedarnih mikrosatelita (nuSSRs) specijalno razvijenih za P. nigra, uraŤena je inter-specijska PCR amplifikacija tri EST-SSRs razvijenih za P. taeda. Iako su dva ESTSSR lokusa uspešno PCR-amplifikovana u našim uzorcima, utvrŤeno je odsustvo varijabilnosti zbog ţega su iskljuţeni iz dalje analize. Od pet testiranih cpDNK SSRs, tri su uspešno PCR-amplifikovana u našim uzorcima, sa 22 detektovana alela koji formiraju 38 haplotipova. Kao što je i oţekivano, s obzirom na intenzivan protok gena posredstvom polena kod ţetinara, utvrŤen je veoma visok nivo cpDNK diverziteta (HT = 0.949) i mali stepen diferencijacije (Fst = 0.017, p = 0.108, Gst = 0.024, Rst = 0.066)... | In this work phytochemical and genetic variability and differentiation of seven native populations belonging to different infraspecific taxa of Pinus nigra (ssp. nigra, var. gocensis, ssp. pallasiana i var. banatica) in Serbia were analyzed. In the needles of 195 trees from seven populations, composition and variability of n-alkanes and terpenes were determined. The size of the n-alkanes found in the needle cuticular waxes of the black pine populations investigated ranged from C16 to C33, with the exception of that of the subspecies nigra, which ranged from C18 to C33. The most abundant n-alkanes were the four odd-numbered n-alkanes C23, C25, C27, and C29 (mean contents of 10.3, 16.5, 15.1, and 10.7%, respectively). In the needle essential oils a total of 58 individual components out of the 75 compounds detected could be identified. The major components of the oils were α-pinene (43.6%) i germakrene D (29.8%), comprising together in average 73.4% of the essential oil composition. Multivariate statistical analysis (PCA, CDA, and HCA) of used phytochemical markers showed, in general, a tendency to the formation of three populations groups (nigra, pallasiana, and banatica). Based on the terpene variability, populations Priboj and Goţ, determined as var. gocensis, showed a moderate tendency of separation from the first group (ssp. nigra), while based on n-alkane composition, population Priboj showed even a greater tendency of separation. It was assumed that taxon gocensis might be a transitional form between taxa nigra occurring in the west and pallasiana growing in the southeast of Serbia. We screened 114 trees from seven studied P. nigra populations from Serbia with nuclear (nrDNA), chloroplast (cpDNA) and mitochondrial (mtDNA) markers which have bi-parental, paternal and maternal inheritance, respectively, and different evolutionary rates to infer levels of genetic diversity and structuring within studied area. Due to the lack of nuclear microsatellites (nuSSRs) specifically developed for P. nigra, we employed three EST-SSRs developed for P. taeda for cross-species amplification in P. nigra. Although two EST-SSR loci were successfuly PCR-amplified in our samples, they were invariable and discarded from further analyses... | false |
Primena Higučijeve fraktalne dimenzije i metode nezavisnih komponenata u analizi spontane bioelektrične aktivnosti Br neurona vinogradskog puža pod delovanjem konstantnog magnetnog polja i uabaina | Application of Higuchi fractal dimension and indepenedent component method in analysis of garden snail Br neuron bursting activity modulated by static magnetic field and ouabain. | Nelinearne i napredne statističke metode, pored linearnih metoda, zauzimaju sve
značajnije mesto u analizi fizioloških signala, posebno u svetlu nelinearnog i haotičnog
ponašanja bioloških sistema. Stoga je zajednička upotreba Higučijeve fraktalne dimenzije i
analize nezavisnih komponentata (ICA), značajan i nov pristup u analizi signala a posebno u
analizi aktivnosti jednog neurona. Najprepoznatljiviji tip spontane bioelektrične aktivnosti
neurona beskičmenjaka i kičmenjaka jeste pojava akcionih potencijala u paketićima.
U radu je po prvi put primenjen, jedinstven i inovativan pristup u razdvajanju
komponenata spontane bioelektrične aktivnosti Br neurona vinogradskog puža i to na akcione
potencijale (AP), intervale između akcionih potencijala (ISI) i tihe intervale bez aktivnosti (IBI)
uz pomoć Higučijeve fraktalne dimenzije
i aproksimacije Gausovim funkcijama. Taj
metodološki pristup je omogućio praćenje uticaja konstantnog magnetnog polja i uabaina
inhibitora Na+/K+ pumpe na promene u kompleksnosti spontane bioelektrične aktivnosti Br
neurona. Sa druge strane, po prvi put je testirana upotreba ICA metode u različitim
eksperimentalnim uslovima po AP, ISI i IBI komponentama spontane bioelektrične aktivnosti.
Na taj način, u ovom radu demonstrirana je snaga zajedničke upotrebe navedenih metoda
uz predlog da se proširi njihova upotreba za potrebe analize spontano aktivnih neurona različitih
vrsta u fiziološkim i patološkim stanjima. | Nonlinear and advanced statistical methods, in addition to linear methods, occupy a
prominent place in the analysis of physiological signals, especially in light of the non-linear and
chaotic behavior of biological systems. Therefore, the use of Higuchi fractal dimension and
independent component analysis (ICA), reperesents a new approach to signal analysis, especially
regarding the activities of one neuron. The most recognizable type of spontaneous bioelectric
activity in neurons of invertebrates as well as vertebrates is the appearance of bursting activity.
This study presents a unique and innovative approach to the separation of the components
of spontaneous bioelectric activity of the garden snail Br neuron - action potential (AP),
interspike interval (ISI) and interburst interval (IBI), by using Higuchi’s fractal dimension and
Gaussian fitting functions. This methodological approach allows monitoring of the effect of
static magnetic field and the inhibitor of the Na+/K+ pump, ouabain, on the changes in the
complexity of the spontaneous bioelectric activity of the Br neuron. On the other hand, for the
first time ICA method was tested in different experimental conditions on AP, ISI and IBI
components of spontaneous bioelectric activity.
This study demonstrates the power of the common use of the above mentioned methods
and proposes to extend their use for the purpose of analyzing spontaneously active neurons of
different species in physiological and patological conditions. | true |
Subsets and Splits