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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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Purification and characterization of selected human microsomal reductase
Skarka, Adam ; Wsól, Vladimír (advisor) ; Živná, Lucie (referee)
Area of human liver microsomal reductases hasn't been fully explored yet. Purification and characterization of still undescribed enzymes is an important step in finishing of this task. There is separation and purification of this reductases described in my work, with usage of hydrophobic interaction chromatography on low pressure chromatography instrument ÄKTA purifier. There were two columns with different substrates used, Phenyl sepharose (low sub) and Octyl sepharose. Separation conditions were the same in both cases. Results obtained by incubation of fractions and measurement on HPLC showed significant differences between both columns and their substrates. But it isn't possible to determine, which column is better for purification, because big area still remain for adjustment and optimization of purification conditions.
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Characterization of human warfarin reductase
Sokolová, Simona ; Malátková, Petra (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. However, the reductive metabolism of warfarin has not been studied in details until now. The reduced metabolite of warfarin, i.e. warfarin-alcohol, is produced by the conversion of the carbonyl group of the side chain. It is known that human liver cytosolic and microsomal fractions exhibit warfarin reductase activity but the specific enzymes catalysing the reduction of warfarin are not known yet. The aim of this study was to identify the enzyme(s) participating in reduction of warfarin and to describe enzyme kinetics. Human liver cytosolic and microsomal fractions and recombinant enzymes AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, CBR1 and CBR3 were incubated with warfarin at various concentrations. The produced warfarin-alcohol was quantified by UHPLC and the specific activities of enzymes and...
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Characterization of human warfarin reductase
Sokolová, Simona ; Malátková, Petra (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. However, the reductive metabolism of warfarin has not been studied in details until now. The reduced metabolite of warfarin, i.e. warfarin-alcohol, is produced by the conversion of the carbonyl group of the side chain. It is known that human liver cytosolic and microsomal fractions exhibit warfarin reductase activity but the specific enzymes catalysing the reduction of warfarin are not known yet. The aim of this study was to identify the enzyme(s) participating in reduction of warfarin and to describe enzyme kinetics. Human liver cytosolic and microsomal fractions and recombinant enzymes AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, CBR1 and CBR3 were incubated with warfarin at various concentrations. The produced warfarin-alcohol was quantified by UHPLC and the specific activities of enzymes and...
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Validation of the method for amiodaron and its metabolite determination by HPLC
Prudková, Marie ; Dršata, Jaroslav (advisor) ; Skarka, Adam (referee)
This thesis introduces a method of quantitative determination of amiodarone and its metabolite desethylamiodarone by high performance liquid chromatography with UV detection using internal standard dronedarone and verification of its use in the analysis of patient samples. The theoretical part describes method of liquid chromatography, amiodarone substance and possibilities of its determination using selected methods. The experimental part of the thesis describes procedures for analysis of amiodarone and desethylamiodarone utilizing high performance liquid chromatography and also includes validation of such method, including the following parameters: LOD, LOQ, linearity, working range, precision and recovery. In the conclusion part, the thesis compares two methods of result evaluation, which were gained from a sample of 41 patients. Reliability of such analytical procedure was verified through validation parameters.
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Biotransformation of selected anthelmintics in Haemonchus contortus
Sečkařová, Anna ; Raisová Stuchlíková, Lucie (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Anna Sečkařová Supervisor: RNDr. Lucie Raisová Stuchlíková, Ph.D. Title of diploma thesis: Biotransformation of selected anthelmintics in Haemonchus contortus Resistance to benzimidazole anthelmintics is one of the main problems of small ruminant producers. The most problematic parasite of goats and sheep is the bloodsucking barber's pole worm, Haemonchus contortus. This parasite is responsible for considerable losses every year. The goal of this thesis was an analysis of the metabolism of a few selected benzimidazoles (albendazole, ABZ; ricobendazole, RCB and flubendazole, FLU) in H. contortus. The metabolism of male and female worms and a susceptible ISE strain (Inbred Susceptible Edinburgh, MHco3) with a resistant IRE strain (Inbred Resistant Edinburgh, MHco5) were compared. For metabolite detection, ultra-high-performance liquid chromatography with mass spectrometry (UHPLC/MS) was used. 12 metabolites of FLU, 9 metabolites of ABZ and 7 metabolites of RCB were identified. From the identified metabolites, schemes of metabolism were proposed. In metabolism comparisons, significant differences between sexes and strains were found. Female worms metabolized tested anthelmintics more...
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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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Characterization of human warfarin reductase
Sokolová, Simona ; Malátková, Petra (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. However, the reductive metabolism of warfarin has not been studied in details until now. The reduced metabolite of warfarin, i.e. warfarin-alcohol, is produced by the conversion of the carbonyl group of the side chain. It is known that human liver cytosolic and microsomal fractions exhibit warfarin reductase activity but the specific enzymes catalysing the reduction of warfarin are not known yet. The aim of this study was to identify the enzyme(s) participating in reduction of warfarin and to describe enzyme kinetics. Human liver cytosolic and microsomal fractions and recombinant enzymes AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, CBR1 and CBR3 were incubated with warfarin at various concentrations. The produced warfarin-alcohol was quantified by UHPLC and the specific activities of enzymes and...
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Cell-free expression of a voltage-gated sodium channel for future 2D IR spectroscopic studies
Kovácsová, Gabriela ; Skálová, Lenka (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Gabriela Kovácsová Supervisor: Dr. Sebastian Peuker, Prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Cell-free expression of a voltage-gated sodium channel for future 2D IR spectroscopic studies Voltage-gated sodium channels (Navs) are membrane proteins from the superfamily of voltage-gated ion channels (VGIC), and are present in every excitable cell where they participate in the propagation of action potentials by changing the Na+ permeability of the cell membrane. Eukaryotic Navs are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1-S6), where S1 to S4 form the voltage-sensing domain and S5 and S6 create the pore domain with the selectivity filter on the extracellular site. Whereas the ion selectivity of the voltage-gated potassium channels has been elucidated on the molecular level in great detail, little is known about this for the voltage-gated sodium channels. To allow future studies of the selectivity filter of Nav by the means of 2D IR spectroscopy, a technique able to provide bond-specific structural information on the picosecond to millisecond time scales, large quantities of the purified channel are needed. Eukaryotic Navs...
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