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basic characterization of human enzymes DHRS7B and DHRS7C
Tučková, Tereza ; Zemanová, Lucie (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Tučková Supervisor: RNDr. Lucie Zemanová, Ph.D. Title: Basic characterization of human enzymes DHRS7B and DHRS7C Human enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily play an important role in wide range of biochemical pathways. They are involved in metabolism of lipids, saccharides, amino acids, steroid hormones, retinoids, prostaglandins etc. Besides physiological processes, they take part in development of several serious diseases, e.g. hormone-dependent cancer, metabolic syndrome, diabetes mellitus. Moreover, SDR enzymes contribute to biotransformation of xenobiotics. Nevertheless, approximately 30 % of SDRs remain completely uncharacterized. Human dehydrogenase/reductase SDR family members 7B (DHRS7B) and 7C (DHRS7C) belong to poorly characterized members of the SDR superfamily. According to in silico predictions, both enzymes are membrane bound and involved in reductive reactions. The aim of this study was to determine their basic biochemical properties. The results show that both enzymes interact with the membrane of endoplasmic reticulum and face cytosol. The pilot screening of enzymatic activity was performed. Reducing activity was detected towards e.g....
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The group of DHRS enzymes and their role in human organism
Zamazalová, Alžběta ; Zemanová, Lucie (advisor) ; Štambergová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Alžběta Zamazalová Supervisor: RNDr. Lucie Zemanová, Ph.D. Title of diploma thesis: The group of DHRS enzymes and their role in human organism Human enzymes from dehydrogenase/reductase SDR family (DHRS) include 17 members that belong to the large short-chain dehydrogenase/reductase superfamily. The short-chain dehydrogenase/reductase superfamily contains 80 human members, some of them are important and well described enzymes such as 11β-hydroxysteroid dehydrogenase, carbonyl reductase 1 and 17β-hydroxysteroid dehydrogenase. However there are many poorly described or undescribed members of this superfamily, including group DHRS. There is just a little information about proteins DHRS1, DHRS12, DHRS13, DHRS4L1, DHRS4L2. Proteins DHRS2, DHRS4, DHRS6, DHRS7, DHRS7B, DHRS7C, DHRS8, DHRS9, DHRS10, DHRS11 and DHRSX are better described because at least subcellular localization and substrate (except DHRSX) is known, and therefore an initial opinion on their possible function can be formed. The DHRS3 is the only one enzyme of group DHRS that has been tested in vivo. DHRS3 could play a significant role in fetal development. For DHRS3, but also for the other DHRS enzymes, first knowledge of...
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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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Co-immunoprecipitation as a tool for the study of protein-protein interactions of DHRS7 enzyme
Fassmannová, Dominika ; Zemanová, Lucie (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Dominika Fassmannová Supervisor: RNDr. Lucie Zemanová, Ph.D. Title of diploma thesis: Co-immunoprecipitace as a tool for the study of protein-protein interactions of DHRS7 enzyme DHRS7 enzyme is a member of short-chain dehydrogenase/reductase superfamily. The enzyme is at least in vitro NADPH-dependent reductase of some substance bearing carbonyl group, including androstenedione or all-trans retinal. DHRS7 is close homolog of well-known 11β-hydroxysteroid dehydrogenase 1. Physiological function of DHRS7 is unknown, the level of the knowledge is quite low. Recently, information about some role of DHRS7 in diseases as prostate cancer or insulin resistance was published. A role of poorly characterized proteins as DHRS7 can be predicted based on evidence of their interaction with a protein with revealed function, because proteins inside cells do not usually work alone, they are part of a huge protein complex known as interactome. Knowledge of such protein-protein interactions help us to understand the function and regulation of the protein inside a cell or organism. The aim of the study is initial investigation of proteins interacting with DHRS7 by co-immunoprecipitation, a basic method for...
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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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