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New aspects of pregnane X receptor function and regulation
Hyršová, Lucie ; Trejtnar, František (advisor) ; Skálová, Lenka (referee) ; Vondráček, Jan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Hyršová, MSc. First Supervisor: doc. PharmDr. František Trejtnar, CSc. Consultant: prof. PharmDr. Petr Pávek, PhD. Title of Dissertation Thesis: New aspects of pregnane X receptor function and regulation The pregnane X receptor belongs to the superfamily of nuclear receptors; it is a ligand dependent transcription factor regulating expression of its target genes. During last two decades, PXR was extensively studied as a xenosensor, i.e. the receptor, which is able to bind xenobiotics including many drugs and to regulate their metabolism by induction of the most important metabolizing enzymes of both phase I. and phase II. Induction, i.e. transcriptional stimulation of expression of the most important cytochrome P450 enzymes, by PXR ligands was described in details at many levels. Within this dissertation thesis, I am dealing with aspects of regulation via PXR, which extend the common understanding of PXR as a receptor whose exclusive function is to up-regulate drug metabolizing enzymes mediated by its agonists. Within the first, project I studied regulation of OCT1 transporter in hepatic cell models, I shown that PXR did not induce, but it rather suppressed the expression of this...
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Determination of plasma protein binding of new radiopharmaceuticals for Alzheimer's disease imaging
Hyršová, Lucie ; Lázníček, Milan (advisor) ; Melicharová, Ludmila (referee)
CHARLES UNIVERSITY IN PRAGUE Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lucie Hyršová Supervisor: Prof. PharmDr. Ing. Milan Lázníček CSc. Title of thesis: Determination of plasma protein binding of new radiopharmaceuticals for Alzheimer`s disease imaging Summary: Determination of plasma protein binding of new potential drugs belongs to basic parameters which are made at early stages of clinical trials. The drug-protein binding affects all the pharmacokinetic factors and it influences pharmacodynamics as well. The aim of this thesis is to examine binding of 99m Tc-5C-2-BOC and 99m Tc-5C-5-BOC which are potential radiodiagnostics of Alzheimer`s disease, is investigated in this work. Blood plasma of four species (namely human, rabbit, bovine and sewer rat`s plasma) is tested and findings are mutually compared. The binding in both cases is determined by means of two methods - equilibrium dialysis and ultrafiltration. The binding of both substances is the lowest in human and bovine plasma with the use of equilibrium dialysis. The binding of 99m Tc-5C-2-BOC determined by ultrafiltration is the lowest in bovine plasma and a slightly higher in human one, the binding of 99m Tc-5C-5-BOC is the lowest in human plasma. These findings show that binding on plasma proteins...
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In vitro testing of cytotoxicity and hematotoxicity of drugs in development
Ďurinová, Anna ; Hyršová, Lucie (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anna Ďurinová Supervisor: PharmDr. Lucie Hyršová, PhD. Title of diploma thesis: In vitro testing of cytotoxicity and hematotoxicity of drugs in development Preclinical drug evaluation is an important stage in development of new drug, it describes testing substances before they may be carried out in humans. This work is focused on the experimental measurement of cytotoxicity and hematotoxicity, which are parts of preclinical research. In vitro cytotoxicity tests are one of the first methods used for investigating novel drugs. Viability was monitored in the HepG2 model cell line. The viability evaluation was performed by obtaining parameter IC50, the concentration of substance at which half of the cultured cells lose their viability. We studied the stability of rat red blood cells after administration of the tested substances, so we observed in vitro hemolysis, which is a good indicator of substances effect and safety. The EC50 value is monitored to determine the hemolytic effect. The EC50 concentration causes a hemolytic effect on 50% of tested erythrocytes. The no observed effective concentration parameter was also observed, when the 10% limit of positive control's hemolytic activity was not...
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Recent knowledge on drug effect on male fertility
Klapková, Tereza ; Trejtnar, František (advisor) ; Hyršová, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Tereza Klapková Supervisor: Assoc. Prof. PharmDr. František Trejtnar, CSc. Title of diploma thesis: Recent knowledge on drug effect on male fertility Among the various types of side effects presented in clinically used drugs, negative effects on male reproductive functions can be find. This issue seems to be important and current especially due to the general trend of the decrease in fertility in men and the increasing drug use in younger age groups. The aim of this diploma thesis was to create an overview summarizing current expert knowledge on the effect of drugs on male fertility. For this purpose, we selected relevant publications in the PubMed database, perform their analysis and create the text ofthe thesis. The review focuses mainly on groups of drugs that are often clinically used and discussed in relation to male fertility, such as drugs acting on the cardiovascular system, antimicrobial drugs, drugs used in pain therapy, antidepressants, antiepileptics, antipsychotics, immunosuppressants and some other drugs. In addition to standard drugs, the review also includes several important natural substances, which are used as adjunctive therapy of various diseases or are important from a...
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Determination of organ toxicity of BRAF inhibitors in vitro.
Miškovčíková, Zuzana ; Maixnerová, Jana (advisor) ; Hyršová, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Miškovčíková Supervisor: RNDr. Jana Maixnerová, PhD. Title of diploma thesis: Determination of organ toxicity of BRAF inhibitors in vitro. Malignant melanoma is one of the most serious skin diseases today. Therapy of advanced melanoma is difficult and often ineffective. BRAF inhibitors (dabrafenib and vemurafenib) have dramatically changed the results of melanoma treatment in the last few years. BRAF inhibitors are one of the most effective drugs against melanoma, but their clinical application is largely limited by drug resistance. Available clinical studies have shown an adverse nephrotoxic effect of BRAF inhibitors, but information on its mechanism is limited. Published studies further suggest that the toxic effect of BRAF inhibitors is primarily directed to podocytes located in the glomerular membrane. Thus, the aim of our study was to assess the cytotoxic effect of BRAF inhibitors on selected model renal cells in vitro in order to confirm the renal target toxicity. The main objective of the study was to analyse whether the nephrotoxic effect of BRAF inhibitors is specifically limited to podocytes or whether it can damage other renal cells. The experiments were performed on human cell lines...
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Study of cytotoxicity of newly prepared bronchodilators in vitro
Havelková, Michaela ; Hyršová, Lucie (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Michaela Havelková Supervisor: PharmDr. Lucie Hyršová, PhD. Title of diploma thesis: Study of cytotoxicity of newly prepared bronchodilators in vitro This diploma thesis is focused on the determination the influence of developmental bronchodilatory active substances on viability of model tumor cell lines of human liver cells HepG2 and human lung cells HCC827. These potential drugs are theoretically suitable for the treatment of asthma bronchiale and COPD. The tested compounds (VN014a, VN015a, VN045b, VN122c) were derived from structure of the quinazoline alkaloids vasicine and vasicinone, which are substances contained in the Justicia adhatoda plant. The plant originated in India is widely used in traditional Ayurvedic medicine besides others to treat respiratory problems. The combination of both alkaloids showed significant bronchodilation activity. Cytotoxic activity of compounds was tested using colorimetric method by measuring cell metabolic activity based on reduction of tetrazolium compound. The IC50 value was determined for each studied compound on both used cell lines to assess the influence on cell viability. Overall, the tested developmental bronchodilators proved cytotoxic effect...
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Study of transport mediated by drug transporters in model cell systems
Znamínková, Jana ; Hyršová, Lucie (advisor) ; Trejtnar, František (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Jana Znamínková Supervisor: PharmDr. Lucie Hyršová, PhD. Title of diploma thesis: Study of transport mediated by drug transporters in model cell systems Liver is the main biotransformation organ of human organism. Not every substance can cross the cell membrane of hepatocytes by simple diffusion. Transport of such substances across the membrane is mediated by specialized transport proteins. Uptake transporters localized on the basolateral membrane of hepatocytes play a crucial role in uptake of compounds from blood into hepatocytes, where the biotransformation takes place. OCT1 transporter belongs between important members of this group, it transports organic cations. Efflux transporters localized on the apical membrane are responsible for transport of substances from hepatocytes to bile canalicular and these transporters mediate their elimination in this way. BCRP is an example of this group of transporters. Drug transporters represent one of the most important mechanisms of drug-drug interactions due to their wide range of substrates. The aim of this study was to evaluate the function of novel cell models using substrate and inhibitors of these transporters. Stably transfected MDCK II cells...
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Alternation of bile production due to iron depletion.
Šimková, Marie ; Prašnická, Alena (advisor) ; Hyršová, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Student: Marie Šimková Supervisor: Mgr. Alena Prašnická Title of diploma thesis: Alternation of bile production due to iron depletion Introduction: Liver has an irreplaceable role in the production and secretion of bile. This body fluid serves as the main excretion way of some endogenous and exogenous substances. Another liver property is the ability to store substances essential for correct functions of the body, e.g. iron. It has been shown that iron could have an impact on the bile production and secretion. Aim: The aim of this diploma thesis was to discover an impact of iron depletion on the bile synthesis and metabolism, especially on bile acids, and the way it affects transporters expression. Methods: Male Wistar rats (n=6 in each group, 250 ± 20 g) were divided into two groups: control group (Chow diet) fed with standard diet and iron depletion group (ID), fed with iron depletion diet for 21 days. To investigate the changes in bile flow, the bile had been collected for 120 min during in vivo clearance study. The analysis of the changes in expression of bile transporters and enzymes responsible for de novo bile acid synthesis was performed at the mRNA (qRT-PCR) and protein (Western blot)...
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Alternation of bile production due to iron depletion.
Šimková, Marie ; Prašnická, Alena (advisor) ; Hyršová, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Student: Marie Šimková Supervisor: Mgr. Alena Prašnická Title of diploma thesis: Alternation of bile production due to iron depletion Introduction: Liver has an irreplaceable role in the production and secretion of bile. This body fluid serves as the main excretion way of some endogenous and exogenous substances. Another liver property is the ability to store substances essential for correct functions of the body, e.g. iron. It has been shown that iron could have an impact on the bile production and secretion. Aim: The aim of this diploma thesis was to discover an impact of iron depletion on the bile synthesis and metabolism, especially on bile acids, and the way it affects transporters expression. Methods: Male Wistar rats (n=6 in each group, 250 ± 20 g) were divided into two groups: control group (Chow diet) fed with standard diet and iron depletion group (ID), fed with iron depletion diet for 21 days. To investigate the changes in bile flow, the bile had been collected for 120 min during in vivo clearance study. The analysis of the changes in expression of bile transporters and enzymes responsible for de novo bile acid synthesis was performed at the mRNA (qRT-PCR) and protein (Western blot)...
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Preparation of genetically modified cell line as a model for accumulation studies
Rückelová, Alexandra ; Hyršová, Lucie (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Alexandra Rückelová Supervisor: PharmDr. Lucie Hyršová, Ph.D. Title of diploma thesis: Preparation of genetically modified cell line as a model for accumulation studies The studied megalin transporter belongs to the group of lipoprotein receptors, namely low density lipoprotein receptor-related protein 2 (LRP2). It is a transporter capable of transporting large organic molecules into the cell through the receptor - mediated endocytosis. Huge variety of peptide compounds including albumin, hemoglobin, vitamins, hormones as well as the amoniglycoside antibiotics (e.g. gentamicine), polymixin B, cadmium and other substrates belong among its substrates. This theses is focused on the creation of cell models for the study of the transport of large organic molecules across the cell membranes. I am dealing with reducing megalin expression in two model cell lines using siRNA against LRP2 gene. The inhibition of expression was determined using the RT-PCR method and subsequently verified on the functional level using known megalin substrate gentamicine. It is an aminoglycoside antibiotic with nephrotoxic action. The principle of these validation experiments was to reduce gentamicine toxic effects in cells...
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