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The European corn borer (Ostrinia nubilalis, Hbn.), enters diapause, a strategy characterized by arrest of development and reproduction, reduction of metabolic rate and the emergence of increased resistance to challenging seasonal conditions as low sub-zero winter temperatures. The aim of this study was to investigate the potential role of inorganic elements in the ecophysiology of O. nubilalis, analysing their content in the whole body, hemolymph and fat body, both metabolically active, non-diapausing and overwintering diapausing larvae by ICP-OES spectrometer following the US EPA method 200.7:2001. O nubilalis as many phytophagous lepidopteran species maintain a very low extracellular sodium concentration and has potassium as dominant cation in hemolymph of their larvae. Changes in hemolymph and the whole body sodium content occur already at the onset of diapause (when the mean environmental temperatures are still high above 0 ºC) and remain stable during the time course of diapause when larvae of this species cope with sub-zero temperatures, it seems that sodium content regulation is rather a part of diapausing program than the direct effect of exposure to low temperatures. Compared to non-diapausing O. nubilalis larvae, potassium levels are much higher in the whole body and fat body of diapausing larvae and substantially increase approaching the end of diapause. The concentration of Ca, Mg, P and S differed in the whole body, hemolymph and fat body between non-diapausing and diapausing larvae without a unique trend during diapause, except an increase in their contents at the end of diapause.

All processes involved in metal homeostasis must be coordinated to provide sufficient, but not toxic, concentrations of important bioelements, and to minimize detrimental effects of toxic metals. Our previous studies dealing with the exposure of O. nubilalis non-diapausing larvae to dietary Cd demonstrated that exposure to higher concentrations of Cd caused delay in the development of larvae, induced oxidative stress and also induced defense mechanisms against the toxic effects of Cd. The aim of the present study was to evaluate how acute and chronic exposure of O. nubilalis larvae to increased concentrations of dietary Cd affected the balance of important bioelements. The concentration of bioelements was analyzed in larvae (after short-term exposure) and pupae (after long-term exposure). The short-term exposure of final instar larvae (L5) to Cd did not affect significantly the concentration of any of the analyzed bioelements, while the long-term exposure of developing larvae to higher concentrations of Cd caused increase in the concentrations of Ca, Mg and Na in pupae. The bioaccumulation factor, calculated for bioelements after long-term exposure to Cd, was higher for the most bioelements in groups fed with diet containing higher concentrations of Cd, except K which displayed the opposite trend. Pearson correlation coefficient showed positive correlations between Cd and Ca, Mg, Na, Fe, Cu and Zn, while negative correlation was observed between Cd and K. The results indicate that impact on the balance of important bioelements might be one of the mechanisms of cadmium toxicity and certainly raise numerous questions for future research.

Evidentne klimatske promene dovode do izmena ne samo u smislu zapremine već i raspodele padavina, pa su posledično sve češće smene perioda ekstremnih vodnih deficita i suficita. Vodni deficit i suficit utiču na rast i razvoj biljaka tako što dovode do inhibicije fotosinteze, oksidativnih oštećenja ćelijskih komponenti te promena u respiraciji. Cilj ove disertacije je bio da se utvrde efekti vodnog suficita i deficita na fiziološke, oksidativne i antioksidativne parametre kod dva hibrida kukuruza (ZP 555 i ZP 606). Vodnom suficitu su podvrgnute biljke u trajanju od 6, 24, 72 i 144 sati, a vodnom deficitu u trajanju od 8 i 15 dana, sa odgovarajućim kontrolnim biljkama. Listovi kontrolnih i tretiranih biljaka su korišteni za određivanje sledećih parametara: fizioloških (relativnog sadržaja vode (RWC), koncentracije fotosintetičkih pigmenata, solubilnih proteina, slobodnog prolina), oksidativnih (koncentracije malondialdehida – MDA i H2O2) i antiokdiativnih (neenzimskih - koncentracija polifenola, i enzimskih komponenti - aktivnosti antioksidativnih enzima: superoksid dismutase (SOD, EC 1.15.1.1), katalaze (CAT, EC 1.11.1.6), askorbat peroksidaze (APX, EC 1.11.1.11) i peroksidaza Klase III (POX, EC 1.11.1.7)). Rezultati dobijeni izlaganjem biljaka vodnom suficitu i deficitu su pokazali različit odgovor hibrida na poplavu i sušu i značajne interhibridne razlike kada je u odgovor na poplavu ili sušu. Promene u procesu fotosinteze su detektovane kroz promene koncentracije fotosintetičkih pigmenata. Pri uslovima vodnog suficita izmeren je porast koncentracije hlorofila a i b, te karotenoida kod hibrida ZP 555. Sa druge strane, smanjena koncentracija hlorofila b i karotenoida izmerena je kod hibrida ZP 606 nakon 24 sata vodnog suficita. Hibrid su imali istovetan odgovor na vodni deficit, gde je detektovano smanjenje koncentracije hlorofila a i karotenoida te povećanje koncentracije hlorofila b. Kao posledica vodnog suficita detektovana je i povećana koncentracij, Plants are continuously challenged by the rising incidence of drought and flooding as a consequence of global warming. These stresses may cause severe depression in the plant’s growth by inhibiting photosynthesis, inducing oxidative cell damage, and cell respiration changes. The present work aimed to study the effect of flooding and drought stress on physiological, oxidative, and antioxidative parameters in leaves of two maize hybrids (ZP 555 and ZP 606). Maize plants`leaves at the stage of three fully developed leaves were harvested after 6, 24, 72, and 144 hours of applied flooding stress and after 8 and 15 days after applied drought stress. Leaves from experimental plants and control plants were used for the determination of physiological (relative water content (RWC), the content of photosynthetic pigments and soluble proteins, proline), oxidative stress parameters (the content of malondialdehyde (MDA) and H2O2), as well as antioxidants (the total polyphenols content (Phe), and activity of antioxidative enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidases (APX, EC 1.11.1.11), and Class III peroxidases (POX, EC 1.11.1.7)]). Results indicated that drought and flooding stress-induced time-dependent changes of measured parameters and those hybrids differ in stress response. Changes in photosynthesis can be seen thought modifications in photosynthetic pigments concentration. An increase in the concentration of chlorophylls and carotenoids have been noted during flooding in ZP 555 hybrids. On the other hand, flooding stress decreased the chlorophyll b and carotenoid concentrations in ZP 606 hybrids. A decrease in the concentration of chlorophyll a and carotenoids and an increase in chlorophyll b concentration were a response of both examined hybrids on drought stress. Also, intensive protein synthesis was noted in both hybrids during flooding. More intensive oxidative stress levels were correlated with an i

Uzimajući u obzir klimatske promjene koje podrazumijevaju smanjenje količine padavina i porast temperatura, suša je postala globalni problem za koji se smatra da će biti aktuelan još dugo vremena u budućnosti. Osim problema koje stvara u brojnim životnim sferama, suša je jedan od najozbiljnijih faktora koji negativno utiče na poljoprivredu, s obzirom da nedovoljno vodosnabdijevanje negativno utiče na rast i razvoj biljaka. U uslovima suše povećana produkcija reaktivnih vrsta kiseonika može da izazove pomjeranje ravnoteže u ćelijskim oksidoredukcionim reakcijama u smjeru oksidacije te da dovede do nastanka oksidativnog stresa. Posljedice oksidativnog stresa su strukturna i funkcionalna oštećenja ćelijskih komponenti koja dovode do poremećaja u metabolizmu i funkcionisanju ćelije.Veoma važnu ulogu u toleranciji biljaka na stres izazvan sušom ima antioksidativni sistem zaštite koji neizbježno uključuje enzime superoksid dismutazu, katalazu, peroksidaze Klase III i askorbat peroksidazu, ali takođe i niskomolekularne antioksidante poput askorbata, glutationa, fenola i karotenoida. Posljednjih godina se naglo povećao interes za korištenje ekološki opravdanih i neinvazivnih sredstava kojima se unaprijeđuje problem vodosnabdijevanja biljaka, a velika pažnja je posvećena primjeni prirodnih aluminosilikata, od kojih je najpoznatiji zeolit. Iako nisu mnogobrojni, postoje radovi koji upućuju da ovaj prirodni mineral može značajno da doprinese poboljšanju vodnog režima biljaka zbog osobine vezivanja te uravnoteženog otpuštanja vode. U ovoj doktorskoj disertaciji predmet istraživanja je uključio dvije biljne vrste različite po tipu fotosintetičkog metabolizma i adaptivnim strategijama na prilagođavanje na sušu, a to su paradajz (Solanum lycopersicum L.) i kukuruz (Zea mays L.). Fokus istraživanja bio je ispitivanje mogućnosti primjene zeolita u svrhu sprječavanja ili ublažavanja posljedica stresa izazvanog sušom kod biljaka paradajza i kukuruza, ali i ispitivanje mogućeg različit, Taking into account climate change, which implies a precipitation decreasing and temperatures rising, drought has become a global problem that is considered to be relevant for a long time to come. In addition to the problems it creates in many spheres of life, drought is one of the most serious factors that negatively affects agriculture, given that insufficient water supply disrupts the growth and development of plants. The production of reactive oxygen species in drought conditions can cause an inbalance of cellular redox homeostasis in the direction of oxidation and lead to the formation of oxidative stress. The consequences of oxidative stress are structural and functional damages of cellular compartments which leads to disturbance of metabolism and cell function. A very important role in plant tolerance to drought stress has the antioxidant protection systems that inevitably include theenzymes superoxide dismutase, catalase, Class III peroxidases and ascorbate peroxidase, but also low molecular weight antioxidants such as ascorbate, glutathione phenol and carotenoids. In recent years, there has been a sharp increase in interest in the use of environmentally sound and non- invasive means to improve the water supply of plants, and much attention has been paid to the use of natural aluminosilicates, the most famous of which is zeolite. Although not numerous, there are studies that suggest that this natural mineral can significantly contribute to the improvement of the water regime of plants due to the binding properties and balanced release of water. In this PhD thesis, the subject of research included two plant species different in type of photosynthetic metabolism and adaptive drought adaptation strategies, namely tomatoes (Solanum lycopersicum L.) and maize (Zea mays L.). The focus of research was to examine the possibility of using zeolite to prevent or mitigate the effects of drought stress in tomato and maize plants, but also to examine the possible diff

Tumori predstavljaju drugi vodeći uzrok smrti u svetu (posle kardiovaskularnih bolesti) i procena je da će učestalost pojave tumora u budućnosti porasti. Hemioterapija je jedna od najčešće korišćenih metoda lečenja tumora koju često prate sporedni sistemski štetni efekti. S obzirom da lečenje tumora jednim terapeutskim agensom često rezultuje ograničenim kliničkim ishodom, kombinovane terapije mogu sinergistički podići nivo antitumorske aktivnosti, dok sniženjem doze svakog pojedinačnog agensa se mogu umanjiti štetni efekti. Istovremena aplikacija različitih terapeutskih agenasa zahteva dostavnu platformu kako bi se omogućila normalizacija farmakokinetike i farmakodinamike agenasa. Fulerenol C60(OH)24 predstavljaju pogodnu platformu za dostavu lekova zbog svojih fizičko-hemijskih i bioloških karakteristika. U vodenom rastvoru fulerenol se nalazi u obliku negativno naelektrisanih polianjonskih nanočestica koje poseduju veliku površinu i imaju mogućnost vezivanja pozitivno naelektrisanih hemioterapeutika kao što je doksorubicin (DOX), kao i pozitivnih jona metala, u ovom slučaju Fe2+. Osnovna biološka karakteristika fulerenola je da ima ulogu antioksidativnog agensa. U ovom radu iskorišćene su fizičkohemijske osobine fulerenolskih nanočestica (FNP), DOX-a i Fe2+ u cilju formiranja stabilnih nanokompozita FNP/DOX i FNP/Fe2+. Nanokompoziti su okarakterisani različitim fizičkohemijskim metodama, pri čemu je ustanovljeno da su čestice nanokompozita FNP/DOX veličine između 20 i 58 nm, naelektrisanja od -6 mV, čestice nanokompozita FNP/Fe2+ veličine između 11 i 60 nm, naelektrisanja od -30.8 mV, i diskoidalne morfologije. Oba nanokompozita imaju dobru stabilnost u vodenom rastvoru, što ih čini biokompatibilnim i pogodnim za primenu u biomedicini. FNP dekorisan DOX-om, u obliku nanokompozita FNP/DOX, omogućava značajno obimniji unos DOXa u ćelije i smanjuje procenat ćelija u G2M fazi ćelijskog ciklusa, u odnosu na samostalnu primenu DOX-a, kod malignih ćelijskih linija dojk, Tumors are the second leading cause of death in the world (following cardiovascular diseases) and it is estimated that the incidence of tumors will increase in the future. Chemotherapy is one of the most common methods of tumor treatment which is often accompanied by systemic side effects. Treatment of tumors with one therapeutic agent often results in limited clinical outcome. Combination therapy, using a variety of agents and modalities of action, can synergistically increase thelevel of antitumor activity, lowering the dose of each single agent and thus reduce adverse side effects. The simultaneous application of various therapeutic agents requires a delivery platform to enable normalization of agents’ pharmacokinetics and pharmacodynamics. Because of its physico-chemical and biological activities,fullerenol C60(OH)24 represents a suitable platform for drug delivery. In the aqueous solution fullerenol is in the form of negatively charged polianionic nanoparticles (FNP) that possess a large surface area and have the ability of binding positively charged chemotherapeutic drug, such as doxorubicin (DOX), as well as positively charged metal ions, particularly Fe 2+ . The main biological activity of fullerenol is manifested through its antioxidant activity. In this study we used physico-chemical properties of fullerenol, doxorubicin and iron (Fe2+ ) in order to form stable fullerenol/doxorubicin (FNP/DOX) nanocomposite and fulerenol/iron (FNP/Fe 2+) nanocomposite. The nanocomposites were characterized by different physical- chemical methods which showed that FNP/DOX has particle size between 20 nm and 58 nm and charge of -6 mV, while FNP/Fe 2+ has particle size between 11 nm and 60 nm and charge of -30.8 mV. Both nanocomposites revealed discoidal morphology. The nanocomposites have also displeyed good stability in aqueous systems, which makethem biocompatible and suitable for use in biomedical applications. FNP decorated with DOX, in the form of FNP/DOX nanocomposite

Medonosna pčela (Apis mellifera Linnaeus, 1758) je široko rasprostranjena vrsta iz grupe socijalnih insekata, glavni oprašivač mnogih divljih biljaka i poljoprivrednih kultura. Pored njene primarne uloge u oprašivanju, koristi se i za dobijanje pčelinjih proizvoda, pre svega meda, voska i propolisa, čime se dodatno naglašava njen privredni značaj. Uzimajući u obzir značaj medonosne pčele, a sa druge strane aktuelan problem pada broja kolonija, postoji potreba za razumevanje uzroka i procenu rizika koji leže u osnovi ovog kompleksnog problema. Stoga, istraživanja u ovoj doktorskoj disertaciji obuhvataju praćenje biohemijskih i fizioloških parametara kod medonosne pčele kao pokazatelja stanja pčelinjih zajednica tokom letnjeg i zimskog perioda na staništima sa različitim antropogenim uticajem. U prvoj fazi istraživanja su izvedeni akutni laboratorijski testovi izlaganja medonosne pčele subletalnim dozama pesticida, tiakloprida i klotianidina, a dobijeni rezultati su ukazali na narušenu neurotransmisiju, na izmene u aktivnosti antioksidativnih enzima i redoks statusa u ćeliji, kao i na imunosupresiju. U drugoj fazi istraživanja su analizirane kolonije medonosne pčele sa različitih lokaliteta na području AP Vojvodine, sa primenom stacionarnog i migratornog tipa pčelarenja, tokom letnjeg i zimskog perioda, a dobijeni rezultati su ukazali na značajne razlike u biohemijskom odgovoru medonosne pčele u zavisnosti od starosti pčela, sezone godine i tipa pčelinjaka. Na osnovu rezultata prethodne dve faze istraživanja, izvršen je odabir uzoraka, kao i izbor parametara za treću fazu istraživanja, gde je praćeno stanje medonosne pčele iz stacionalnih pčelinjih zajednica, tokom letnjeg aktivnog perioda, kao i početkom i krajem zimskog perioda mirovanja pčela, sa tri lokaliteta u Republici Srbiji sa različitim stepenom urbanizacije i industrijalizacije: Beograd kao urbano, Susek kao ruralno i Zajača kao industrijsko područje. Dobijeni rezultati su ukaza, The honey bee (Apis mellifera Linnaeus, 1758) is a‚widespread social insect species and the main pollinator of various wild plants and agricultural crops.In addition to its primary role in pollination, the honey bee is also used for obtaining honeybee products, mostly honey, wax, and propolis, with a significant role in the economy. Number of honey bee colonies is declining, thus necessitating the assessment of underlying reasons and risks. Therefore, in this doctoral dissertation, biochemical and physiological parameters in the honey bee were monitored as indicators of the colonies’ condition during the summer and winter period in habitats with different anthropogenic impacts. In the first phase of the research, acute laboratory tests of honey bee exposure to sublethal doses of pesticides (thiacloprid and clothianidin) were performed and the obtained results indicated impaired neurotransmission, changes in the activity of antioxidant enzymes, and redox status in the cell, as well as immunosuppression. In the second phase of the research, honey bee colonies from different localities in the area of AP Vojvodina were analyzed, using the stationary and migratory types of beekeeping, during the summer and winter period, and the obtained results indicated significant differences in the biochemical response of the honey bee depending on the age of the bees, the season of the year, and the type of apiary. Based on the results of the previous two phases of the research, the selection of samples was performed, as well as the selection of parameters for analysis within the third phase of the research, where the condition of honey bees from stationary bee communities was monitored during the summer active period, as well as the beginning and end of winter dormancy of bees, from three localities in the Republic of Serbia with diffe

U organizmu se, pod fiziološkim uslovima, produkuju slobodni radikali. Iako se u organizmu nalaze u veoma niskoj koncentraciji, slobodni radikali mogu ispoljiti toksične efekte. Težeći da spare elektrone, u hemijskoj reakciji oksidacije, dolazi do brzog i nepredvidivog vezivanja za susedne molekule, proteine, lipide, ugljene hidrate i nukleinske kiseline od kojih su sačinjeni strukturni elementi ćelije, pokrećući unutrašnji put apoptoze. Antioksidansi su supstance koje sprečavaju ili značajno smanjuju oksidaciju biomolekula. Oksidativni stres je stanje koje nastaje kada produkcija slobodnih radikala premaši kapacitete antioksidativnih enzima da ih neutrališu. U antioksidativne enzime spadaju: superoksid dismutaza (SOD), katalaza (CAT), glutation peroksidaza (GPx), glutation reduktaza (GR) i glutation-S-transferaza (GST). Lipidna peroksidacija (LP) je proces oksidacije višestruko nezasićenih masnih kiselina od strane slobodnih radikala. Malondialdehid predstavlja biohemijski marker pomoću kog je moguće meriti stepen oksidativnog oštećenja ćelijskih membrana. Oksidativna modifikacija DNK dovodi do promene strukture DNK koje rezultuju genetskim oštećenjima. Najčešće korišćen marker oksidativnog stresa je urinarni 8-hidroksi-2-deoksiguanozin (8-OHdG). Oštećenja proteina, lipida, DNK čine važan osnov mnogih oboljenja kao što su ateroskleroza, neurodegenerativna oboljenja, dijabetes, gojaznost, proces starenja, retinopatija, hronične inflamatorne bolesti i karcinom. Polazeći od hipoteze da su ovi biomolekuli različiti u različitim stadijumima bolesti, oni bi mogli predstavljati prognostički marker proširenosti bolesti. Cilj istraživanja je bio da se ispita da li postoje razlike između kontrolne grupe (zdravih žena), pacijentkinja sa prekanceroznim lezijama na grliću materice (HSIL), pacijentkinja sa lokalno ograničenim (FIGO Ia-Ib) i pacijentkinja sa lokalno uznapredovalim karcinomom grlića materice (IIa-IV) u pokazateljima oštećenja DNK (određivanjem vrednosti 8-OHdG), p, Free radicals are produced in our body under physiological conditions. Although in very low concentrations, they can show some toxic effects. While trying to bind electrons, in the chemical reaction of oxidation, they rapidly and unpredictably bind to adjacent molecules- proteins, lipids, carbohydrates and nucleic acids from which the structural elements of the cell are made, triggering the internal pathway of apoptosis. Antioxidants are substances that prevent or significantly reduce the oxidation of biomolecules. Oxidative stress is a condition that occurs when the production of free radicals exceeds the capacity of antioxidant enzymes to neutralize them. The antioxidant enzymes include: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST). Lipid Peroxidation (LP) is the process of oxidation of polyunsaturated fatty acids by free radicals. Malondialdehyde is a biochemical marker by which it is possible to measure the degree of oxidative damage of cell membranes. The oxidative modification of DNA leads to a change in DNA structure that results in genetic damage. The most commonly used marker of oxidative stress is urinary 8-hydroxy-2-deoxiguanosine (8-OHdG). The damage to proteins, lipids and DNA is an important basis for many diseases such as atherosclerosis, neurodegenerative diseases, diabetes, obesity, aging, retinopathy, chronic inflammatory disease and cancer. Starting from the hypothesis that these biomolecules are different at different stages of the disease, they could represent a prognostic marker of the progression of the disease. The aim of the study was to examine whether there were differences between the control group (healthy women), the patients with precancerous lesions on the cervix (HSIL), the patients with early stage cervical cancer (FIGO Ia-Ib) and the patient with locally advanced cervical cancer (IIa - IV) in the indicators of DNA damage (determining the va

Истраживање има фокус на хуманим ензимима из суперфамилије алдо-кето редуктаза, које имају велики метаболички значај за хомеостатско функционисање организма. Неки од чланова подфамилије 1С алдо-кето редуктаза (AKR1C) имају улогу у развоју одређених патолошких стања, као што су леукемија, тумори простате, дојке и ендометријума, као и у смањивању ефекта хемотерапија. До сада није регистрован лек који директно утиче на протеине ове групе и самим тим је акценат на изналажењу специфичних лиганада (супстрата, инхибитора), који би могли да имају фармаколошку примену, али и на утврђивању везе између структуре и функције испитиваних лиганада према ензиму. Теза је имала фокус на протеину AKR1C3. У овој дисертацији је представљена оптимизација ензимског есеја и испитивање потенцијалних лиганада и њиховог ефекта на ензимску активност одређених хуманих изоформи протеина из подфамилије AKR1C. Тестирана су синтетисанa стероиднa jeдињења, комерцијална једињења и биљни екстракти. Стероидни лиганди (AKR-1, -2, -3, -7, -9, -19 и -22) који су показали добре инхибиторне карактеристике су детаљније описани одређеним добијеним кинетичким параметрима и затим су кокристализовани са протеином и кофакторм. Од 7 различитих комплекса протеина са најбољиминхибитором, за два комплекса су добијене дифракције са инхибитором и решене кристалне структуре са лигандом у везном месту и врло добром резолуцијом, AKR-7: 1.7 Å, AKR -19: 1.6 Å. Ови резултати представљају прве протеинске кристале чију су структуру решили истраживачи из Србије, а у научном смислу и одличну основу за даљи дизајн и тестирање једињења и кокристализације., Istraživanje ima fokus na humanim enzimima iz superfamilije aldo-keto reduktaza, koje imaju veliki metabolički značaj za homeostatsko funkcionisanje organizma. Neki od članova podfamilije 1S aldo-keto reduktaza (AKR1C) imaju ulogu u razvoju određenih patoloških stanja, kao što su leukemija, tumori prostate, dojke i endometrijuma, kao i u smanjivanju efekta hemoterapija. Do sada nije registrovan lek koji direktno utiče na proteine ove grupe i samim tim je akcenat na iznalaženju specifičnih liganada (supstrata, inhibitora), koji bi mogli da imaju farmakološku primenu, ali i na utvrđivanju veze između strukture i funkcije ispitivanih liganada prema enzimu. Teza je imala fokus na proteinu AKR1C3. U ovoj disertaciji je predstavljena optimizacija enzimskog eseja i ispitivanje potencijalnih liganada i njihovog efekta na enzimsku aktivnost određenih humanih izoformi proteina iz podfamilije AKR1C. Testirana su sintetisana steroidna jedinjenja, komercijalna jedinjenja i biljni ekstrakti. Steroidni ligandi (AKR-1, -2, -3, -7, -9, -19 i -22) koji su pokazali dobre inhibitorne karakteristike su detaljnije opisani određenim dobijenim kinetičkim parametrima i zatim su kokristalizovani sa proteinom i kofaktorm. Od 7 različitih kompleksa proteina sa najboljiminhibitorom, za dva kompleksa su dobijene difrakcije sa inhibitorom i rešene kristalne strukture sa ligandom u veznom mestu i vrlo dobrom rezolucijom, AKR-7: 1.7 Å, AKR -19: 1.6 Å. Ovi rezultati predstavljaju prve proteinske kristale čiju su strukturu rešili istraživači iz Srbije, a u naučnom smislu i odličnu osnovu za dalji dizajn i testiranje jedinjenja i kokristalizacije., This research focuses on human enzymes of the aldo-keto reductase superfamily, whose functions have a significant metabolic impact on organism homeostasis. Some members of the 1C aldo-keto reductase (AKR1C) subfamily play role in the development of specific pathological conditions, such as leukaemia, prostate cancer, breast cancer and endometrial cancer, as well as reducing the effectivness of chemotherapy. However, currently there are no approved and registered drugs that directly affect proteins from this subfamily. Therefore our main aim was to screen for specific ligands (substrates, inhibitors) with potential pharmacological applications, and to establish structure-activity relationships for these ligands and enzymes. This thesis mainly focuses on isoform AKR1C3. In this dissertation, optimization of an enzymatic assay and testing of potential ligands and their effects on the enzymatic activity of specific human isoforms of proteins from subfamily AKR1C are presented. Tested ligands include synthetic steroidal compounds, commercial compounds and plant extracts. Steroid compounds, AKR-1, -2, -3, -7, -9, -19 and -22, were found to be good inhibitors of AKR1C3, and further kinetic studies were conducted. Finally, cocrystalization of protein AKR1C3 with cofactor and these inhibitors was accomplished. From 7 different complexes of protein with inhibitors, two structures were solved to very high resolution, AKR-7: 1.7 Å, AKR -19: 1.6 Å. These results represent the first protein crystal structures solved by researchers from Serbia, and results provide an excellent basis for further design and testing of new inhibitors.

Over the past decades, the number of managed honey bee Apis mellifera L. (Hymenoptera: Apidae) colonies have been decreasing. The majority of losses occur during winter, suggesting that overwintering honey bees are more susceptible to adverse factors. We focused on the oxidative status of overwintering honey bees, particularly at the beginning (November) and end (March) of the wintering period. Colonies from three locations with different anthropogenic influences were selected: Belgrade, an urban zone, Zajača, an industrial zone, and Susek, a rural area. We measured levels of malondialdehyde (MDA), as a marker of lipid peroxidation, as well as the expression and activity of select antioxidative enzymes: Superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). Our results show that enzyme activity and gene expression of antioxidative enzymes are influenced by both sample location and the time of sampling. The majority of analyzed genes had significantly reduced expression, at the end of the overwintering period when higher activities of antioxidative enzymes were also recorded. Among the analyzed parameters, SOD activity and gene expression of microsomal GST isoforms were more affected by local environmental conditions, suggesting the complex role of these enzymes in antioxidative defense and detoxification. The higher MDA levels observed at the end of overwintering for all three locations likely reflects accumulated oxidative damage which could be associated with the aging process, brood rearing and/or the onset flying activity.

Over the past decades, the number of managed honey bee Apis mellifera L. (Hymenoptera: Apidae) colonies have been decreasing. The majority of losses occur during winter, suggesting that overwintering honey bees are more susceptible to adverse factors. We focused on the oxidative status of overwintering honey bees, particularly at the beginning (November) and end (March) of the wintering period. Colonies from three locations with different anthropogenic influences were selected: Belgrade, an urban zone, Zajača, an industrial zone, and Susek, a rural area. We measured levels of malondialdehyde (MDA), as a marker of lipid peroxidation, as well as the expression and activity of select antioxidative enzymes: Superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). Our results show that enzyme activity and gene expression of antioxidative enzymes are influenced by both sample location and the time of sampling. The majority of analyzed genes had significantly reduced expression, at the end of the overwintering period when higher activities of antioxidative enzymes were also recorded. Among the analyzed parameters, SOD activity and gene expression of microsomal GST isoforms were more affected by local environmental conditions, suggesting the complex role of these enzymes in antioxidative defense and detoxification. The higher MDA levels observed at the end of overwintering for all three locations likely reflects accumulated oxidative damage which could be associated with the aging process, brood rearing and/or the onset flying activity.

Ugljenične nanocevi (UNC) imaju sve veću primenu u elektronici, kompjuterskoj i optičkoj industriji, kao i u biomedicini. Dok proizvodnja jednoslojnih UNC nanocevi beleži sve veći rast poslednjih godina, rizik koji nosi izlaganje ovom nanomaterijalu ostaje nerazjašnjen. Oskudni i često kontradiktorni podaci o toksičnim efektima jednoslojnih UNC ukazuju na potrebu za daljim ispitivanjima. U našim istraživanjima ispitivane su promene u ćelijskom odgovoru kao i morfološke promene nakon delovanja jednoslojnih ugljeničnih nanocevi na ćelijskoj liniji humanih fetalnih fibroblasta pluća MRC-5 i ćelijskoj liniji humanog adenokarcinoma pluća A549. U ovoj studiji korišćene su jednoslojne ugljenične nanocevi koje su sadržale rezidualne nečistoće poput gvožđa. Citotoksičnost jednoslojnih UNC (engl. single-walled carbon nanotubes – SWCNT) je ispitivana kolorimetrijskim MTT testom. Tokom 24 i 48h niske koncentracije jednoslojnih ugljeničnih nanocevi (<250 μg/mL) pokazale su nisku toksičnost na proliferaciju i vijabilnost u obe ispitivane ćelijske linije. Ipak, pri visokim koncentracijama UNC (250-750 μg/mL) antiproliferativni efekat je bio blizu IC50 vrednostima. Na osnovu rezultata dobijenih MTT testom može se zaključiti da su maligne A549 ćelije osetljivije na delovanje jednoslojnih UNC u odnosu na normalne MRC-5 ćelije. Kombinacija ugljeničnih nanocevi sa prirodnim polifenolima (resveratrolom i proantocijanidolima) nije značajno uticala na citotoksičnost u MRC-5 ćelijama, za razliku od A549 ćelija gde je tretman kombinacijama umanjio toksičnost ugljeničnih nanocevi. Transmisionom elektronskom mikroskopijom ispitivan je efekat jednoslojnih ugljeničnih nanocevi na ćelijsku morfologiju i preživljavanje. Intracelularni agregati ugljeničnih nanocevi primećeni su u obe ćelijske linije, čime je potvrđeno da ugljenične nanocevi ulaze u ćelije. Imajući u vidu da nanomaterijali poput ugljeničnih nanocevi indukuju oksidativni stres i njime posredovanu apoptozu, na protočnom citometru je, Carbon nanotubes are being actively introduced in electronics, computer science, and optics as well as for various biomedical applications. While production of single-walled carbon nanotubes (SWCNT) has escalated in recent years, the knowledge on risk associated with exposure remains unclear. Contradictory data on the toxic effects of single-walled carbon nanotubes highlights the urgent need for further studies. In this study we investigated the alterations in cellular response along with morphological changes induced by single-walled carbon nanotubes in human lung fibroblast cell line MRC-5 and adenocarcinoma human alveolar basal epithelial cells A549. In this study we used SWCNT containing large amounts of residual metallic impurities such is iron, and the iron concentration increased in dose dependent manner in cells exposed to SWCNT. Cytotoxicity was evaluated by MTT assay and SWCNT showed little cytotoxic effect on the proliferation and viability of two cell lines tested at the concentrations used (<250 μg/mL) within 24 and 48h. However exposing both cell lines to high concentrations (250-750 μg/mL) resulted in near IC50 values. Based on MTT test SWCNT were more cytotoxic to A549 cell line. Cytotoxicity of SWCNT in combination with natural polyphenols (resveratrol and proanthocyanidins) did not noticeably affect the cytotoxicity of SWCNT to MRC-5 cells. However introduction of polyphenols did reduce the cytotoxicity of SWCNT to A549 cells. Transmission electron microscopy was used to complement cytotoxicity assays and to investigate the pathological effect of internalized SWCNT on cell morphology and survival. Intracellular bundles of CNTs, possibly aggregated/agglomerated were observed in both cell lines, confirming internalization after 24h exposure. Since nanoparticles like carbon nanotubes are toxic mainly because they cause oxidative stress, often associated with an increased apoptosis we checked for apoptotic and necrotic cells using flow cytometry. Incub

Larvae of the European corn borer (Ostrinia nubilalis Hubn.) were cold acclimated during different phases of diapause to determine if changes in the fatty acid composition lipids occur as part of a programmed diapause strategy, or as a response to low temperatures during winter. Cold acclimation of fifth instar larvae of O. nubilalis during diapause had modest effects further on the readjustments in fatty acid composition of triacylglycerols and phospholipids. Overall, FA unsaturation (UFAs/SFAs ratio) was stable, with the exception of the triacylglycerols fraction after exposure to −3 and −10 °C in mid-diapause (MD) when it significantly increased. Differential scanning calorimetry (DSC) was used to examine phase transitions of total body lipid of cold-acclimated larvae in diapause. Thermal analysis indicated that changes in the melt transition temperatures of whole body total lipids were subtle, but consistent with the modest changes in the level of FA unsaturation observed. We conclude that lipid rearrangements are a function of the endogenous “diapause program” rather than a direct effect of low temperatures, which proved to have limited impact on lipid changes in diapausing larvae of O. nubilalis.

Istraživanja u ovoj doktorskoj disertaciji su bila usmerena ka razumevanju molekularnih mehanizama koji se aktiviraju kod medonosne pčele (Apis mellifera, L.) kao odgovor na stres izazvan jonima teških metala, što zbog važnosti medonosne pčele kao oprašivača ima poseban značaj i očekuje se da će doprineti očuvanju ove vrste. Istraživanja su bila podeljena u tri faze. U prvoj fazi za analize su korišćene populacije pčela sa lokaliteta sa različitim antropološkim uticajem i izmerena je koncentracija metala u pčelama, pergi i medu, kao i relativna genska ekspresija i aktivnost antioksidativnih enzima. U drugoj fazi pčele su u kontrolisanim laboratorijskim uslovima bile izložene subletalnim dozama jona teških metala (bakra, kadmijuma i olova), nakon čega su izmereni parametri koji ukazuju na redoks status i nivo oksidativnog stresa. Rezultati prve dve faze su pokazali da se ekspresija gena i aktivnost antioksidativnih enzima (superoksid dismutaze, katalaze i glutation S-transferaze) razlikuje u zavisnosti od stepena urbanizacije i industrijalizacije, dok je izlaganje bakru i kadmijumu u kontrolisanim uslovima u trajanju od 48 h dovelo samo do promene u ekspresiji gena i u većini slučajeva ekspresija je bila dozno zavisna od koncentracije metala. Olovo je uzrokovalo promene u koncentraciji glutationa i sulfhidrilnih grupa proteina, što govori o tome da helacija olova može da bude prvi mehanizam odbrane od toksičnih efekata ovog metala. U trećoj fazi bioinformatičkom analizom je pronađen metalotionein medonosne pčele i ispitana je njegova funkcija u zaštiti od toksičnih efekata jona teških metala. Utvrđeno je da pčele poseduju jedan gen za metalotionein, koji kodira mali protein sa regionima bogatim cisteinom za koje mogu da se vežu joni metala. Indukcija genske ekspresije metalotioneina medonosne pčele nakon izlaganja metalima i povećana tolerancija bakterija koje ekspresuju rekombinantni metalotionein na metale je potvr, Research in this doctoral thesis focuses on understanding the molecular mechanisms activated in the honey bee (Apis mellifera L.) as a response to stress caused by exposure to heavy metal ions. Because of the importance of honeybees as pollinators, this has special significance and is expected to contribute to the conservation of this species. Studies have been divided into three phases. In the first phase, bee populations from three localities under different anthropological influence were used and the concentrations of metals in the bees, honey and bee bread (perga), as well as relative gene expression and activity of antioxidant enzymes were measured. In the second phase, bees were exposed to sublethal doses of heavy metal ions (copper, cadmium and lead) under controlled laboratory conditions, after which parameters that indicate redox status and oxidative stress were determined. The results of the first two phases showed that gene expression and activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione S-transferase) varies depending on the degree of urbanization and industrialization, while exposure to copper and cadmium in controlled conditions for 48 h resulted only in a change in gene expression in the majority of cases, and the expression was dose-dependent on the concentration of the metal. Lead has caused changes in the concentration of glutathione and sulfhydryl groups of proteins, which indicates that chelation may be the first defense mechanism against the toxic effects of this metal. In the third stage, honeybee metallothionein was identified by bioinformatic analysis and its function in protection against the toxic effects of heavy metal ions was examined. It has been found that honeybees have one metallothionein gene, which encodes a small protein with cysteine-rich regions that may bind metal ions. The induction of metallothionein gene expression after exposure of honeybees to metals and increased tolerance of