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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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Optimization of purification of human membrane-bound carbonyl reductase.
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Bílková, Zuzana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Rudolf Andrýs Supervisor: prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Optimalization of purification of a human membrane-bound carbonyl reductase Carbonyl reductases are enzymes participating in metabolic pathways of various eobiotics and xenobiotics. Of all known enzymes metabolizing xenobiotics only 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is found in the microsomal membrane. It also contributes to metabolism of prochiral anticancer drug oracin, which main metabolic pathway is a carbonyl reduction on the position 11 leading to two enantiomers of (+) an (-) 11-dihydrooracin (DHO). Based on the discrepancy between microsomes and 11β-HSD1 stereospecifity of oracin reduction exist a hypothesis of participation an unknown microsomal enzyme in oracin metabolism. The aim of this study is to purify a new microsomal carbonyl reducing enzyme contributing in the biotransformation of oracin. Human liver microsomes were solubilised and desalted. The prepared sample was used for the first purification step on Q-Sepharose. Captured flow through fraction Q2 was loaded on Phenyl-sepharose. Captured suitable fraction P11 was used for third purification step by gel filtration. All...
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Nephrotoxicity testing in vitro
Handl, Jiří ; Roušar, Tomáš (advisor) ; Patočka, Jiří (referee) ; Andrýs, Rudolf (referee)
2020 UNIVERSITY OF PARDUBICE FACULTY OF CHEMICAL TECHNOLOGY Doctoral Thesis: NEPHROTOXICITY TESTING IN VITRO Author: Mgr. Jiří Handl, Ph.D. Supervisor: doc. RNDr. Tomáš Roušar, Ph.D. Abstract The doctoral thesis deals with in vitro testing of nephrotoxic effects of substances in immortalized human proximal tubular cells HK-2. The basic aim of the thesis was to introduce and optimize the cultivation of HK-2 cells under different culture conditions, to characterize the cultivation and growth stability of HK-2 cells and last but not least to characterize the nephrotoxic effect of cadmium in HK-2 cell line using bioanalytical and biochemical methods. The introduction summarizes the basic rules and conditions for long-term cultivation of cell lines. Furthermore, HK-2 cells and their importance in nephrotoxicity testing are characterized in detail. The thesis also mentions methods for monitoring toxic effects of substances with focus on measuring cell viability and oxidative stress. The outcomes of the thesis are characterizing nephrotoxic effects of CdCl2 in HK-2 cells. The results part describes obtained findings and the discussions follows. Keywords Nephrotoxicity; In vitro cultivation; HK-2 cell line; Cell viability; Cadmium.
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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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Optimization of purification of human membrane-bound carbonyl reductase.
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Bílková, Zuzana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Rudolf Andrýs Supervisor: prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Optimalization of purification of a human membrane-bound carbonyl reductase Carbonyl reductases are enzymes participating in metabolic pathways of various eobiotics and xenobiotics. Of all known enzymes metabolizing xenobiotics only 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is found in the microsomal membrane. It also contributes to metabolism of prochiral anticancer drug oracin, which main metabolic pathway is a carbonyl reduction on the position 11 leading to two enantiomers of (+) an (-) 11-dihydrooracin (DHO). Based on the discrepancy between microsomes and 11β-HSD1 stereospecifity of oracin reduction exist a hypothesis of participation an unknown microsomal enzyme in oracin metabolism. The aim of this study is to purify a new microsomal carbonyl reducing enzyme contributing in the biotransformation of oracin. Human liver microsomes were solubilised and desalted. The prepared sample was used for the first purification step on Q-Sepharose. Captured flow through fraction Q2 was loaded on Phenyl-sepharose. Captured suitable fraction P11 was used for third purification step by gel filtration. All...
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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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