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Introduction of an afinity separation to purification protocol for human membrane carbonyl-reducing enzymes
Štýbnarová, Radka ; Zemanová, Lucie (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Radka Štýbnarová Supervisor: RNDr. Lucie Zemanová, Ph.D. Title of diploma thesis: Implementation of affinity separation step into the purification protocol of human membrane-bound carbonyl-reducing enzymes Carbonyl reducing enzymes play important role in the metabolism of various eobiotic (e.g. steroids, prostaglandins) and xenobiotic (e.g. doxorubicin, daunorubicin, oracin, NNK, haloperidol) compounds. Due to their substrate specificity, they also play a role in development of some diseases like hormone-dependent cancers and metabolic syndrome. While cytosolic carbonyl reducing enzymes are well characterised the knowledge about membrane-bound types is quite poor because their study is demanding. Actually, until today there are only three described microsomal carbonyl reducing enzymes participating in the metabolism of xenobiotic compounds (11β-HSD1, DHRS7 and DHRS3) of which only the 11β-HSD1 is well characterized. However, based on the research of anticancer drug oracin reduction stereospecificity, there were predicted other microsomal carbonyl reducing enzymes involved in its metabolism and inactivation. In order to isolate these "unknown" enzymes the in-house developed affinity...
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Effect of monepantel on expression of biotransformation enzymes in sheep
Štolcová, Tereza ; Skálová, Lenka (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Štolcová Supervisor:prof. RNDr. Lenka Skálová Ph.D. Title of diploma thesis: Effect of monepantel on expression of biotransformation enzymes in sheep Monepantel (MOP) represents a new anthelmintic drug from the amino-acetonitrile derivatives group with the different mode of action in comparison with commonly used broad-spectrum drugs. It is used for the treatment and control of gastrointestinal nematode infection in sheep and cattle. This study focuses on the effect of MOP on expression of selected cytochromes P450 (CYP) in sheep in vivo and in vitro. The sheep treated with the recommended therapeutic dose of MOP was used for the in vivo experiment. Non-treated animals represented the control group. Changes of mRNA expression of selected CYPs in primary cultures of ovine hepatocytes were observed after 24 h incubation with MOP (10 μM) in the in vitro experiment. The qPCR method (real time quantitative PCR) was used for the quantification of transcription levels of mRNA. Both in vivo as well as in vitro results showed a significant up-regulation in CYP3A24 expression. The induction effect of MOP was even stronger than induction effect of well-known CYP3A inducer rifampicin....
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Function and determination of sulfotransferase in the skin
Hekrlová, Jana ; Zemanová, Lucie (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jana Hekrlová Supervisor: Mgr. Martina Moravcová, Ph.D. RNDr. Lucie Zemanová, Ph.D. Title of diploma thesis: Function and determination of sulfotransferase in the skin Sulfotransferases (SULTs) are enzymes that are expressed in small intestine, muscles, prostate and also in smaller amounts in placenta, trachea, lungs and skin, where they participate on sulfonation of steroid hormones, neurotransmiters as well as other endogenous molecules. SULTs are important for biotransformation processes of many xenobiotics. Cholesterol sulfotransferase (SULT2B1b) is one of the significant transferases in the skin, whose function is to catalyze transfer of sulfo group to cholesterol molecule. SULT2B1b plays an important role in skin barrier formation and also in skin aging. Cholesterol sulphate (CHS) is a product of sulfonation of cholesterol by SULT2B1b in epidermis. CHS belongs to epidermal lipids. CHS participates on forming of skin barrier and on desquamation. The aim of study was to find an optimal method and conditions for measuring the activity of SULT2B1b. It was necessary to find a method for determination of enzyme activity in also acellular and cellular systems. Two types of reaction from...
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A competitivefighting on the railwey Prague-Ostrava in the czech daily newspapers
Zemanová, Lucie ; Jirků, Jan (advisor) ; Vysloužilová, Kristýna (referee)
The thesis titled A Competitive Fighting on the Railway Prague-Ostrava in the Czech Daily Newspapers deals with the way the media informed about what was happening on the railway during the onset of the third carrier on this route in the period from 1. 10. 2012 to 1. 2. 2013. The author draws on theories about the objectivity and impartiality of the media, which assumes that the media will be informed about what is happening on the railways from different perspectives and give equal space to comment that to all carriers. In the introduction of the thesis the essential concepts of the subject, such as competition and competitiveness, were defined as well as the theoretical concepts of objectivity and impartiality of the press, which the research was based on. The profiles of competitive transport Czech Railways, STUDENT AGENCY and LEO Express are parts of the work as well as the significant events on the railways in the period examined. Research methodology, which the research techniques have been identified in and the analysed material have been specified, belongs to the important theoretical parts of the thesis. The thesis is based on the quantitative analysis of sixty-one relevant articles dedicated to rail between 1. 10. 2012 and 1. 2. 2013. Contributions by MF DNES, Lidové noviny and...
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Optimization of human carbonyl reductase overexpression in Escherichia coli
Ježková, Veronika ; Novotná, Eva (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Veronika Ježková Supervisor: RNDr. Eva Novotná, PhD. Title: The optimization of expression of the selected human carbonyl reductases in Escherichia coli The present thesis called The optimization of expression of the selected human carbonyl reductases in Escherichia coli is divided into two parts. The first one is theoretical and the second one is experimental. The theoretical part presents a brief information summary about selected enzymes: carbonyl reductases 1 (CBR1), quinone oxidoreductase 1 (NQO1) and DHRS2 (Hep27). It also includes a theoretical summary of laboratory methods which were used in the experimental part. The experimental part aims to optimize the express conditions of the selected enzymes in the bacterial expression system Lemo21(DE3). The plasmid pET-28b(+), which encodes the selected enzymes, is transformed by a thermal shock into competent bacterial cells Lemo21(DE3). In these cells a protein expression was carried out at different concentrations of L-rhamnose. We also purified the expressed recombinant protein NQO1 by the affinity chromatography method. The success rate of protein expression was examined by SDS-PAGE or Western Blot. Based on the obtained results...
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Reference gene selection for mRNA quantification in Heamonchus contortus
Růžičková, Michaela ; Matoušková, Petra (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Michaela Růžičková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Reference gene selection for mRNA quantification in Haemonchus contortus Haemonchus contortus is a parasitic nematode, whose multiresistance to ant- helmintics means global problem, threatening mostly small ruminants farming. Genom and transcriptom have been published in 2013, allowing gene expression studies to be conducted. Reference genes for this studies have not been validated yet. Use of suitable reference genes is essential for accurate normalization of gene expression levels. Aim of this work was to identify and validate potential reference genes for gene expression studies in H. contortus adults. Eleven genes were chosen, stability of their expression was assessed in males and females of two H. contortus strains, one drug-susceptible (ISE) and one multi-drug-resistant (WR). Total RNA was extracted and reverse trancribed to cDNA. cDNA was diluted and analyzed using quantitative real-time PCR with SYBR Green I detection. Expression stability was evaluated by computer programs BestKeeper, geNorm, NormFinder and the comparative CT method. Ncbp, ama, sodc, gapdh and farb were found to be most stable...
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Detection of oxidative DNA damage by comet assay
Kúdelová, Terézia ; Szotáková, Barbora (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Terézia Kúdelová Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Consultant: Prof. RNDr. Rudolf Štětina, CSc. Title of diploma thesis: Detection of the oxidative damage of DNA by the Comet Assay The reparative mechanisms have a very important role in organisms. Imbalance develops due to disruption of these mechanisms, which can lead to induction of many disorders. The aim of this diploma thesis was to study protection of DNA against oxidative damage. The ability of A549 cell line to cope with this damage was studied in vitro. Oxidative damage was induced by hydrogen peroxide (H2O2), which initiated induction of single strand breaks (SSBs) and at the same time oxidation of pyrimidine and purine bases. Mechanism of damage and reparation was detected by two methods. In the first case, cells were treated with antioxidant and then influenced by H2O2. In the second case, cells were influenced by H2O2 and then let to repair in the presence of antioxidants. Tested antioxidants were epigallocatechin gallate (EGCG), glutathione (GSH) and chemical modifications of GSH - S-allyldithioglutathione (GSH-A) and S-allyltrithioglutahione (GSH-B). The extent of damage was assessed using by alkaline version...
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Immunochemical determination of DHRS8 and DHRS12 enzymes in human tissues
Svobodová, Markéta ; Novotná, Eva (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Markéta Svobodová Supervisor: RNDr. Eva Novotná, Ph.D Title of diploma thesis: Expression of DHRS8 and DHRS12 enzymes in human tissues Dehydrogenase/reductase (SDR family) member 8 (DHRS8, SDR16C2) and dehydrogenase/reductase (SDR family) member 12 (DHRS12, SDR40C1) are human microsomal enzymes belonging to the superfamily of short-chain dehydrogenases/reductases (SDR). This superfamily represents one of the largest protein groups. SDR enzymes participate in the metabolism of various xenobiotic and endogenous compounds and are involved in physiological and pathological processes. However, there are still many enzymes which are only poorly characterised. To this date, the expression on mRNA level and catalytic activity toward 5α- androstane-3α,17β-diol are the only available information about DHRS8. Moreover, there is still no published information (apart from the prediction) regarding DHRS12. The aim of this study was to examine the protein expression of DHRS8 and DHRS12 enzymes in various human tissues. The tissue samples were collected from four middle aged male subjects after the sudden death without apparent disease. Proteins of interest were detected using western blotting and...
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