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Gljive iz roda Trichoderma spp. su kosmopolitske filamentozne gljive koje se pojavljuju kao dominantne komponente zemljišnog ekosistema. Ekološka i biološka uloga ovih gljiva je veoma raznovrsna. Kao endofitni simbionti biljaka imaju primenu u kontroli biljnih bolesti čime pospešuju biljni rast i prinos. Takođe, poznata je i njihova sposobnost da indukuju otpornost kod biljaka koje su izložene biotičkom ili abiotičkom stresu. Pored važne primene u poljoprivredi one nalaze primenu i u različitim granama industrije jer proizvode mnogobrojne enzime i metabolite. Obzirom da su mnoge vrste u okviru ovog roda ne mogu razlikovati na morfološkom nivou, neophodna je njihova identifikacja na molekularnom nivou.Cilj ove teze bio je utvrđivanje najvažnijih faktora koji utiču na varijabilnost roda Trichoderma u različitim tipovima zemljišta. Molekularna identifikacija gljiva roda Trichoderma, izolovanih iz različitih tipova zemljišta (rendzina, sirozem na lesu, ritska crnica, fluvisol, ranker, gajnjača, černozem sa različitim podtipovima i smonica) i iz rizosfere različitih biljnih kultura urađena je analizom sekvenci ITS i TEF1-α gena. Uzorci zemljišta ispitani su u funkciji fizičko-hemijskih i mikrobioloških karakteristika, kao i sadržaja ukupnih i lakopristupačnih metala (Cr, Co, Ni, Cu, Zn, Cd, Pb i Mn). U cilju ispitivanja primene ovih gljiva kao antagonista fitopatogenih mikroorganizama, testirani su odabrani izolati prema Fusarium solani species complex (FSSC), Alternaria alternata i Phoma cucurbitacearum. Enzimska karakterizacija izolata odabranih za test antagonizma, urađena je semikvantitativnim testom API-ZYM. Kako bi se ispitao efekat ovih gljiva i na abiotski stres izazvan povećanim sadržajem metala u zemljištu, između ostalih, odabrani su izolati koji su identifikovani iz zemljišta u kojima je određena povećana koncentracija metala, bakra(II) i nikla(II). Na osnovu in vitro testa, gajenjem izolata na povećanim koncentracijama bakra(II) i nikla(II), odabran je izola, Fungi of the genus Trichoderma spp. are cosmopolitan species present in different ecosystems. with important ecological and biological roles.. Beneficial effects of this fungi are responsible for their use in various fields of biotechnology and agriculture. As plant endophytes they are involved in control of plant deseases. They are known to induce plant tolerance to various biotic and abiotic stresses. Known to produce different enzymes, they can be used in preparations of commercial products, such as biopesticides. However, for their adequate use and application their identification on the molecular level is of great importance.The aim of this thesis was to investigate the most important factors that influence Trichoderma variabilty in different soil types. Molecular identification of Trichoderma spp. from different soil types (Rendzic Leptosol, Regosol (Calcaric, Arenic), Calcic Gleysol, Fluvisol, Dystric Leptosol, Eutric Cambisol, Chernozem on loess terrace Chernozem (Arenic) and Vertisol) and plant rhizosphere of different crops was done based on their ITS and TEF1-α sequences. Variability was investigated in function of physical, chemical, microbiological properties of the soil, as well as total and extractable metal (Cr, Co, Ni, Cu, Zn, Cd, Pb and Mn) content in the soil. In order to examine the application of these fungi as antagonists of phytopathogens, selected strains of Trichoderma species were tested against Fusarium solani species complex (FSSC), Alternaria alternata and Phoma cucurbitacearum. The enzyme characterization of selected strains was performed with semiquantitative test API-ZYM. Furthermore, the selected strains were tested for tolerance to various concentrations of copper and nickel. The strain T. brevicompactum SZMC 22661 which showed the best tolerance to metals was applied in the examination of drought effect on tomato plants...

Objective: The objective of the present study was to determine if vitamin D intake and status are associated with pre-eclampsia in a country without a vitamin D fortification policy. Design: A case-control study of pregnancies with (case) and without (control) pre-eclampsia was conducted from January to April when UVB is minimal. Maternal and cord blood obtained at delivery were measured for plasma 25-hydroxycholecalciferol (25-OH-D-3), 3-epimer of 25-OH-D-3 (3-epi-25-OH-D-3) and 24,25-dihydroxycholecalciferol (24,25-(OH)(2)D-3) by LC-MS/MS and maternal 1,25-dihydroxyvitamin D (1,25-(OH) 2D). Differences between groups were tested with ANOVA and Bonferroni post hoc tests (P lt 0.05). Setting: Clinical Center of Serbia. Subjects: Pregnant women with and without pre-eclampsia (n 60) and their infants. Results: Exogenous vitamin D intake (0.95-16.25 mu g/d (38-650 IU/d)) was not significantly different between groups. Women with pre-eclampsia delivered infants at an earlier gestational age and had significantly lower mean total plasma 25-hydroxyvitamin D (25-OH-D; case: 11.2 (SD 5.1); control: 16.1 (SD 5.7) ng/ml; P=0.0006), 25-OH-D-3 (case: 10.0 (SD 4.9); control: 14.2 (SD 5.8) ng/ml; P=0.002), 3-epi-25-OH-D-3 (case: 0.5 (SD 0.2); control: 0.7 (SD 0.2) ng/ml; P=0.0007) and 1,25-(OH)(2)D (case: 56.5 (SD 26.6); control: 81.0 (SD 25.7) pg/ml; P=0.018), while 24,25-(OH)(2)D-3 was not different between groups. Infants did not differ in total plasma 25-OH-D, 25-OH-D-3, 3-epi-25-OH-D-3 and 24,25-(OH)(2)D-3, but the mean proportion of 3-epi-25-OH-D-3 was higher in the infant case group (case: 7.9 (SD 1.1); control: 7.0 (SD 1.4) % of total 25-OH-D-3; P=0.005). Conclusions: A high prevalence of vitamin D deficiency, as defined by plasma 25-OH-D lt 12 ng/ml, was observed in 47 % of all mothers and 77 % of all infants. These data underscore the need for prenatal vitamin D supplementation and a food fortification policy in Serbia.

Humani serum albumin (HSA) je najzastupljeniji protein plazme sa udelomod oko 50 do 60 % svih proteina plazme. Vezuje i transportuje mnoge endogene iegzogene molekule, i doprinosi antioksidativnom kapacitetu seruma jer na površinimolekula poseduje jednu slobodnu tiolnu grupu ostatka Cys34. Slobodne masne kiseline(MK), koje transportuje HSA, i karbonilni stres mogli bi, kroz promenu reaktivnostitiolne grupe, uticati na njen potencijal kao hvatača reaktivnih karbonilnih vrsta. Stogasu ciljevi ove teze bili: in vitro ispitivanje uticaja 1) vezivanja MK, različite dužinelanca i zasićenosti na (i) reaktivnost Cys34-SH grupe, (ii) njen potencijal kao hvatačareaktivnih α-oksoaldehida, tj. na stepen karbonilacije i (iii) reaktivnost tiolne grupeHSA karbonilovanog metilglioksalom (model-sistem za molekule HSA modifikovane ukarbonilnom stresu); 2) vezivanja MK ribljeg ulja na reaktivnost Cys34-SH grupe(sagledavanje mogućnosti modulacije svojstava HSA pomoću suplemenata); 3)određivanje sadržaja Cys34-SH grupe, korelacija sa sadržajem HbA1c i glukoze ukarbonilnim stresom; 4) određivanje reaktivnosti Cys34-SH grupe HSA izolovanog izseruma dijabetičara i zdravih osoba; 5) razvijanje qTLC metode za određivanje sadržajaMK, vezanih za HSA, koji je izolovan iz realnih uzoraka.Vezivanje MK (različite dužine lanca i zasićenosti: miristinske (MYR),palmitinske (PLM), stearinske (STE), oleinske (OLA), eikozapentenske (EPA) idokozaheksaenske kiseline (DHA)) za HSA in vitro, dovodi do povećanja vrednostikonstanti brzine reakcije (k) Cys34 tiolne grupe (i DTNB) (k vrednosti kompleksa HSAMK:od 14,58±0,19 x 10-3 do 26,02±1,06 x 10-3 s-1 u odnosu na k HSA: 7,52±0,04 x 10-3 s-1), odnosno njene reaktivnosti za 2 do 3,5 puta. STE i OLA ispoljavaju slične efektena reaktivnost HSA-SH, a najjači efekat ispoljava polinezasićena EPA. Od zasićenihMK, najveća vrednost k dobijena je pri vezivanju MYR za HSA, što može biti posledicaizuzetne komplementarnosti njenog molekula sa vezivnim mestima na molekulu, Human serum albumin (HSA) is the most abundant protein of humanplasma, accounting for 50-60% of total plasma proteins. HSA binds and transport manyendogenous and exogenous substances, and contributes to the antioxidative pool inserum because of Cys34 free thiol group on the surface of its molecule. Free fatty acids(FAs), transported by HSA, and carbonyl stress could influence, through changedreactivity of the thiol group, on its potential as the scavenger of the reactive carbonylspecies. Therefore, the aims of this thesis were: in vitro examination of the influence 1)of different (in terms of chain length and saturation) FAs binding on the: (i) reactivity ofthe Cys34-SH group, (ii) its potential as the scavenger of the reactive α-oxoladehydes,and (iii) on the reactivity of the thiol group of HSA carbonylated with methylglyoxal(the model-system for HSA molecules modified during carbonyl stress); 2) of FAsfrom fish oil on the Cys34-SH group reactivity (overviewing the possibility of themodulation of the HSA scavenger properties with supplements); 3) determination of theCys34-SH group content, correlation with the HbA1c and glucose level of persons withelevated carbonyl stress; 4) determination of the Cys34-SH group reactivity of the HSAisolated from the sera of diabetic patients and healthy persons; 5) development of qTLCmethod for determination of the FAs content, bound to the HSA, which was isolatedfrom sera samples.Binding of FAs of different chain lengths and saturation (myristic (MYR),palmitic (PLM), stearic (STE), oleic (OLA), eicosapentaenoic (EPE) anddocosahexaenoic acid (DHA)) to the HSA in vitro resulting in increasing of the kineticsconstant (k) of the Cys34 thiol group reaction (with DTNB)(k values for HSA-FAscomplexes: from 14.58±0.19 ×10-3 to 26.02±1.06 × 10-3 s-1 comparing to k of the HSAsolely 7.52 ± 0.04 × 10-3 s-1), i.e. its reactivity for 2 to 3.5 times. STE and OLA showsimilar effects to the HSA-SH reactivity, and the strongest effect shows polyuns

Objective: The objective of the present study was to examine the external validity of an FFQ designed to estimate dietary vitamin D intake compared with a plasma biomarker and three repeated 24 h dietary recalls in women of reproductive age in Serbia, where there is no exposure to food fortified with vitamin D. The method of triads was applied. Design: In a cross-sectional study, 422 women completed the Women and Reproductive Health FFQ (WRH-FFQ) during the winter months. From a representative subgroup (n 44), three 24 h dietary recalls and anthropometric parameters were collected as well as a fasting blood sample for vitamin D biomarker analyses. Correlation coefficients were calculated between each of the dietary methods. Validity coefficients, as a correlation between the measured and estimated 'true' exposure, were calculated using the method of triads. Bland-Altman plots were also constructed. Setting: Three major universities in Serbia. Subjects: Healthy young women (n 422) aged 18-35 years. Results: The WRH-FFQ estimate of vitamin D intake for all participants was 4.0 (SD 3.3) mu g/d and 3.1 (SD 2.3) mu g/d for the subgroup. Bland-Altman plots for these intakes showed high agreement. Validity coefficients for the FFQ, 24 h recall and biomarker were. rho(QI) = 0.847 (95 % CI 0.564, 0.928), rho(RI) = 0.810 (95 % CI 0.537, 0.997) and rho(BI) = 0.499 (95 % CI 0.190, 0.840), while the correlation coefficients were 0.686, 0.422 and 0.404. Conclusions: The FFQ applied in the present study is a valid tool for assessing dietary vitamin D intake in women living in Serbia, a region without mandatory vitamin D food fortification.

Non-esterified fatty acids bound to the human serum albumin (HSA) contribute to several HSAs properties of special concern in pathologies, for instance to the reactivity of the free HSA-Cys34 thiol group (important antioxidative thiol pool in plasma), and to the affinity for binding of molecules and ions (for example cobalt as a prominent biomarker in heart ischemia). Therefore, the method for determination of FAs bound to HSA was developed. FAs were released from HSA (previously isolated from serum by ammonium sulfate precipitation) using acidic copper(II) sulfate in phosphoric acid, extracted by n-heptane-chloroform (4:1, v/v) mixture, spotted on TL silica-gel and then developed with n-heptane-chloroform-acetic acid (5:3:03, v/v/v). Common office flatbed scanner and software solution for densitometric image analysis, developed in R, were used. The linearity of calibration curve in concentration range from 0.1 to 5.0 mmol/L stearic acid was achieved. The method was proved to be precise (with RSD of 1.4-4.7%) and accurate. Accuracy was examined by standard addition method (recoveries 97.2-102.5%) and by comparison to results of GC. The method is sample saving, technically less demanding, and cheap, and therefore suitable for determination of FAs/HSA ratio when elevated concentrations of free FAs are reliable diagnostic/risk parameter of pathological states. (C) 2016 Elsevier B.V. All rights reserved.

This paper describes the results achieved by high school seniors on an item which involves translation of the equation P = kT into a corresponding pictorial external representation. The majority of students (the classes of 2011, 2012 and 2013) did not give the correct answer to the multiple choice part of the translation item. They chose pictorial representations of the other gas laws (P = k/V, or V = kT) instead. Failure to choose the correct answer was surprising considering that the symbol for volume was absent which should have been the key clue. Through the analysis of students' explanations (the classes of 2011 and 2012) and interviews (the class of 2013) we considered the reasoning applied by students who chose the correct answer or distractors for the multiple choice part of the item. Among the students who answered correctly there were explanations which contained misconceptions. Several factors that lead to the unsuccessful translation between external representations have been discovered. Students interpreted the change in one quantity based on the notation for the change in another one because of deep rooted cognitive schemas about changing two quantities (volume and pressure, pressure and temperature, temperature and volume), without consideration that for such changes to be valid for gases all three quantities need to be considered for a certain amount of substance. Those cognitive schemas interfered with mathematical reasoning, i.e. students possessed limited understanding of the equations.

Vitamin D je od izuzetne važnosti u trudnoći pre svega za normalan rast kostiju i zacelokupan razvoj bebe. Pokazano je međutim da 40-80 % trudnica ima nizak nivo (< 20ng/ml) 25-hidroksivitamina D (25(OH)D) u serumu kao i da nizak nivo vitamina D utrudnoći povećava rizik za pojavu preeklampsije (PE) i prevremenih porođaja. U Srbiji,gde hrana nije obogaćena vitaminom D, stopa preeklampsije je veća u zimskom periodu.Odgovor na suplemenatciju vitaminom D dodatno se usložnjava prisustvom C-3α-epimera 25(OH)D3 (3-epi-25(OH)D3), čiji udeo u ukupnom nivou 25(OH)D u krvi možebiti i do 60 % kod beba, a do 17 % kod odraslih. Prisustvo 3-epi-25(OH)D3 u krvi pupčanevrpce ukazuje da postoji prenos sa majke na novorođenče. Međutim, i dalje je nepoznatokoja je uloga povišenog nivoa 3-epi-25(OH)D3 kod novorođenčadi, kako se nivo menjatokom prvih nedelja života novorođenčadi kao i kakav efekat 3-epi-25(OH)D3 ima na rastkostiju beba rođenih iz fizioloških trudnoća u odnosu na trudnoće sa komplikacijama kaošto je preeklampsija. Stoga su ciljevi ove teze: 1) validacija upitnika o učestalostikonzumiranja namirnica (WRH-FFQ) koji bi omogućio procenu unosa vitamina D kodmladih žena u reproduktivnom dobu u Srbiji; 2) sagledavanje povezanosti niskog unosa istatusa vitamina D sa pojavom preeklampsije, kao i određivanje udela 3-epi-25(OH)D3 uukupnom sadržaju 25(OH)D3 u odnosu na zdravstvene ishode majki i novorođenčadi; 3)određivanje nivoa 3-epi-25(OH)D3 tokom prvih nedelja života novorođenčadi isagledavanje njegovog efekta na rast kostiju beba rođenih iz fizioloških trudnoća uodnosu na trudnoće sa komplikacijama kao što je preeklampsija; i 4) ispitivanje biološkogodgovora kostiju na izloženost endogenom i egzogenom izvoru 3-epi-25(OH)D3 naanimalnom modelu.Studija validacije upitnika WRH-FFQ za procenu unosa vitamina D kod mladih žena ureproduktivnom dobu u Srbiji izvedena je od novembra do aprila, kada je smanjeno UVBzračenje. Upitnik WRH-FFQ adekvatno su popunile 422 žene od kojih su nasumič, Vitamin D exposure during pregnancy is important for skeletal growth and developmentin the offspring. However, 40-80% of expecting mothers have low (< 20 ng/ml) serum25-hydroxyvitamin D (25(OH)D) level . A recent meta-analysis demonstrated that lowvitamin D status in pregnancy increases the risk of preeclampsia (PE) and preterm birth.In Serbia where there is no mandatory vitamin D food fortification policy, rates of PEare higher in winter. Response to vitamin D supplementation is complicated by thepresence of C-3α epimer of 25(OH)D3 (3-epi-25(OH)D3) representing up to 60% oftotal 25(OH)D in infants and 17% in adults, raising questions regarding its biologicalderivation and function. The presence of cord 3-epi-25(OH)D3 suggests maternal-fetaltransfer. However, it is still unknown whether 3-epi-25(OH)D3 increases in the first fewweeks after birth and how it effects bone mineral accretion in infants of healthy andcomplicated pregnancies. Therefore, sequential objectives of this thesis are to: 1)validate a food frequency questionnaire (WRH-FFQ) for assessing vitamin D intake inyoung women of reproductive age in Serbia; 2) determine if low vitamin D intake andstatus is associated with PE in Serbia; and describe the proportion of total 25(OH)D inthe 3-epi-25(OH)D3 form in relation to maternal and neonatal outcomes; 3) establish if3-epi-25(OH)D3 in preterm and term infants of mothers with and without PE ispredictive of bone mass at term age using a nested case vs. control design; and 4) testthe biological response of bone to endogenous and exogenous 3-epi-25(OH)D3 in ananimal model.The Women and Reproductive Health FFQ (WRH-FFQ) was completed by 422 women.A cross-sectional study, was conducted from November to April when UVB in Serbia isminimal. On a representative sub-sample (n=44) three 24 hour dietary recalls andanthropometric parameters were collected. Fasting blood samples for vitamin Dbiomarkers analyses were available for 23 women...

Poznato je da su semenke lana, susama i golice bogate esencijalnim masnim kiselinama ida predstavljaju značajni dijetarni izvor vitamina E i polifenola. Kod osoba sa hroničnombubrežnom insuficijencijom, na programu hemodijalize, aktuelna su ispitivanja dijetarnih mera ucilju smanjenja incidence kardiovaskularnih oboljenja. Iz navedenog proizišao je cilj ovedisertacije da se ispitaju efekti suplementacije ishrane smešom semenki lana, susama i golice kodbolesnika na hemodijalizi.Antioksidativna uloga humanog serum-albumina (HSA) u značajanoj meri određujeukupnu antioksidativnu aktivnost u plazmi, a analiza HSA pokazala je da je kod bolesnika naprogramu hemodijalize smanjen sadržaj redukovane Cys34 tiolne grupe (od koje potičeantioksidativni potencijal HSA) i povećan stepen karbonilacije HSA. Stoga je drugi cilj ovedisertacije bio da se in vitro ispita uticaj vezivanja komponenti ekstrakta smeše semenki lana,susama i golice, ferulinske kiseline (FE) i metabolita lignana (enterolaktona (EL) i enterodiola(ED), koji se povećavaju u cirkulaciji nakon konzumiranja namirnica bogatih biljnim lignanima),kao i masnih kiselina, na promenu konformacije HSA i reaktivnosti (antioksidativnogpotencijala) Cys34 tiolne grupe, a da bi se sagledali mogući mehanizmi kojima komponente lana,susama i golice ostvaruju svoje in vivo efekte, pre svega antioksidativno dejstvo.Analizom masnokiselinskih profila ukupnih lipidnih ekstrakata smeše semenki gasnotečnomhromatografijom potvrđeno da je u semenkama lana najzastupljenija α-linolenskakiselina (ALA), a u semenkama susama i golice linolna kiselina (LA). Hladnoceđena uljaispoljila su različitu antioksidativnu aktivnost (rafinisano suncokretovo ulje>maslinovo ulje>uljelana>ulje golice> ulje susama) pri DPPH testu, u opsegu od 970 do 2000 μmola TE/1000 g ulja.Sadržaj hidrofilnih antioksidanasa u hladnoceđenim uljima je veoma nizak, sem za hladoceđenomaslinovo ulje. Pored lipofilnih antioksidanasa, pokazano je da metanolni ekstraktiobezmašćenih se, It is known that seeds of flax, sesame, and pumpkin are reach in essential fatty acids, andrepresent significant dietary source of vitamin E and polyphenols. In patients with chronic renalinsufficiency, on hemodialysis treatment, research topics about dietary supplementations withaims to reduce incidence of cardiovascular diseases, are actual. Therefore, the goal of thisdissertation was to investigate the effects of diet supplementation with the mixture of flax,sesame, and pumpkin seeds in patients on hemodialysis.The total antioxidative activity in plasma is significantly determined by antioxidative roleof human serum albumin (HSA), and analysis of the HSA in patients on hemodialysis showedthat the content of the free Cys34 thiol group (from which the HSA antioxidative potentialoriginates) is reduced and on the other hand, degree of HSA carbonylation is increased. Thus, thesecond goal of this dissertation was in vitro investigation of the binding of flax, sesame, andpumpkins seeds extracts components, ferulic acid (FE) and lignan metabolites (enterolactone(EL), enterodiol (ED), which are increased in circulation after consumption of food reach withplants lignan), as well as fatty acids, on the conformational change of the HSA and on thereactivity (antioxidative potential) of Cys34 thiol group, with the final goal to perceive thepossible mechanisms with which the flax, sesame, and pumpkin seeds explore in vivoantioxidative effects.Analysis of fatty acids profiles of total lipid extracts of seeds mixture using gas-liquidchromatography, confirmed that flaxseeds are the most abundant with α-linolenic acid (ALA),and sesame and pumpkin with linoleic acid (LA). Cold pressed oils showed differentantioxidative activity (refined sunflower oil>olive oil>flaxseed oil>pumpkin oil>sesame oil) inthe DPPH test (ranged from 970 to 2000 μmol TE/1000g oil). Content of hydrophilicantioxidants in cold pressed oils was extremely low, except for cold pressed olive oil. Besideslipophilic

Funkcije membranskih proteina su brojne: međućelijska komunikacija, adhezija, signalna transdukcija. Većina membranskih proteina je glikozilovana i N-glikani imaju važnu ulogu u formiranju trodimenzionalne strukture membranskih proteina, kao i u ispoljavanju njihove funkcije. Receptori za insulin (IR) i faktor rasta sličan insulinu tip 1 (IGF1R) su transmembranske tirozin-kinaze sa visokim stepenom homologije (u nekim domenima čak i 80%). Familija IGF receptora, pored IR i IGF1R, uključuje i receptor za faktore rasta slične insulinu tip 2 (IGF2R). Sva tri receptora (IR, IGF1R i IGF2R) su glikozilovani i obilno prisutni u placenti, gde imaju važne uloge u njenom razvoju i funkcionisanju.Placenta raste i razvija se da bi ispunila različite potrebe fetusa, pa se struktura i funkcija placente menjaju tokom gestacije. Znajući da su proteini odgovorni za biološke funkcije placente pretpostavljeno je da tokom gestacije može doći i do promene u sadržaju različitih tipova N-glikana prisutnih na membranskim proteinima. U ovoj tezi analizirani su tipovi N-glikana koji se mogu naći u sastavu membranskih glikoproteina, odnosno membranski N-glikom proteina humane placente. Ispitana je podložnost membranskog N-glikoma individualnim varijacijama i uticaju starosti majke/trudnice, kao i uticaj gestacije na membranski N-glikom. Znajući da je izmenjena glikozilacija često povezana sa izmenjenom funkcijom, kao i da izmenjena funkcija jednog ili više proteina može biti uzrok bolesti, ispitane su potencijalne promene membranskog N-glikoma u patološkim trudnoćama (kod preeklampsije) i trudnoćama komplikovanim patologijom majki (dijabetes). Uporedo je ispitan i uticaj starosti, gestacije i patologije na tip i zastupljenost različitih N-glikana koji ulaze u sastav receptora IGF sistema. Cilj je bio da se ispita da li promene na ukupnim membranskim proteinima (na N-glikomu) prate promene N-glikana prisutnih na pojedinačnim membranskim glikoproteinima važnim za rast i funkcionisanje placente.

The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 thiol group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1 : 1 and 4 : 1, were investigated by the determination of the pseudo first order rate constants (k') for the thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 x 10(4) M-1 and ED: 5.59 x 10(4) M-1 at 37 degrees C) and the lowest for S/HSA 4 : 1-enterolignan complexes (EL: 2.43 x 10(4) M-1 and ED: 1.92 x 10(4) M-1). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.

The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 thiol group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1 : 1 and 4 : 1, were investigated by the determination of the pseudo first order rate constants (k') for the thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 x 10(4) M-1 and ED: 5.59 x 10(4) M-1 at 37 degrees C) and the lowest for S/HSA 4 : 1-enterolignan complexes (EL: 2.43 x 10(4) M-1 and ED: 1.92 x 10(4) M-1). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.

Biljke izabrane kao eksperimentalni modeli za ovu disertaciju bile su panaširanemuškatle (Pelargonium zonale) i tamjanike (Plectranthus coleoides). Zeleno-belopanaširano lišće predstavljalo je odličan sistem za proučavanje uticaja fotosinteze naantioksidativni i fenolni metabolizam, kao i za istraživanje metaboličkih interakcijaizmeđu autotrofnog, „izvor“, tkiva, i heterotrofnog, „uvir“, tkiva unutar istog lista.U svom prirodnom staništu biljke su izložene visokom intenzitetu vidljive svetlostii UV-B zračenju (280-315 nm). Visok intenzitet fotosintetski aktivnog zračenja (PAR,400-700 nm) može da nadjača kapacitet fotosintetske potrošnje i procese energetskograsipanja i da prouzrokuje inhibiciju fotosinteze, CO2 asimilacije i pojačanu akumulacijureaktivnih kiseoničnih vrsta (eng. reactive oxygen species, ROS). Stoga su biljke razvilebrojne mehanizme zaštite, koji uključuju i postojanje specijalizovanih enzimskih ineenzimskih antioksidanata.Cilj istraživanja ove teze bio je određivanje konstitutivnih komponenataantioksidativnog sistema i njihove distribucije u fotosintetski aktivnom i fotosintetskineaktivnom tkivu listova panaširanih vrsta. Pri optimalnim svetlosnim uslovima za rast,aktivnosti enzima askorbat-glutationskog (Asc-GSH) ciklusa, kao i Cu/Zn i Mnsuperoksid-dismutaze su bile veće u belom tkivu, dok su aktivnosti katalaze (CAT) itilakoidne askorbat-peroksidaze (APX) bile veće u zelenom tkivu listova biljaka P. zonale.Biohemijskim i imunocitohemijskim analizama, pomoću transmisione elektronskemikroskopije, pokazano je da je Asc dvostruko više zastupljen u mezofilnim ćelijamazelenog tkiva (u peroksizomima, mitohondrijama i jedru), a GSH u mezofilnim ćelijamabelog tkiva (u mitohondrijama, jedru i citosolu). S obzirom na doprinos fotosinteze ustvaranju reaktivnih kiseoničnih vrsta (eng. reactive oxygen species, ROS), praćen jeodgovor enzimskih komponenata Asc-GSH ciklusa, CAT i peroksidaza III klase u belom izelenom tkivu listova u uslovima stimulisane Melerove, In this thesis, variegated geranium (Pelargonium zonale) and Swedish ivy(Plectranthus coleoides) were used as model plants. Green-white variegated leaves are anexcellent model system for investigating the effects of photosynthesis on antioxidative andphenolic metabolism, and “source”-“sink” interactions within the same leaf.In their natural habitat, plants are exposed to high light intensity and to UV-Bradiation (280-315 nm). High levels of photosynthetically active radiation (PAR) however,might exceed the the photosynthetic assimilation capacity and energy dissipation processesand provoke inhibition of photosynthesis and enhanced accumulation of reactive oxygenspecies (ROS). Therefore, plants have developed numerous protection mechanisms,including specialized enzymatic and non-enzymatic antioxidants.The aim of this study was to determine constitutive components of antioxidativesystem and their distribution in photosynthetically active and non-active tissues ofvariegated plants. Under optimal light conditions, activities of ascorbate-glutathione (AscGSH)cycle enzymes, Cu/Zn and Mn superoxide dismutases were higher within white leaftissue of P. zonale, while thylakoid ascorbate peroxidase (APX) and catalase (CAT)activities were higher in green leaf tissue. Biochemical and immunocytochemical analysisusing transmission electron microscopy revealed two-fold higher Asc content in mesophyllcells of green leaf tissue (in peroxisomes, mitochondria, nucleus), while GSH was moreabundant in mesophyll cells of white leaf tissue (in mitochondria, nucleus, cytosol). Sincephotosynthesis is the main source of ROS in the leaves, the second aim in this thesis wasto analyze the response of Asc-GSH cycle components, CAT and class III peroxidases ingreen and white leaf tissues, under the conditions of stimulated Mehler reaction during theexposure of P. zonale to high PAR and paraquat. These treatments induced greaterresponse of Asc-GSH cycle enzymes in white leaf tissue, compared with g

U okviru ove doktorske disertacije uraĎena je hemijska derivatizacija polisaharida biljnog i mikrobnog porekla u cilju dobijanja novih funkcionalizovanih polimera. Polisaharidi gumiarabika, pululan i levan korišćeni su za sintezu dve grupe konjugata. U prvom delu ovog rada raĎene su oksidativne transformacije polisaharida sa ciljem povećanja njihove reaktivnosti. Tretiranjem polisaharida natrijum-perjodatnim reagensom u vodenom rastvoru uvedene su reaktivne aldehidne grupe u polimerni niz glikana. Perjodatnom oksidacijom gumiarabike i pululana u kontrolisanim uslovima dobijeni su polialdehidni derivati, a variranjem koncentracija polisaharida i oksidacionog sredstva, kao i reakcionog vremena, dobijeni su uzorci različitog stepena oksidacije. Kuplovanjem ovako dobijenih polialdehida gumiarabike i pululana sa polienskim antibioticima u adekvatnim reakcionim uslovima sintetisane su hemijske formulacije nistatina i amfotericina B, sa ciljem povećanja rastvorljivosti ovih antibiotika u vodi. Ispitivanjem antifungalne aktivnosti sintetisanih konjugata dokazano je zadržavanje antimikrobne aktivnosti kuplovanih antibiotika. U drugom delu ovog rada raĎena je hemijska derivatizacija polisaharida pululana i levana kuplovanjem molekula reaktivne antrahinonske boje Remazol Brilliant Blue R za polisaharidne nizove, u baznim uslovima. Sintetisani proizvodi su upotrebljeni kao hromogeni supstrati za odreĎivanje aktivnosti enzima koji selektivno hidrolizuju glikozidne veze karakteristične za ciljne polisaharide, kao i za „screening” mikroorganizama na odreĎenu enzimsku aktivnost direktnom selekcijom na agarnim podlogama..., Within the scope of this Doctoral Dissertation the chemical derivatization of polysaccharides of plant and microbial origin was done for the purpose of getting new functionalized polymers. Polysaccharides gum Arabic, pullulan and levan were used for the synthesis of two groups of conjugates. The first section of this paper deals with the oxidative transformation of the polysaccharides aiming to increase their reactivity. By treating the polysaccharides with sodium periodate reagent in aqueous solution the reactive aldehyde groups are introduced into the polymer chain of glycan. Periodate oxidation of gum Arabic and pullulan under the controlled conditions resulted in formation of polyaldehide derivatives, and with the varying concentrations of polysaccharides and oxidizing agent as well as the reaction rate, samples with various levels of oxidation were obtained. By coupling gum Arabic and pullulan polyaldehide, formed in such way, with polyene antibiotics under the adequate reaction conditions, chemical formulations of nystatin and amphotericin B have been synthesized, with the purpose of increasing solubility of these antibiotics in water. The investigation of antifungal activites of the synthesized conjugate has proved that the antimicrobial activity of the coupled antibiotics has been preserved.The second section of this paper describes the chemical derivatization of pullulan and levan polysaccharides by coupling molecules of the reactive Remazol Brilliant Blue R antraquinone dye to polysaccharide chains, under base conditions. Synthesized products were used as chromogenic substrates for the determination of the enzyme activity that selectively hydrolize glycosidic linkages which are typical for the target polysaccharides, as well as for the screening of microorganisms for the specific enzyme activity through direct selection in agar substrates...

U okviru ove doktorske disertacije uraĎena je hemijska derivatizacija polisaharida biljnog i mikrobnog porekla u cilju dobijanja novih funkcionalizovanih polimera. Polisaharidi gumiarabika, pululan i levan korišćeni su za sintezu dve grupe konjugata. U prvom delu ovog rada raĎene su oksidativne transformacije polisaharida sa ciljem povećanja njihove reaktivnosti. Tretiranjem polisaharida natrijum-perjodatnim reagensom u vodenom rastvoru uvedene su reaktivne aldehidne grupe u polimerni niz glikana. Perjodatnom oksidacijom gumiarabike i pululana u kontrolisanim uslovima dobijeni su polialdehidni derivati, a variranjem koncentracija polisaharida i oksidacionog sredstva, kao i reakcionog vremena, dobijeni su uzorci različitog stepena oksidacije. Kuplovanjem ovako dobijenih polialdehida gumiarabike i pululana sa polienskim antibioticima u adekvatnim reakcionim uslovima sintetisane su hemijske formulacije nistatina i amfotericina B, sa ciljem povećanja rastvorljivosti ovih antibiotika u vodi. Ispitivanjem antifungalne aktivnosti sintetisanih konjugata dokazano je zadržavanje antimikrobne aktivnosti kuplovanih antibiotika. U drugom delu ovog rada raĎena je hemijska derivatizacija polisaharida pululana i levana kuplovanjem molekula reaktivne antrahinonske boje Remazol Brilliant Blue R za polisaharidne nizove, u baznim uslovima. Sintetisani proizvodi su upotrebljeni kao hromogeni supstrati za odreĎivanje aktivnosti enzima koji selektivno hidrolizuju glikozidne veze karakteristične za ciljne polisaharide, kao i za „screening” mikroorganizama na odreĎenu enzimsku aktivnost direktnom selekcijom na agarnim podlogama..., Within the scope of this Doctoral Dissertation the chemical derivatization of polysaccharides of plant and microbial origin was done for the purpose of getting new functionalized polymers. Polysaccharides gum Arabic, pullulan and levan were used for the synthesis of two groups of conjugates. The first section of this paper deals with the oxidative transformation of the polysaccharides aiming to increase their reactivity. By treating the polysaccharides with sodium periodate reagent in aqueous solution the reactive aldehyde groups are introduced into the polymer chain of glycan. Periodate oxidation of gum Arabic and pullulan under the controlled conditions resulted in formation of polyaldehide derivatives, and with the varying concentrations of polysaccharides and oxidizing agent as well as the reaction rate, samples with various levels of oxidation were obtained. By coupling gum Arabic and pullulan polyaldehide, formed in such way, with polyene antibiotics under the adequate reaction conditions, chemical formulations of nystatin and amphotericin B have been synthesized, with the purpose of increasing solubility of these antibiotics in water. The investigation of antifungal activites of the synthesized conjugate has proved that the antimicrobial activity of the coupled antibiotics has been preserved. The second section of this paper describes the chemical derivatization of pullulan and levan polysaccharides by coupling molecules of the reactive Remazol Brilliant Blue R antraquinone dye to polysaccharide chains, under base conditions. Synthesized products were used as chromogenic substrates for the determination of the enzyme activity that selectively hydrolize glycosidic linkages which are typical for the target polysaccharides, as well as for the screening of microorganisms for the specific enzyme activity through direct selection in agar substrates...

In order to improve the ability to apply knowledge of chemistry (acquired in the existing educational system) in real life, the model for consideration of ecological issues was developed and applied in high school. The model consists of a continuous text "It Happened, What's the Problem?" and a test with non-continuous text "A Guide Through the Problem", which were prepared for consideration of the problem of eutrophication. All results obtained (average achievement of 70.9 +/- 14.3 %) showed that the application of the model enabled: understanding of an ecological problem based on scientific representations of the term eutrophication given in the continuous text, realization that pollution of the environment may be directly related to modern life, application of acquired knowledge of chemistry to observe and understand the cause and effect of eutrophication in the environment, to draw a scientific conclusion, and understanding the importance of science and technology discoveries for solving ecological problems. In addition, the model contributed to the development of student's environmental literacy (ecological knowledge and cognitive skills), ability to think critically, and provided possibilities for classroom knowledge to become applicable in real life.

The potential of carbonylation with methylglyoxal to alter HSA's binding affinity for copper(II) ions and its influence on the release of copper(II) ions from copper-HSA complexes were studied. The affinity of HSA to coordinate copper(II) decreased upon carbonylation of the Cys34-SH group. Carbonylation of copper-HSA complexes caused a decrease in Cys34-SH content, conformational changes and the release of copper(II) ions. The ratio between the percentage of reduction in the Cys34-SH group content and the percentage of release of copper(II) from complexes is 2.12 +/- 0.28. Because the same ratio (1.96 +/- 0.36) was obtained upon oxidation of the Cys34-SH group (with no changes in HSA conformation), the binding/release of copper (II) by HSA depended mainly on the redox state of the Cys34-SH group. The contents of Cys34-SH and HSA-bound copper(II) ions in the diabetic group (0.457 +/- 0.081 mol SH per mol HSA, 10.7 +/- 0.01 mmol per mol HSA, resp.) were significantly lower (p lt 0.01) compared to the control group (0.609 +/- 0.027 mol SH per mol HSA; 13.4 +/- 0.01 mmol per mol HSA, resp.). Very strong correlations between the values for HSA-SH and glycated haemoglobin, HbA1c, (R = -0.803, p lt 0.01), and between the values for the HSA-bound copper(II) content and HSA-SH content (R = 0.841, p lt 0.002) were found in the diabetic group. Thus, HSA carbonylation leads to decrease in HSA-SH content and to the impairment of its copper(II) binding capacity that could contribute to further enhancement of oxidative and carbonyl stress in diabetes (as well as in other diseases with carbonyl stress).

Kontrakcije uterusa su važne za regulaciju brojnih reproduktivnihfunkcija. Neregularna i neadekvatna aktivnost uterusa povezana je sa brojnimpatološkim stanjima: neplodnost, neadekavtna implantacija, dismenoreja, kasnetrudnoće i prevremeni porođaji. Kontraktilnost uterusa regulisana jekompleksnom elektrofiziološkom mrežom koja može biti modulirana različitimfarmakološkim i signalnim molekulima.Uterus se karakteriše izuzetnom osobinom, brzom adaptacijom iz stanjamirovanja u oscilatorne cikluse jakih kontrakcija. Ovo je omogućeno zahvaljujućiaktivnosti ćelija koje su sposobne da remodeliraju svoje signalne puteve.Endogeno-sintetisani vodonik-sulfid (H2S), koji je zajedno sa azotmonoksidomi ugljen-monoksidom prepoznat kao gasoviti transmiter, imasignalnu ulogu i u uterusu. Vodonik-sulfid redukuje kontrakcije uterusa iprepoznat je kao agens velikog potencijala u tretmanu oboljenja uterusa. Do sadanisu urađene detaljnije studije, koje su dale uvid u mehanizme njegovogrelaksantnog dejstva na uterusu, te je to od značajnog interesa. Nije poznatoda li subiološki efekti sulfida posredovani njegovim molekulskim oblikom (H2S) ilitiolatnim anjonom (HS−).Rezultati ove disertacije implicirali su da molekulska i anjonska forma nemogu imati identične farmakološke efekte, uključujući i regulaciju konraktilnosti.Efekti H2S/HS− na relaksaciju uterusa upoređeni su sa efektom metantiol (CH3SH,koji „imitira“ H2S). Uočene razlike mogu biti posljedica specifične aktivnosti HS−jona. Ujedno, ovo upoređivanje može biti jedan od načina za razlikovanje efekataH2S i HS−. Metabolizam CH3SH je povezan sa sulfidom, budući da se H2S metiluje doCH3SH i ova metilacija može biti novi način modulacije H2S efekata in vivo.Ispitivano je učešće jonskih kanala (K+, Ca2+, Cl−), kao posrednika u relaksacijiuterusa indukovanoj sa H2S/HS−. Nađeno je da sulfid izaziva reverzibilnukoncentracijski-zavisnu relaksaciju koja je posredovana DIDS-osjetljivim Cl−kanalom. Dodatno, pokazano je da je DIDS-osjetljivi CaCC k, Uterine contractions are important in many reproductive functions.Improper or irregular uterine activity may underlie the common pathologicaldisorders such as infertility, improper implantation, dysmenorrhea, weak uterinecontraction during labor and preterm labor. The contractile activity of the uterus isregulated by the complex electrophysiologic network which is highly sensitive tovarious pharmacological and signaling molecules. Remarkably, uterus is able toswitch from quiescence to oscillatory cycles of strong contractions. This isdependent on the activity of the cells being able to remodel their signallingsystems.Endogenously produced hydrogen sulphide (H2S), which, together withNO and CO, is a recognized gasotransmitter, also appears to be a signallingmolecule in rat uterus. Hydrogen sulphide reduces uterine contractility, and it isrecognized as a promising treatment for uterine disorders. There were no studiesabout mechanistic insights of its relaxatory effect, done so far, and thus is ofpotential interest. It is not currently known whether the biological effects of H2Sare mediated directly by H2S or by its derivatives, the most important being thethiolate anion HS−. The results presented in this thesis imply that it appearsunlikely that molecular and anionic forms have pharmacologically identicalactions, including the regulation of contractility. Pharmacological effects ofH2S/HS− were compared to effects of methanethiol (CH3SH, mimicks H2S), andobserved differences might be a consequence of the specific actions of HS−. Thiscomparison may represent a useful tool for discrimination between differentactions of H2S and HS−. The metabolism of CH3SH is intertwined with hydrogensulfide (H2S), since the later is methylated to CH3SH and this methylation may be anew way of modulation of H2S effects in vivo.We investigated the involvement of ion channels (K+, Ca2+, Cl−) in therelaxation of rat uteri induced by H2S/HS−. We found that sulphide induces...