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Synthesis of epitestosterone and 4-hydroxyestrone glucuronides using recombinant human UDP-glucuronosyltransferases.
Kašparová, Michaela ; Skálová, Lenka (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Michaela Kašparová Supervisors: Prof. RNDr. Lenka Skálová, Ph.D., Erkka Järvinen, M.Sc. (Pharm.) Title of diploma thesis: Synthesis of epitestosterone and 4-hydroxyestrone glucuronides using recombinant human UDP-glucuronosyltransferases Steroid hormones constitute an important part of the human endocrine system and are involved in a variety of physiological and pathological processes. Phase I and phase II biotransformation reactions convert these lipophilic, biologically active compounds to inactive, water-soluble metabolites that are readily excretable into bile or urine. One of the most common phase II biotransformation reactions is conjugation with glucuronic acid that is enabled by the catalytic activity of UDP-glucuronosyltransferases (UGTs). This study is focused on development and optimization of an enzymatic method for producing conjugates of glucuronic acid and two naturally occurring steroid hormones, epitestosterone and 4-hydroxyestrone. Recombinant human UGT2B7 was employed as a catalyst for the production of epitestosterone 17-glucuronide and 4-hydroxyestrone 4- glucuronide while synthesis of 4-hydroxyestrone 3-glucuronide was accomplished by UGT1A10 isoform. The synthesis reactions...
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Genetic markers for monitoring post-transplant chimerism
Řehounková, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Řehounková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Genetic markers for monitoring post-transplant chimerism The aims of the thesis: Data processing of patients, who underwent allogenic hematopoietic stem cell transplantation in a period from 2010 to 2014 in University Hospital Hradec Kralove and whose state of chimerism was monitored at the Section of Molecular Biology at the Institute of Clinical Biochemistry and Diagnostics. Consequently, analysis of the possible relationship between selected clinical parameters and used genetic markers for chimerism quantification was carried out after the processing of acquired data. Finally, the possible influence of treatment success and mortality by chosen clinical parameters was evaluated. Methods: Analysis of short tandem repeat loci, which uses genetic variability between donor and recipient of transplanted graft, was employed for quantification of post- transplant chimerism. DNA of donor and recipient was isolated by QIAmp DNA Blood Mini Kit (QIAGEN, Germany), amplified by AmpFlSTR Identifier Kit (Applied Biosystems, USA) and separated by capillary electrophoresis (analyzer ABI 3130-4, Applied...
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Modulatory effect of humulene, caryophyllene and caryophyllene oxide on selected biotransformation enzymes in human liver cells
Trnčáková, Veronika ; Boušová, Iva (advisor) ; Matoušková, Petra (referee)
1 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Bc. Veronika Trnčáková Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Title of diploma thesis: Modulatory effect of humulene, caryophyllene and caryophyllene oxide on selected biotransformation enzymes in human liver cells Sesquiterpenes are substances of natural origin, produced mainly by higher plants. They are mainly found in food supplements and natural medicines. Large structural diversity is a feature of these substances. The anti-inflammatory, antiparasitic and anti-cancer effects were also reported. The aim of this diploma thesis was to investigate the effect of selected sesquiterpenes, α-humulene (HUM), β-caryophyllene (KAR) and β-caryophyllene oxide (CAO), on gene and protein expression of selected phase I drug-metabolizing enzymes, which were carbonyl reductase 1 (CBR1), aldo/keto reductase 1C (AKR1) and several cytochrome P450 isoforms, namely CYP3A4, CYP2B6 and CYP2C. The effect of sesquiterpenes on the gene and protein expression of enzymes was studied using precision-cut human liver slices (8 mm diameter, 150-180 µm thickness). Liver samples were received from hepatic surgery of five subjects of both sexes and at 58-69 years of age. Liver slices were incubated with studied compounds (HUM, KAR,...
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HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone
Novák, Filip ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Filip Novák Supervisor: Ing. Petra Matoušková, Ph.D. Consultant: prof. Seppo Auriola Title of Diploma thesis: HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone Scoparone is an active ingredient of Artemisia scoparia, a medicinal plant used in traditional Chinese medicine. It has been studied for various pharmacological effects such as upregulation of conjugation enzymes included in excretion of bilirubin, reduction of proinflammatory cytokines, lowering of plasma lipids levels and inhibition of platelet aggregation. In this thesis, metabolism of scoparone was studied by LC-MS method using Q-ToF device. Scoparone was incubated with liver microsomes obtained from 6 different mammal species to study in vitro oxidation. In total, six metabolites were detected in the incubation samples. Scopoletin and isoscopoletin were identified as major metabolites in every species, however, the rates of scoparone oxidation as well as a ratio of formed isoscopoletin and scopoletin varied. Furthermore, in vivo metabolites in human were studied in urine samples obtained from...
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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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IN VITRO assays for investigating nucleic acid delivery.
Mihaličoková, Dajana ; Jirkovská, Anna (advisor) ; Matoušková, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Dajana Mihaličoková Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ.Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: In vitro assays for investigating nucleic acid assay Keywords: transfection, splice correction, BCA assay, polyplexes One of the most important tasks of biochemical research is to find out the right way how to cure cancer, genetic disorders and other illnesses which are still not curable. Towards this, gene therapy is emerging as a potential treatment owing to its ability to deliver genetic material inside the cell. Reporer gene based transfection process can be used to study gene expression. Transfection is mediated by vectors, either of viral or non-viral origin. Non-viral vectors offer several advantages over the viral counterparts like easier to synthesize, relatively cheap and the most important is their non-immunogenicity. Cationic polymers based on polyethylenimine form complexes with plasmid DNA reffered to as...
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Study of protein-protein interaction of DHRS7 enzyme by pull-down assay
Káchová, Kateřina ; Novotná, Eva (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Biochemical Sciences Candidate: Kateřina Káchová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of protein-protein interaction of DHRS7 enzyme by pull-down assay Dehydrogenase/reductase (SDR family) member 7 (DHRS7) is one of the less studied enzymes of SDR superfamily. It has been proven that this enzyme is in vitro involved in reductive metabolism of various compounds, such as steroids, retinoids and xenobiotics. Recently results pointing out to possible role of this enzyme in the pathogenesis of prostate cancer or other diseases has been published. It would be suitable to better characterize this enzyme to clarifying its patho/physiological role in the organism. Because protein-protein interactions seem to be important for the function of proteins, the aim of this study was to identify interaction partners of the DHRS7, and thus contribute to the improvement of understanding of this enzyme. For our experiments, pull-down assay, in vitro method was utilized. The first step was immobilization of DHRS7 enzyme (bait protein) to suitable carrier (His Mag Sepharose Ni particles and nonmagnetic Protino Ni-IDA particles). Subsequently, the carrier with immobilized DHRS7 was incubated with the lysate of Hep G2...
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HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone
Novák, Filip ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Filip Novák Supervisor: Ing. Petra Matoušková, Ph.D. Consultant: prof. Seppo Auriola Title of Diploma thesis: HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone Scoparone is an active ingredient of Artemisia scoparia, a medicinal plant used in traditional Chinese medicine. It has been studied for various pharmacological effects such as upregulation of conjugation enzymes included in excretion of bilirubin, reduction of proinflammatory cytokines, lowering of plasma lipids levels and inhibition of platelet aggregation. In this thesis, metabolism of scoparone was studied by LC-MS method using Q-ToF device. Scoparone was incubated with liver microsomes obtained from 6 different mammal species to study in vitro oxidation. In total, six metabolites were detected in the incubation samples. Scopoletin and isoscopoletin were identified as major metabolites in every species, however, the rates of scoparone oxidation as well as a ratio of formed isoscopoletin and scopoletin varied. Furthermore, in vivo metabolites in human were studied in urine samples obtained from...
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Modulatory effect of humulene, caryophyllene and caryophyllene oxide on selected biotransformation enzymes in human liver cells
Trnčáková, Veronika ; Boušová, Iva (advisor) ; Matoušková, Petra (referee)
1 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Bc. Veronika Trnčáková Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Title of diploma thesis: Modulatory effect of humulene, caryophyllene and caryophyllene oxide on selected biotransformation enzymes in human liver cells Sesquiterpenes are substances of natural origin, produced mainly by higher plants. They are mainly found in food supplements and natural medicines. Large structural diversity is a feature of these substances. The anti-inflammatory, antiparasitic and anti-cancer effects were also reported. The aim of this diploma thesis was to investigate the effect of selected sesquiterpenes, α-humulene (HUM), β-caryophyllene (KAR) and β-caryophyllene oxide (CAO), on gene and protein expression of selected phase I drug-metabolizing enzymes, which were carbonyl reductase 1 (CBR1), aldo/keto reductase 1C (AKR1) and several cytochrome P450 isoforms, namely CYP3A4, CYP2B6 and CYP2C. The effect of sesquiterpenes on the gene and protein expression of enzymes was studied using precision-cut human liver slices (8 mm diameter, 150-180 µm thickness). Liver samples were received from hepatic surgery of five subjects of both sexes and at 58-69 years of age. Liver slices were incubated with studied compounds (HUM, KAR,...
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Effect of anthelmintics on expression of selected plant enzymes
Navrátilová, Martina ; Matoušková, Petra (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Martina Navrátilová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of anthelmintics on expression of selected plants enzymes Fenbendazole (FEN) is a broad-spectrum benzimidazole anthelmintic that is widely used to prevent and treat helminthoses in farm animals. The use of anthelmintics is associated with the risk of their transfer to the environment, where they can potentially affect non-target organisms, including plants. They come into contact with anthelmintics and their metabolites mainly due to the fertilization of farmland with manure and/or come directly into contact with the excrements of treated animals on pastures. There have not been many studies focused on the cell physiology and cell metabolism of plants affected by anthelmintics, which lead to our participation in a study which dealt, in part, with the influence of FEN on the transcriptome and gene expression in model plant Arabidopsis thaliana. A. thaliana is a monocotyledonous plant, which is a very common model organism of higher plants. Its genome was also published in 2000, allowing extensive research into gene expression. The aim of this work was to monitor expression of selected A....
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