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A role of microRNAs in the regulation of hepatic drug-metabolizing enzymes
Sedlmajer, Štěpán ; Smutný, Tomáš (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Štěpán Sedlmajer Supervisor: PharmDr. Tomáš Smutný, Ph.D. Title of diploma thesis: A role of microRNAs in the regulation of hepatic drug-metabolizing enzymes The liver is the main biotransformation organ in the body. Cytochromes P450 and conjugation enzymes are essential enzymes in the metabolism of drugs and other xenobiotics. Recently, there is growing evidence of post-transcriptional regulation of these enzymes through microRNA molecules. MicroRNAs are epigenetic regulators of expression, which usually bind to fully or partially complementary sequences within the 3'UTR (3' untranslated) regions of mRNA molecules. MicroRNAs have a role in interindividual variability of expression and activity of metabolic enzymes. Understanding their role and mechanisms of action may contribute to the development of new drugs and tailored pharmacotherapy. The work summarizes current selected achievements in the research on regulation of mainly hepatic cytochromes P450 and conjugation enzymes through microRNAs. The impact of RNA editing, single nucleotide polymorphisms and pseudogenes is also described.
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Development of novel Constitutive androstane receptor (CAR) ligands
Dušek, Jan ; Pávek, Petr (advisor) ; Hudeček, Jiří (referee) ; Chládek, Jaroslav (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Jan Dušek Supervisor Prof. PharmDr. Petr Pávek, Ph.D. Title of Doctoral Thesis Development of novel Constitutive androstane receptor (CAR) ligands Constitutive androstane receptor is nowadays known as the established nuclear receptor that regulates the expression of several key cytochrome P450 enzymes, predominantly CYP3A4 and CYP2B6. Recently it has been shown that CAR has also essential role in the regulation of endogenous metabolism of glucose, lipids, cholesterole or bile acids. Simultaneously, this receptor is considered to have proliferative effect on human hepatocytes and protective effects against toxic and dietary damage of liver parenchyme. Given the possible therapeutic utilization of CAR, its therapeutic options are being intensively studied. Unfortunately, currently known ligands of human or mouse CAR are either poorly selective or indirect. The aim of my doctoral thesis was to find new ligands of mouse and human CAR, that would enable more detailed study of the receptor.
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Bioinformatic aspects of nuclear receptors in pharmacotherapy
Škoda, Josef ; Pávek, Petr (advisor) ; Kollár, Peter (referee) ; Matoušková, Petra (referee)
IN ENGLISH LANGUAGE Candidate: Mgr. Josef Škoda Supervisor: Prof. PharmDr. Petr Pávek, PhD. Title of the doctoral thesis: Bioinformatical aspects of nuclear receptors in pharmacotherapy The liver plays a central role in energy homeostasis via processing post-prandial excess energy into storage molecules and reusing stored energy via gluconeogenesis during the fasting period. This tight energy balance is maintained by a myriad of regulating processes. Dysfunction of metabolic control is a key event of severe diseases in today's population, starting with obesity, insulin resistance, and metabolic syndrome, proceeding to end-stage complications in type 2 diabetes, whole organ malfunctions, or even tumor diseases. Master players in metabolic regulation are nuclear receptors (NR) activated by endogenous stimuli or scavenging for nutritional or toxicological signals. NRs regulate gene transcription activation and therefore maintain liver metabolic plasticity. In this thesis, modern molecular biology approaches were used to study ligands of NRs and the effects of their treatment. After the typical activation of a NR, hundreds of genes are regulated, which is beyond the ability to study with conventional biology methods. For this purpose, omics methods are an ideal solution. They are characterized by...
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Interactions of resveratrol derivatives with PXR receptor
Marešová, Veronika ; Pávek, Petr (advisor) ; Trejtnar, František (referee)
Charles University in Prague Department of Pharmacology & Toxicology Student: Supervisor: Prof Title of diploma thesis: Interactions of resveratrol derivatives with PXR receptor Derivatives of resveratrol belong between natural substances contained mainly in red wine and grapefruit. These compounds with stilbene-like structure show antioxidant activity. Pregnane X receptor (PXR) regulates expression of the most important biotransformation enzymes in human liver and intestine including cytochrome P450 3A4 (CYP3A4). Interaction of resveratrol and PXR have already been studied, but published data are contradictory. In my diploma thesis, I studied interaction of resveratrol and its derivatives with PXR. For this purpose, gene reporter assay with quantitative real-time polymerase chain reaction (qRT-PCR) was introduced. It was not possible to conduct experiments in standard way because both resveratrol and its derivatives inhibit lusiferase activities. I studied also effects of resveratrol and its derivatives on expression of CYP3A4 mRNA on differentiated HepaRG cell line. New method gene reporter method with qRT-PCR detection of luciferase was established on hepatoma cell line HepG2 transfected with CYP3A4 reporter construct. Concertation dependent effects of resveratrol and its derivatives were...
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Molecular mechanisms of interactions- interactions of constitutive androstane receptor with selected stilbene compounds
Linhartová, Lenka ; Pávek, Petr (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...
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Pharmacological interventions of nuclear receptors in diabettes mellitus
Draský, Jakub ; Pávek, Petr (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Draský Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Pharmacological influence of nuclear receptors in diabetes mellitus therapy Nuclear receptors belong to the superfamily of transcription factors, their main functions include regulating the expression of target genes. In my work I focused mainly on the group of orphan receptors, namely the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). A common feature of these receptors is their activation by a specific ligand. Both CAR and PXR have an essential function as biological sensors of hydrophobic xenobiotics when they induce enzymes I and II. phase of metabolism. They are also essential in the regulation of gluconeogenesis, insulin response, adipogenesis, cholesterol homeostasis, fatty acids, triglycerides and glycogen. The aim of this experimental work was to introduce a luciferase reporter assay method for two DNA constructs containing the promoter region of the PEPCK and CYP7A1 genes. We used the known agonist rifampicin and the antagonist SPA70 to activate/deactivate PXR. We used CITCO as a CAR receptor agonist. We first verified the functionality of the luciferase reporter gene assay...
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Mechanisms of potential toxic effects of bisphenol A on the endocrine system
Zachardová, Lenka ; Novotný, Jiří (advisor) ; Rupert, Marian (referee)
Bisphenol A (BPA) has already been used in the chemical industry for several decades. Since that time, a huge amount of in vitro and in vivo studies have been accumulated summarizing the molecular mechanisms of BPA causing disruption in endocrine homeostasis in all kinds of biological models. It enters the body from external environment; however, the main source of penetration is through consumption of food and liquids that are in contact with materials containing the mentioned xenoestrogen. The hydroxyl group within BPA phenol ring enables binding to transmembrane G- protein-coupled estrogen receptors and a number of nuclear receptors affecting hormonal system and resulting in alterations in signalling pathways. BPA induces oxidative stress and interferes with regulation factors functions at gene, cell and tissue level. Disruptive effects are associated with developmental, reproductive, cardiovascular, immune and neurobiological disorders. This thesis is aiming to sum up existing findings about the molecular mechanisms of BPA toxicity in the endocrine system mainly using Mus musculus model. Keywords: BPA, endocrine system, nuclear receptors, GPER, oxidative stress.
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Inhibitory effect of SPA70 on hPXR activation
Dohnalová, Klára ; Pávek, Petr (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Klára Dohnalová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Inhibitory effect of SPA70 on hPXR activation This work focuses on pregnane X receptor (PXR) and its antagonists. PXR is a ligand-activated nuclear receptor that plays a major role in detoxification of xenobiotics and protecting the organism from their toxic effects. Recent evidence also shows endogenous action of PXR in the metabolism of lipids, glucose and bile acids. However, PXR activation could be harmful, since induction of biotransformation enzymes by PXR agonists may result in reduced treatment efficacy, increased toxicity of drug metabolites and resistance to chemotherapeutic agents. Recent research has been intensively focused on PXR antagonists capable of abolishing these unfavourable effects. Recently discovered human PXR antagonist SPA70 has a promising potential for future usage. In this study, we investigated the inhibitory effect of SPA70 on activated PXR. To activate PXR we used agonists binding directly to PXR (rifampicin, hyperforin, SR12813) and also agonists activating PXR indirectly via cell signalling pathways (U0126, PD184352, PD0325901). Experiments were performed using luciferase...
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Interactions of resveratrol derivatives with PXR receptor
Marešová, Veronika ; Pávek, Petr (advisor) ; Trejtnar, František (referee)
Charles University in Prague Department of Pharmacology & Toxicology Student: Supervisor: Prof Title of diploma thesis: Interactions of resveratrol derivatives with PXR receptor Derivatives of resveratrol belong between natural substances contained mainly in red wine and grapefruit. These compounds with stilbene-like structure show antioxidant activity. Pregnane X receptor (PXR) regulates expression of the most important biotransformation enzymes in human liver and intestine including cytochrome P450 3A4 (CYP3A4). Interaction of resveratrol and PXR have already been studied, but published data are contradictory. In my diploma thesis, I studied interaction of resveratrol and its derivatives with PXR. For this purpose, gene reporter assay with quantitative real-time polymerase chain reaction (qRT-PCR) was introduced. It was not possible to conduct experiments in standard way because both resveratrol and its derivatives inhibit lusiferase activities. I studied also effects of resveratrol and its derivatives on expression of CYP3A4 mRNA on differentiated HepaRG cell line. New method gene reporter method with qRT-PCR detection of luciferase was established on hepatoma cell line HepG2 transfected with CYP3A4 reporter construct. Concertation dependent effects of resveratrol and its derivatives were...
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Molecular mechanisms of interactions- interactions of constitutive androstane receptor with selected stilbene compounds
Linhartová, Lenka ; Pávek, Petr (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...
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