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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

Honeybee colony losses have been a focus of research in the last years, due to the importance of managed honeybee colonies for economy and ecology. Different unfavorable conditions from the outside environment have a strong impact on the hive health. The majority of losses occur mainly during winter and the exact reason is not completely understood. Only a small number of studies are dealing with content of bioelements, their function and influence on honeybee physiology. The aim of the present study was to determine seasonal and spatial variations in content of bioelements and non-essential elements, in hemolymph and whole body of honeybees originating from three regions with different degrees of urbanization and industrialization. Concentrations of 16 elements were compared: macroelements (Ca, K, Mg, Na), microelements (Cu, Fe, Mn, Zn) and non-essential elements (Al, Ba, Cd, Co, Cr, Ni, Pb, Sr) in samples collected from 3 different environments: Golija (rural region), Belgrade (urban region) and Zajača (industrial region). Content of bioelements and non-essential elements in honeybees was under noticeable influence of the surrounding environment, season and degree of honeybee activity. Hemolymph was proven to be helpful in differentiating air pollution from other sources of honeybee exposure. The results of our study demonstrated that bees can be successfully used as biomonitors since we have observed statistically significant differences among observed locations, but unless compared locations are exposed to excessively different pollution pressures, it is essential that all bees should be collected at the same season.

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

Radioterapija (RT) je jedan od najvažnijih modaliteta lečenja solidnih malignih tumora i koristi je više od 50% pacijenata (52,3%) sa malignim tumorima. Nauka koja proučava efekte elektromagnetnog zračenja na biološke sisteme naziva se radiobiologija. Radiobiologija se fokusira na odgovor ćelija, tkiva i organizma kao celine na jonizujuće zračenje i proučava mehanizme radiobiološkog odgovora. Izlaganje ćelija niskim dozama JZ koje su nakon određenog vremenskog intervala praćene uobičajenim radioterapijskim dozama naziva se radioadaptivno zračenje. Adaptivni odgovor u sebi može da sadrži nekoliko fenomena: hiperradiosenzitaciju/radiorezistenciju, “bystander” efekat i radioadaptivni efekat u užem smislu. O molekularnim mehanizmima koji stoje iza navedenih efekata ne zna se dovoljno. U ovom radu ispitivan je odgovor malignih i zdravih ćelija na različite modalitete jonizujućeg zračenja u cilju boljeg poznavanja puteva ćelijske smrti i preživljavanja. Potpuno razumevanje molekularnih puteva koji vode u apoptozu ili u preživljavanje ćelija nakon izlaganja jonizujućem zračenju moglo bi koristiti u iznalaženju novih i efikasnijih strategija i modaliteta lečenja malignih tumora u cilju njihove potpune eredikacije. U istraživanju su korištene dve humane ćelijske linije ćelijska linija humanog kolorektalnog karcinoma HT-29 i ćelijska linija humanih fetalnih fibroblasta pluća MRC-5. Ćelije su zračene u dva režima različitim pre-iradijacionim dozama(0,03; 0,05 i 0,07Gy) i istom kurativnom dozom (2Gy) tokom 4 dana. Višekratna primena niskih doza JZ nije značajno smanjila vijabilnost HT-29 ćelija, dok su dve radioadaptivne doze (0,05+2Gy i 0,07+2Gy), adekvatne doze JZ za radioterapijski postulat poštede zdravih ćelija i bolji antitumorski efekat u odnosu na neradioadaptivno zračenje od 2Gy u toku 4 dana. Pokazana je mogućnost modulisanja ćelijskog odgovora na JZ uz pomoć niskih doza JZ koje su praćene dozom od 2Gy (radioadaptivni tip zračenja) u oba dizajnirana režima zračenja. S, Radiotherapy (RT) is one of the most important treatment modality for solid malignant tumors and it is applied in more than 50% of the patients (52.3%). Radiobiology id scientific discipline which studies the effects of electromagnetic irradiation on biological systems. Radiobiology focuses on the response of the cells, tissues and the organism as a whole to ionizing radiation and studying the mechanisms of radiobiological response. Exposure of cells to low-dose irradiation (priming dose) followed by challenging doses is called radioadaptive radiation. Adaptive response is described as several phenomena: hyperradiosensitivity / radiorezistence, "bystander" effect and radioadaptive effect in sensu strict. Molecular mechanisms underlying the above effects are not sufficiently known. In this study, the response of malignant and healthy cells on various modalities of ionizing radiation is explored in order to improve knowledge of pathways of cell death and survival. Fully understanding the molecular pathways leading to apoptosis or cell survival after exposure to ionizing radiation may be used in finding new and more effective strategies and modalities for the treatment of malignant tumors. The study used two human cell lines: human colorectal cancer HT-29 cell line and the human fetal lung fibroblast MRC-5. The cells were irradiated in two modalities using different pre-irradiation doses (0.03, 0.05 and 0,07Gy) and the same challenging dose (2Gy) for 4 days. Everyday use of low-dose did not significantly reduce the viability of HT-29 cells, while two radioadaptive doses (0.05 + 2Gy and 0.07+2Gy), are adequate doses for sparing healthy cells with better anti-tumor effects. The possibility of modulating the cellular response to the ionizing radiation was shown using low-doses followed by 2Gy (radioadaptive radiation) in both designed regimes of radiation. The level of chromosomal damage showed a dose-dependent trend. Dose-dependent damage to the genetic material caused b

Identifikacija genomskih regiona na koje je delovala selekcija tokom stvaranja elitnih sorti soje (Glycine max (L.) Merr.) može ukazati na pozicije gena koji determinišu važna agronomska svojstva ili su odgovorni za adaptabilnost. U radu su sagledani efekti oplemenjivanja u agroklimatskim uslovima centralne i istočne Evrope koristeći pristup “hitchhiking” mapiranja i analizu pedigrea. U ovu svrhu su primenjeni molekularni markeri, mikrosateliti i principi populacione genetike, koristeći više različitih pristupa za identifikaciju selektivno značajnih lokusa. Analiza je obuhvatila populacije soje koje su se sastojale od predačkih genotipova i elitnih genotipova koji su nastali u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Analizom pedigrea je potvrđena uska genetička osnova sorti soje elitne populacije. Kao posledica dugogodišnjeg oplemenjivanja, svi analizirani parametri su ukazali na statistički značajno smanjenje genetičkog diverziteta elitne populacije u odnosu na predačku. Usled specifične strukture populacija soje, koja je u velikoj meri bila pod uticajem pedigrea elitnih genotipova, uočen je nizak nivo genetičke diferencijacije među ispitivanim populacijama. Primenom najmanje dva različita pristupa identifikovano je devet mikrosatelitskih lokusa koji su ukazivali na regione genoma na koje je delovala selekcija, a koji su bili uključeni u proces adaptacija u agroklimatskim uslovima centralne i istočne Evrope. U elitnoj populaciji je potvrđeno povećanje stope gametskog disekvilibrijuma, najverovatnije kao posledica delovanja selekcije. „Bottleneck” test je ukazao na značajno smanjenje diverziteta samo kod lokusa na koje je delovala selekcija u elitnoj populaciji, što najverovatnije nije uzrokovano demografskim faktorima, nego takođe predstavlja posledicu delovanja selekcije. Analizom kolokacije poznatih QTL regiona i identifikovanih, selektivno značajnih genomskih regiona, uočeno je ukupno 264 QTL-ova, od kojih su najzastupljeniji bili l, The identification of genomic regions affected by selection during breeding of soybean (Glycine max (L.) Merr.) may indicate the positions of important agronomic traits genes or genes underlying adaptation to a specific target environment. This study investigated the effects of breeding in Central-East European environments by a hitchhiking mapping approach and pedigree analysis. Population genetic principles were applied to microsatellite markers using multiple outlier detection tests. The analysed populations comprised ancestral genotypes and elite varieties, developed at the Institute of Field and Vegetable Crops, Novi Sad. The pedigree analysis confirmed narrow genetic base of elite genotypes. As a result of long-term breeding, all analysed parameters showed significant reduction in genetic diversity in the elite population, compared to the ancestral. Specific population structure of analysed varieties, which has been largely influenced by the pedigree, probably caused a low level of genetic differentiation between the populations. Using at least two approaches, nine markers were considered as strong positive selection candidates, indicating regions involved in the adaptation to Central-East Europe environments. Also, an excess of linkage disequilibrium was confirmed in the elite population, probably caused by selection. Bottleneck tests provided evidence of population bottlenecks only for the candidate positive selection loci in the elite population, suggesting that selection might shaped the pattern of genetic diversity in these regions. The co-localisation analysis of the candidate positive selection loci and previously mapped quantitative trait loci (QTLs), identified in total 264 QTLs in selectively important genomic regions. The highest number of identified QTLs had impact on the reproductive period. In silico analysis revealed a high level of agreement between the identified QTLs and the traits expected to be under selection during soybean breeding, indic

Seasonal changes in the FA composition of triacylglycerols and phospholipids prepared from the whole body of non-diapausing and diapausing fifth instar larvae of Ostrinia nubilalis, Hubn. (Lepidoptera: Crambidae) were determined to evaluate the role of these lipids in diapause. Substantial changes in the FA composition of triacylglycerols and phospholipids were triggered by diapause development. This led to a significant increase in the overall FA unsaturation (UFAs/SFAs ratio), attributable to an increase in the relative proportion of MUFAs and the concomitant decrease in PUFAs and SFAs. In triacylglycerols, the significant changes in FAs composition is the result of an increase in the relative proportions of MUFAs, palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), and a concomitant reduction in composition of SFAs and PUFAs, mainly palmitic acid (16:0) and linoleic acid (18:2n-6), respectively. Changes in the composition of phospholipids were more subtle with FAs contributing to the overall increase of FA unsaturation. Differential scanning calorimetry (DSC) analysis revealed that the melt transition temperatures of total lipids prepared from whole larvae, primarily attributable to the triacylglycerol component, were significantly lower during the time course of diapause compared with non-diapause. These observations were correlated to the FA composition of triacylglycerols, most likely enabling them to remain functional during colder winter conditions. We conclude that O. nubilalis undergoes remodelling of FA profiles of both energy storage triacylglycerols and membrane phospholipids as an element of its overwintering physiology which may improve the ability to cold harden during diapause.

Identifikacija genomskih regiona na koje je delovala selekcija tokom stvaranja elitnih sorti soje (Glycine max (L.) Merr.) može ukazati na pozicije gena koji determinišu važna agronomska svojstva ili su odgovorni za adaptabilnost. U radu su sagledani efekti oplemenjivanja u agroklimatskim uslovima centralne i istočne Evrope koristeći pristup “hitchhiking” mapiranja i analizu pedigrea. U ovu svrhu su primenjeni molekularni markeri, mikrosateliti i principi populacione genetike, koristeći više različitih pristupa za identifikaciju selektivno značajnih lokusa. Analiza je obuhvatila populacije soje koje su se sastojale od predačkih genotipova i elitnih genotipova koji su nastali u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Analizom pedigrea je potvrđena uska genetička osnova sorti soje elitne populacije. Kao posledica dugogodišnjeg oplemenjivanja, svi analizirani parametri su ukazali na statistički značajno smanjenje genetičkog diverziteta elitne populacije u odnosu na predačku. Usled specifične strukture populacija soje, koja je u velikoj meri bila pod uticajem pedigrea elitnih genotipova, uočen je nizak nivo genetičke diferencijacije među ispitivanim populacijama. Primenom najmanje dva različita pristupa identifikovano je devet mikrosatelitskih lokusa koji su ukazivali na regione genoma na koje je delovala selekcija, a koji su bili uključeni u proces adaptacija u agroklimatskim uslovima centralne i istočne Evrope. U elitnoj populaciji je potvrđeno povećanje stope gametskog disekvilibrijuma, najverovatnije kao posledica delovanja selekcije. „Bottleneck” test je ukazao na značajno smanjenje diverziteta samo kod lokusa na koje je delovala selekcija u elitnoj populaciji, što najverovatnije nije uzrokovano demografskim faktorima, nego takođe predstavlja posledicu delovanja selekcije. Analizom kolokacije poznatih QTL regiona i identifikovanih, selektivno značajnih genomskih regiona, uočeno je ukupno 264 QTL-ova, od kojih su najzastupljeniji bili lokusi koji su deter, The identification of genomic regions affected by selection during breeding of soybean (Glycine max (L.) Merr.) may indicate the positions of important agronomic traits genes or genes underlying adaptation to a specific target environment. This study investigated the effects of breeding in Central-East European environments by a hitchhiking mapping approach and pedigree analysis. Population genetic principles were applied to microsatellite markers using multiple outlier detection tests. The analysed populations comprised ancestral genotypes and elite varieties, developed at the Institute of Field and Vegetable Crops, Novi Sad. The pedigree analysis confirmed narrow genetic base of elite genotypes. As a result of long-term breeding, all analysed parameters showed significant reduction in genetic diversity in the elite population, compared to the ancestral. Specific population structure of analysed varieties, which has been largely influenced by the pedigree, probably caused a low level of genetic differentiation between the populations. Using at least two approaches, nine markers were considered as strong positive selection candidates, indicating regions involved in the adaptation to Central-East Europe environments. Also, an excess of linkage disequilibrium was confirmed in the elite population, probably caused by selection. Bottleneck tests provided evidence of population bottlenecks only for the candidate positive selection loci in the elite population, suggesting that selection might shaped the pattern of genetic diversity in these regions. The co-localisation analysis of the candidate positive selection loci and previously mapped quantitative trait loci (QTLs), identified in total 264 QTLs in selectively important genomic regions. The highest number of identified QTLs had impact on the reproductive period. In silico analysis revealed a high level of agreement between the identified QTLs and the traits expected to be under selection during soybean breeding, indic

Metabolic syndrome is a complex polygene multifactor metabolic disorder. By knowing genetic predisposition, environment factors which are subject to change can be corrected, which allows the disorder to be postponed or prevented. Determining genetic background of metabolic syndrome is one of the necessary steps in preventing the disorder; it would save the cost of medical treatments as well as help in designing personalized therapy. Experiment goals are determining the connection between individual alleles of apoE and LRPI genes on one side and the appearance of metabolic syndrome on the other; the analysis of correlation between apoE and LRPI genes polymorphism and each individual anthropometric and biochemical parameter in both tested groups; as well as establishing the correlation of every composite genotype of apoE and LRPI genes with presence of metabolic syndrome, and the connection with each anthropometric and biochemical parameter in both tested groups. This paper has demonstrated the importance ofapoE gene and LRPI gene in the metabolism of lipids and in all other anthropometric and biochemical parameters tested and the impact of e4 allele form of apoE gene and T allele form of LRPI gene individually, as well as the impact of composite genotypes both of the above mentioned genes on the emergence and development of the metabolic syndrome. It has been determined that the most common allele form of apoE gene are e3, e4 and e2 subsequently, while the most common allele forms of LRP 1 gene is C in both test groups. It is concluded that in the experimental group more fi-equent is e4 allele of apoE gene and T allele ofLRPl gene, which confirms negative impact of stated alleles onto tested anthropometric and biochemical parameters. Acquired results show that presence ofe4 allele ofapoE gene increases the chance 11.5 times fur the metabolic syndrome compared to the carriers of e2 and e3 alleles, while T allele of LRPl gene increases the chance 4.76 times in comparis

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, 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...

This study compares the composition and biophysical properties of lipids in non-diapausing and diapausing fifth instar larvae of Ostrinia nubilalis Hubn. (Lepidoptera: Crambidae). The majority of fat body lipids in both of these physiological states were comprised of ~ 90% triacylglycerols (TAGs), whereas the haemolymph contained a more even distribution of all lipid classes. The fatty acid composition and biophysical properties of the fat body lipids differed markedly between non-diapausing and diapausing larvae. Diapause was associated with a dramatic increase in the proportions of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), with concurrent reductions in palmitic acid (16:0) and linoleic acid (18:2n-6). The increase in the level of unsaturation of the fat body lipids, which caused a marked shift in their phase transitions to lower temperatures, was triggered by diapause rather than low temperatures. Adjustments of fatty acid compositions are likely to be an important component of winter diapause mechanisms, possibly maintaining the fluidity of cell membranes and the functionality of the organism during lower winter temperatures.

The Arctic springtail, Megaphorura arctica, survives sub-zero temperatures in a dehydrated state via trehalose-dependent cryoprotective dehydration. Regulation of trehalose biosynthesis is complex; based in part on studies in yeast and fungi, its connection with oxidative stress caused by exposure of cells to oxidants, such as hydrogen peroxide (H2O2), or dehydration, is well documented. In this respect, we measured the amount of H2O2 and antioxidant enzyme activities (superoxide dismutases: copper, zinc—CuZnSOD and manganese containing–MnSOD, and catalase—CAT), as the regulatory components determining H2O2 concentrations, in Arctic springtails incubated at 5 °C (control) versus −2 °C (threshold temperature for trehalose biosynthesis). Because ecdysone also stimulates trehalose production in insects and regulates the expression of genes involved in redox homeostasis and antioxidant protection in Drosophila, we measured the levels of the active physiological form of ecdysone—20-hydroxyecdysone (20-HE). Significantly elevated H2O2 and 20-HE levels were observed in M. arctica incubated at −2 °C, supporting a link between ecdysone, H2O2, and trehalose levels during cryoprotective dehydration. CAT activity was found to be significantly lower in M. arctica incubated at −2 °C versus 5 °C, suggesting reduced H2O2 breakdown. Furthermore, measurement of the free radical composition in Arctic springtails incubated at 5 °C (controls) versus −2 °C by Electron Paramagnetic Resonance spectroscopy revealed melanin-derived free radicals at −2 °C, perhaps an additional source of H2O2. Our results suggest that H2O2 and ecdysone play important roles in the cryoprotective dehydration process in M. arctica, linked with the regulation of trehalose biosynthesis.

The Arctic springtail Megaphorura arctica (Tullberg, 1876) employs a strategy known as cryoprotective dehydration to survive winter temperatures as low as -25 degrees C. During cryoprotective dehydration, water is lost from the animal to ice in its surroundings as a result of the difference in vapour pressure between the animal's supercooled body fluids and ice (Worland et al., 1998; Holmstrup and Somme, 1998). This mechanism ensures that as the habitat temperature falls, the concentration of solutes remains high enough to prevent freezing (Holmstrup et al., 2002). In M. arctica, accumulation of trehalose, a cryo/anhydro protectant, occurs in parallel with dehydration. Recent studies have identified a number of genes and cellular processes involved in cryoprotective dehydration in M. arctica (Clark et al., 2007; Clark et al., 2009; Purac et al., 2011). One of them includes late embryogenesis abundant (LEA) proteins. This study, together with that of Bahrndorff et al. (2008), suggests that LEA proteins may be involved in protective dehydration in this species.

Arthropods have evolved a number of different adaptations to survive extreme environmental temperatures including, in some regions, over-wintering temperatures well below 0°C. One of the less common adaptations to surviving cold is that of cryoprotective dehydration, where the animal becomes almost anhydrobiotic with the loss of virtually all osmotically active water. In this chapter, we describe integrated studies utilising physiology, biochemistry and molecular biology to understand this phenomenon in the Arctic springtail (Megaphorura arctica) (formerly Onychiurus arcticus). These studies concentrate on the action of trehalose as a cryoprotectant, the production of antioxidants to reduce cell damage and changes in membrane composition.

Sposobnost insekata da se prilagode različitim ekološkim uslovima je veoma dobro dokumentovana; oni predstavljaju najrasprostranjeniju grupu životinja na planeti, sa vrstama koje naseljavaju različita kopnena i vodena staništa, od tropskih predela do polova. Razumevanje mehanizama koji omogućavaju insektima da prežive ekstremne temperature i zadrže vitalne funkcije tokom dugog perioda dormancije je kao model sistem od interesa za mnoge naučne oblasti. Na osnovu načina na koji preživljavaju temperature ispod 0ºC insekte možemo podeliti u tri grupe: i) insekti koji tolerišu formiranje leda u ekstraćelijskom prostoru,ii) insekti koji ne tolerišu zamrzavanje i moraju da ga izbegnu, a to čine superhlađenjem svojih telesnih tečnosti i iii) insekti koji preživljavaju zahvaljujući gubitku vode kroz permeabilnu kutikulu, što je nazvano krioprotektivna dehidratacija. Zajednička odlika organizama sa različitim mehanizmima adaptacije na niske temperature je setbiohemijskih jedinjenja čija se fiziološka funkcija razlikuje u zavisnosti da li organizam pripada grupi koja toleriše ili ne toleriše zamrzavanje. To su nukleatori kristalizacije leda, krio/anhidroprotektanti i antifriz proteini. Cilj ovih istraživanja je bio ispitivanje molekularne osnove otpornosti na niske temperature dve vrste polarnih kolembola Onychiurus arcticus i Cryptopygus antarcticus kombinujući fiziološki, biohemijski i molekularno biološki pristup. Ispitivane vrste izbegavaju zamrzavanje svojih telesnih tečnosti primenjujući različite strategije preživljavanja. Za antarktičku vrstu C. antarcticus karakteristična je brza promena tačke superhlađenja, kao i njena bimodalna distribucija tokom leta, kada neke jedinke mrznu na višim temperaturama (manje otporne na hladnoću), a druga na nižim (otpornije na hladnoću). Ova bimodalna distribucija tačke superhlađenja je dobro dokumentovana, ali slabo razjašnjena na molekularnom nivou. Druga, arktička vrsta O. arcticus koristi strategiju preživljavanja zimskih, The ability of insects to adapt to diverse ecological conditions iswell documented; they are the most diverse fauna on earth, with different species occupying arange of terrestrial and aquatic habitats from the tropics to the poles. Understanding the mechanisms by which insects survive such extreme temperatures and retain viability for longperiods in the dormant state is of great interest to many scientific fields. Insects have evolved three main strategies to survive sub-zero temperatures: i) freeze tolerance, ii) freeze avoidance and iii) cryoprotective dehydration. The main biochemical compounds involved in surviving sub-zero temperatures are same for different strategies but their physiological role is different. They include: ice nucleating agents (INAs), cryo/ anhydroprotectants, and antifreeze proteins (AFPs). The aim of this study was to determine molecular adaptations to extreme cold environments, combining physiology, biochemistry and molecular biology pproaches, in thePolar Collembola: Cryptopygus antarcticus and Onychiurus arcticus. Both species are freeze avoiding but employ different strategies for surviving low temperatures. The Antarctic springtail C. antarcticusis capable of rapid cold hardening with a bi-modal distribution of super cooling points (SCP) with high (less cold-hardened) and low (more cold-hardened) groups of animals present even during the growing season in summer. This bimodal distribution has been well documented, but is poorly understood. The Arctic springtail O. arcticusemploys the strategy known as cryoprotective dehydration to survive winter temperatures as low as -25ºC. With this technique, the amount ofavailable water in the body is reduced to almost zero and also there is an accumulation of trehalose, which acts as a cryo/anhydroprotectant. Although cryoprotective dehydration has been described in other insects, the molecular mechanisms behind this phenomenon are poorly understood. A total of 16,379 EST clone

Background: Insects provide tractable models for enhancing our understanding of the physiological and cellular processes that enable survival at extreme low temperatures. They possess three main strategies to survive the cold: freeze tolerance, freeze avoidance or cryoprotective dehydration, of which the latter method is exploited by our model species, the Arctic springtail Megaphorura arctica, formerly Onychiurus arcticus (Tullberg 1876). The physiological mechanisms underlying cryoprotective dehydration have been well characterised in M. arctica and to date this process has been described in only a few other species: the Antarctic nematode Panagrolaimus davidi, an enchytraied worm, the larvae of the Antarctic midge Belgica antarctica and the cocoons of the earthworm Dendrobaena octaedra. There are no in-depth molecular studies on the underlying cold survival mechanisms in any species. Results: A cDNA microarray was generated using 6,912 M. arctica clones printed in duplicate. Analysis of clones up-regulated during dehydration procedures (using both cold-and salt-induced dehydration) has identified a number of significant cellular processes, namely the production and mobilisation of trehalose, protection of cellular systems via small heat shock proteins and tissue/cellular remodelling during the dehydration process. Energy production, initiation of protein translation and cell division, plus potential tissue repair processes dominate genes identified during recovery. Heat map analysis identified a duplication of the trehalose-6-phosphate synthase (TPS) gene in M. arctica and also 53 clones co-regulated with TPS, including a number of membrane associated and cell signalling proteins. Q-PCR on selected candidate genes has also contributed to our understanding with glutathione-S-transferase identified as the major antioxdidant enzyme protecting the cells during these stressful procedures, and a number of protein kinase signalling molecules involved in recovery. Conclu

Background Ice, snow and temperatures of -14°C are conditions which most animals would find difficult, if not impossible, to survive in. However this exactly describes the Arctic winter, and the Arctic springtail Onychiurus arcticus regularly survives these extreme conditions and re-emerges in the spring. It is able to do this by reducing the amount of water in its body to almost zero: a process that is called "protective dehydration". The aim of this project was to generate clones and sequence data in the form of ESTs to provide a platform for the future molecular characterisation of the processes involved in protective dehydration. Results Five normalised libraries were produced from both desiccating and rehydrating populations of O. arcticus from stages that had previously been defined as potentially informative for molecular analyses. A total of 16,379 EST clones were generated and analysed using Blast and GO annotation. 40% of the clones produced significant matches against the Swissprot and trembl databases and these were further analysed using GO annotation. Extraction and analysis of GO annotations proved an extremely effective method for identifying generic processes associated with biochemical pathways, proving more efficient than solely analysing Blast data output. A number of genes were identified, which have previously been shown to be involved in water transport and desiccation such as members of the aquaporin family. Identification of these clones in specific libraries associated with desiccation validates the computational analysis by library rather than producing a global overview of all libraries combined. Conclusion This paper describes for the first time EST data from the arctic springtail (O. arcticus). This significantly enhances the number of Collembolan ESTs in the public databases, providing useful comparative data within this phylum. The use of GO annotation for analysis has facilitated the identification of a wide variety of ESTs associate