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prof. Ing. Adriana Kolesárová, PhD.
Identifikačné číslo: 1505
Univerzitný e-mail: adriana.kolesarova [at] uniag.sk
 
profesorka CSc./PhD. - Katedra fyziológie živočíchov (FBP)
dekanát - centrum - Fakulta biotechnológie a potravinárstva
Prodekanka - Fakulta biotechnológie a potravinárstva

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Základné informácie o záverečnej práci

Typ práce: Dizertačná práca
Názov práce:Fuzariotoxínmi indukované signálne dráhy v granulóznych bunkách vaječníkov ošípaných
Autor: Ing. Marína Medveďová, PhD.
Pracovisko: Katedra fyziológie živočíchov (FBP)
Vedúci práce: prof. Ing. Adriana Kolesárová, PhD.
Oponent 1:Ing. Alexander Makarevič, DrSc.
Oponent 2:doc. RNDr. Radoslav Omelka, PhD.
Oponent 3:prof. MVDr. Imrich Maraček, DrSc.
Stav záverečnej práce:Záverečná práca bola úspešne obhájená


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Jazyk spracovania záverečnej práce:anglický jazyk

slovenský jazyk        anglický jazyk

Názov práce:Fusariotoxins induced porcine ovarian granulosa cells signaling pathways
Abstrakt:A major goal of toxicological research is to enhance the ability to predict and evaluate potential risk of the enviroment to human health. A toxicological approach utilizing in vitro and whole animal studies of suspected hazards can provide specific rationale of toxicity to reproductive function by examining dose -- responses, routes of exposure, and cellular/molecular mechanisms in cell model. In -- vitro culture systems and studies extrapolating the effects of the high doses used in animal studies to lower doses or the actual exposure levels that humans are likely to encounter is complex. Interpretation of results is further complicated because particular reproductive endpoints may be difficult to identify and observe in animals, and numerous variables such as age at exposure, and lifestyle, may affect responses in humans. Extensive research over the past decade has identified a rapidly growing list of environmental contaminants that disrupt reproductive processes in vertebrates primarily by mimicking or opposing the actions of endogenous steroid hormones. Aim of this dissertation work was examination of the secretory activity, markers of proliferation and apoptosis expression in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) and zearalenone (ZEA) exposure. Ovarian GCs were incubated with DON/ZEA for 24h at 10, 100, 1000, 2000, 3000 and 5000 ng.ml-1, while the control group was without presence of DON/ZEA. The secretion of progesterone (P4) and 17beta-estradiol (E2) was determined by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). An expression of proliferation peptides (cyclin B1 and PCNA) and apoptotic peptides (Bax and caspase-3) was determined by immunocytochemistry and fluorescent immunocytochemistry. Experiments were accomplished in several institutions such as: SUA (Slovak University of Agriculture, Department of animal physiology), APRC Nitra (Animal Production Research Centre Nitra, Lužianky), UWM (University of Warmia and Mazuria, Department of animal physiology, Olsztyn, POL) and MSU (Michigan State University, Department of food science and human nutrition, East Lansing, MI, USA). Hormone production as one of the main functions of GCs was examined by ELISA/RIA after DON/ZEA treatment. Our results reveal that: (1) DON decreased P4 secretion at doses of 100, 3000 and 5000 ng.ml-1 in GCs from cyclic gilts; (2) DON increased concentrations of P4 production in GCs from non-cyclic gilts at doses of 1000, 2000, 3000 and 5000 ng.ml-1; (3) DON promoted FSH induced P4 secretion at all experimental doses in GCs from non-cyclic gilts (10, 100, 1000, 2000, 3000 and 5000 ng.ml-1); (4) DON sustain production of E2 by GCs from non-cyclic gilts treated at doses of 2000, 3000 and 5000 ng.ml-1; (5) DON reduced FSH induced E2 secretion by GCs from non-cyclic gilts at doses of 10, 100, 1000 and 5000 ng.ml-1; (6) ZEA had small effect on the dose-response P4 production by GCs from non-cyclic gilts in all used doses; (7) ZEA increased FSH induced P4 secretion in all used doses by GCs from non-cyclic gilts; (8) ZEA promoted P4 hormone levels by GCs from cyclic gilts at doses of 3000 and 5000 ng.ml-1; (9) ZEA in GCs from non-cyclic gilts did not affected E2 production; (10) ZEA at doses of 10, 100, 3000 and 5000 ng.ml-1 had inhibitory effect on FSH induced E2 secretion in GCs from non-cyclic gilts; (11) in the dose combinations with DON and ZEA was not affected P4 secretion in groups A, C and D (Table 5) in GCs from non-cyclic gilts; (12) in GCs from non-cycling gilts DON and ZEA combinations significantly increased P4 production in groups B, E, F, G, H, I and J (Table 5); (13) E2 secretion by GCs from non-cyclic gilts was not affected by any combinations of DON and ZEA (Table 6). To assess cellular proliferation in gilts GCs after exposure to DON/ZEA, selected proliferation (cyclin B1 and PCNA) -- associated peptides were ascertain in our experiments. The expression of peptides after DON/ZEA treatment determined by fluoroimmuno- and immuno- cytochemistry revealed that: (1) cyclin B1 was overexpressed at doses of 1000, 2000, 3000 and 5000 ng.ml-1 DON in GCs from non-cyclic gilts; (2) DON had no effect on cyclin B1 expression in GCs from cyclic gilts; (3) expression of PCNA protein was stimulated at doses of 100, 1000, 2000, 3000 and 5000 ng.ml-1 DON in GCs from non-cyclic gilts; (4) cyclin B1 expression after the ZEA treatment at doses of 100, 1000 and 5000 ng.ml-1 significantly increased in GCs from non-cyclic gilts; (5) in the GCs from cyclic gilts ZEA did not have effect on cyclin B1 expression; (6) significant stimulation of PCNA expression by ZEA exposure was observed in doses of 1000, 3000 and 5000 ng.ml-1 in GCs from non-cyclic gilts. In order to gain further insight into mechanism of DON/ZEA-mediated cytotoxicity, we observed the molecular event in cell death triggered by DON/ZEA. Our results showed a dose-dependent cytotoxic effect of DON/ZEA as revealed by fluoroimmuno- and immuno- cytochemistry that: (1) DON affected caspase-3 expression in all used doses in GCs from non-cyclic gilts; (2) caspase-3 expression in GCs from cyclic gilts was inhibited significantly at doses of 10, 1000, 3000 and 5000 ng.ml-1 DON; (3) expression of pro-apoptotic factor Bax was upregulated significantly at the DON doses of 100 and 1000 ng.ml-1 by GCs from non-cyclic gilts; (4) in the GCs from non-cyclic gilts caspase-3 expression significantly increased at doses of 10, 100, 1000 and 5000 ng.ml-1 ZEA; (5) ZEA stimulated significantly caspase-3 expression in GCs from cyclic gilts at doses of 10, 100, 1000, 3000 and 5000 ng.ml-1; (6) Bax expression was found to be significantly higher at doses of 10 and 1000 ng.ml-1 ZEA in GCs from non-cyclic gilts. The study of potentially hazardous compounds on the female reproductive system is therefore requiring the assessment of their specific interaction with each follicular component at each stage of development. Research in the field during the next decade is likely to see many more studies like those decribed here that will investigate cellular and molecular events involved in effects caused by a wide variety of reproductive toxicants. As a result, peptide adducts can be identified in mixtures of proteins from biological sample. Thus, such a method offers us new approaches for the identification of proteins targeted by reactive chemicals in toxic responses to xenobiotics. Mycotoxins can be acutely and/or chronically toxic, depending on the toxin and the toxin dose. In terms of acute toxicity, the mycotoxins most commonly encountered in food are about a factor of a million times less toxic than the most virulent botulism toxin. It is the long term toxicity which is of special concern because certain mycotoxins ingested dialy in small quantities for an extended period are known to be carcinogenic and to influence the immune response of humans and animal species. It is concluded that mycotoxins constitute a significant problem for the animal feed industry and an ongoing risk to feed supply. Our data suggest a possible underlying molecular mechanisms for DON and ZEA to induces the proliferation and apoptosis signaling pathway, as well as these mycotoxins could be possibly encountered as endocrine disrupting chemicals in steroidogenic pathway in porcine ovarian granulosa cells.
Kľúčové slová:zearalenone, progesteron, 17beta-estradiol, proliferation, apoptosis, deoxynivalenol, cyclin B1, PCNA, Bax, caspase-3, steroidogenesis, granulosa cells

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