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Effect of inflammation on placental metabolism of tryptophan
Orbisová, Anna ; Štaud, František (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Anna Orbisová Supervisor: Prof. PharmDr. František Štaud, Ph.D. Consultant: Dr. Cilia Abad, Ph.D. Title of diploma thesis: Effect of inflammation on placental metabolism of tryptophan Maternal inflammation throughout pregnancy has been firmly linked to the development of neuropsychiatric disorders in the offspring. Furthermore, intrauterine infections, whether viral or bacterial, are widely recognized as significant risk factors for conditions like autism and schizophrenia. Tryptophan metabolism has been suggested as a plausible pathway through which maternal inflammation during gestation can hinder fetal brain development and programming. This occurs as tryptophan is metabolized through the serotonin (5-HT) pathway, producing metabolites that exert direct effects on the development of the fetal brain. In this work, using an ex vivo model, we assess the impact of bacterial (LPS) and viral (poly I:C) placental infection on the 5-HT pathway. Human term placenta explants were treated with LPS or Poly I:C for different times (4 or 18 hours). Subsequently, the impact on gene and protein expressions of the key enzymes within the 5-HT pathway, along with their functional enzymatic activities, was...
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Comparison of the effect of bisphenols on parental and differentiated H9c2 cells
Macáková, Barbora ; Jirkovský, Eduard (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Barbora Macáková Supervisor: PharmDr. Eduard Jirkovský, Ph.D. Title of diploma thesis: Comparison of the effect of bisphenols on parental and differentiated H9c2 cells Bisphenol is compound largely found in products and environment around us. Its toxicity and danger for human health is still being studied, meanwhile direct influence of environmentally relevant doses was not discovered. These endocrine disruptors are affecting variousreceptorsin bodiesand effecton cardiovascularsystem isnotexcludedeither.The most extensively studied substance is BPA, which is gradually being replaced by its analogs, although these are not extensively researched to the same extent. In this work we focused on influence of bisphenol BP, M and S on differentiation of H9c2 cells, serving as a model with potential negative effects on heart development and its regeneration after damage. H9c2 cell line has an undifferentiated phenotype but can be easily directed towards differentiation under certain conditions Research was focused on impact of bisphenol on differentiated cells and its changes in the expression of selected differentiation markers (GATA4, Hand2, Tnnt2, Myog) (transcription factors). Set protocols were...
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Endocrine disruptors as ligands of nuclear receptors.
Tóthová, Veronika ; Pávek, Petr (advisor) ; Jirkovský, Eduard (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Veronika Tóthová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Endocrine disruptors as ligands of nuclear receptors Endocrine disruptors are synthetic substances used in many areas of industry or agriculture that negatively affect and interfere with the functions of the endocrine system. Their negative effects have been mentioned since the beginning of the 20th century, but only recently have more attention been paid to them due to their adverse effects on the human organism and the environment. In the thesis, we summarized the mechanisms of action of the most well-known endocrine disruptors on selected nuclear receptors. Specifically, it was bisphenol A and phthalate-type compounds. From nuclear receptors, we focused on PPAR (Peroxisome Proliferator-Activated Receptors), Pregnane X receptor (PXR) and Aryl androstane receptor (AHR). From the information obtained, it follows that we are in constant contact with these substances, and therefore we should devote more attention to studying their effects, because not all mechanisms of action and effects of these compounds are completely known. In this way, we could increase the safety of using these substances,...
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The evaluation of senescence markers in the H9c2 cell line after cobalt exposure
Rottenbornová, Lucie ; Jirkovský, Eduard (advisor) ; Hrubša, Marcel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lucie Rottenbornová Supervisor: PharmDr. Eduard Jirkovský, Ph.D. Title of diploma thesis: The evaluation of senescence markers in the H9c2 cell line after cobalt exposure Introduction and aim: Cellular senescence is the response of a cell to a physiological or stressful stimulus. Cobalt is an element that simulates hypoxia under normoxic conditions through hypoxia-inducible factors. One of the factors that can induce cellular senescence is hypoxia. The aim of the work was to evaluate p16INK4a and Ki67 proteins as markers of cell senescence in an H9c2 cell line after exposure to CoCl2. Methods: Cytotoxicity was measured using the MTT assay on the H9c2 cell line after 24 and 48 hours of exposure to CoCl2. Subsequently, relative gene expression of genes associated with hypoxia (HIF1α and BNIP3) and senescence (p16INK4a and Ki67) in H9c2 after exposure to cobalt was investigated using the RT-PCR method. Results: Exposure of H9c2 cells to CoCl2 induced dose- and time-dependent toxicity. After a 24-hour exposure, CoCl2 toxicity was not observed up to a concentration of 100 μM, the IC50 value was 500.5 μM. After 48 hours of exposure, higher toxicity was observed already at 100 μM concentration and...
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Analysis of the molecular pathways and interactome of FUBP3
Brychta, David ; Jirkovský, Eduard (advisor) ; Karahoda, Rona (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: David Brychta Supervisor: Assist. Prof. Eduard Jirkovský, PharmD, PhD.; Assist. Prof. Nika M. Lovšin, M.Chem., PhD Title of Diploma Thesis: Analysis of the molecular pathways and interactome of FUBP3 The far upstream element-binding protein 3 (FUBP3) has been recognized by genome-wide association studies to be associated with a higher risk of osteoporotic fracture. The knowledge about this protein and its role in bone biology is quite limited, though. Therefore, we aimed to broaden the horizons and created an overview of FUBP3 protein-protein interactors and possible pathway involvement. The interacting proteins were gathered across multiple databases. Their association with osteoporosis (OP) and bone mineral density (BMD) was assessed using an online tool - OpenTargets Platform. Twelve hits associated with either were then used for qPCR analysis to investigate the influence of FUBP3 knockout. Among FUBP3 interactors were also proteins of cytosolic membraneless organelles - stress granules (SG). Stress conditions were induced and co-localization of SG markers PABPC1 and G3BP1 was carried out using immunostaining and fluorescence microscopy. We were able to identify 75 protein interactors...
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Characterization of different differentiation protocols of H9c2 cardiomyoblasts
Gardianová, Anna ; Jirkovský, Eduard (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anna Gardianová Supervisor: PharmDr. Eduard Jirkovský, Ph.D. Title of diploma thesis: Characterization of different differentiation protocols of H9c2 cardiomyoblasts Cell differentiation is a common process in the development of multicellular organisms leading to an alteration of cell's appearance and function. The process of differentiation plays a significant role in the reparation of damaged tissues too. Some chemicals may have negative effects on these processes and therefore affect human health. This thesis focuses on characterization of differentiation processes of H9c2 cell line using various published differentiation protocols, and to describe changes over time in the morphology (occurrence of multinucleated and elongated cells) and mRNA expression of selected markers typical for the cardiac (cTnT, Hand2, GATA4) or skeletal (myogenin) muscle phenotype. We monitored the process of the differentiation for 15 days and the individual protocols differed by the addition of fetal bovine serum (FBS, 1 % or 10 %) and all-trans- retinoic acid (ATRA, 10 nmol/L) to the growth medium Dulbecco's Modified Eagle's Medium. From what we have observed, the results show that all of the utilized protocols...
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The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase
Sobolová, Zuzana ; Mladěnka, Přemysl (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Sobolová Supervisor: Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Consultant: Marcel Hrubša, MS.c. Title of diploma thesis: The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase Inhibitors of alcohol dehydrogenase play a key role in the treatment of poisoning caused by methanol, ethanol, ethylene glycol, and their toxic metabolites. Such treatment can therefore be considered as life-saving. In clinical practice, a single substance, fomepizole, is used. This thesis is aimed at investigation of the effect of eleven experimental chelators from the 4- acylpyrazol-5-ones group on equine and yeast alcohol dehydrogenases, and the possible definition of structural features that would be the most suitable for interaction with this enzyme. Due to the ability of these substances to chelate zinc ions, it is believed that they could inhibit an enzyme that has a zinc molecule in the structure. The measurement itself is based on principle of increase in absorbance during the enzymatic reaction, which is caused by the formation of NADH. This measurement is relatively simple, efficient and, due to used kinetic method, also less susceptible to be affected by...
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Placental transport of dopamine and norepinephrine
Turková, Helena ; Štaud, František (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Helena Turková Supervisor: prof. PharmDr. František Štaud, Ph.D. Title of diploma thesis: Placental transport of dopamine and norepinephrine During the whole course of pregnancy, it is important to maintain proper monoamine homeostasis, namely serotonin (5-HT), norepinephrine (NE), and dopamine (DA), which are crucial for proper placental function and fetal development. Monoamines are important neuromodulators, involved in cell proliferation, and differentiation. and neuronal migration. High fetal monoamine secretion during gestation demands a responsible clearance mechanism, as disruption of their balance may lead to long-lasting changes in brain structure and function, provoking a higher risk of attention deficit hyperactivity disorder (ADHD), autism or depression. However, uptake of NE a DA through the fetoplacental unit has not been fully and in detail described. Therefore, in this diploma thesis, we focus on the uptake of NE and DA through ex vivo isolated vesicles of microvillous (MVM) and basal (BM) membranes from healthy human term placentas. Our results show that NE and DA uptake is mediated via high-affinity and low-capacity serotonin (SERT) and norepinephrine (NET) transporters in...
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