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Role of disulphide bonds in hA2A subtype adenosine receptor
Zappe, Lukáš ; Matoušková, Petra (advisor) ; Bárta, Pavel (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lukáš Zappe Supervisor: Ing. Petra Matoušková Ph.D., PD Dr. Anke C. Schiedel Title of diploma thesis: Role of disulphide bonds in hA2A subtype adenosine receptor The adenosine A2A receptor belongs to the G protein coupled receptor family (GPCR). GPCRs are the targets of almost 40% of the drugs on the market. GPCRs are characterized by seven transmembrane helices, which are linked by three extracellular and three intracellular loops (ECL and ICL). The structure of the receptor has been revealed by crystallography, hence we know that ECL1 and ECL2 are connected by several disulphide bonds. The ECL2 is believed to be involved in ligand binding and recognition. In order to understand the relevance of those disulphide bonds involved in this process, four adenosine A2A receptor mutants were generated by one-site direct mutagenesis, in which the cysteine residues were replaced with serine residues (C146S, C159S, C166S and C146S- C159S). These receptor mutants were expressed in the mammalian cell line, CHO K1(Chinese Hamster Ovary) and the receptor expression was tested with ELISA (Enzyme- linked immunosorbent assay). The determination of ligand binding has been carried out by radioligand...
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The pharmacokinetics of ivermectin in the feces of Ovis ammon
Lokvencová, Kateřina ; Raisová Stuchlíková, Lucie (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Kateřina Lokvencová Tutor: RNDr. Lucie Raisová Stuchlíková, Ph.D. Title of Thesis: The pharmacokinetics of ivermectin in the feces of Ovis ammon Keywords: Anthelmintics, ivermectin, sheep, pharmacokinetics, excretion Infection with internal parasites is one of the most common diseases in sheep worldwide and causes significant economic losses. The nematode (Haemonchus contortus) is among to the particularly problematic representatives. Anthelmintics are used to treat helmintoses and they are divided into the several classes. The class of macrocyclic lactones includes ivermectin (IVE), which is especially popular for wide range of efficacy and low toxicity. However, the study of IVE pharmacokinetics is difficult, because it depends on many factors. Considerable attention is directed to the elimination of IVE, because its residuals in dung can adversely affect the environment and thus promote the emergence of resistance. The aim of this study was study of the elimination of IVE in sheep, especially the study of excretion profile. IVE (dose 0.2 mg/kg of body weight) was subcutaneously administered to sheep, followed by sampling of faeces at specified intervals. Ultra-high performance...
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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 impact of selected prenylflavonoids on the effect of anticancer therapy in cancer cell lines
Pešková, Klára ; Matoušková, Petra (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Pešková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: The impact of selected prenylflavonoids on the effect of anticancer therapy in cancer cell lines Prenylflavonoids 6-prenylnaringenin and 8-prenylnaringenin and flavonoid naringenin are substances with anti-proliferative properties. Prenylflavonoids are present mostly in hop and in beer. Main source of naringenin are primarily citrus fruits. In this diploma thesis the effect of compounds was tested by neutral red uptake test in cell lines SW480 and SW620. These substances were tested individually and then in combination with cytostatic agent oxaliplatin. Oxaliplatin is used in treatment of cancer disease. 6-prenylnaringenin was the most effective compound in both cell lines. Statistical evaluation of the effect was carried out by Graphpad using the method One-way ANOVA in comparison with control. Combination index was determined by software program Compusyn using the method by Chou-Talalay.
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Quantification of selected microRNAs-potential biomarkers of cardiotoxicity
Vernerová, Andrea ; Matoušková, Petra (advisor) ; Hanušová, Veronika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Andrea Vernerová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Determination of selected microRNA - potential cardiotoxicity biomarkers Cardiotoxicity is adverse reaction of chemotherapy that causes damage to the heart. Monitoring of potential biomarkers of cardiotoxicity could have a positive effect on the elimination of drugs' toxicity on heart tissue. Therefore, at present, interest in the potential microRNA (miRNA) as biomarkers for cardiotoxicity is rising. MiRNA is very stable short noncoding RNA that has the ability to post-transcriptionally regulate gene expression. From bioinformatic analyses miRNAs are able to regulate more than half of human genes. Various studies have shown miRNAs to be much more specific and rapid diagnostic biomarkers in comparison with troponins. MiRNAs as biomarkers are not established in routine clinical practice yet. So far, all studies of miRNAs are in the process of search and methods optimization. In my thesis selected miRNAs for detection of doxorubicin (DOX) cardiotoxicity in vivo in mouse cardiac tissue samples and in vitro on rat cardiomyocytes were investigated. RNA isolation from biological samples and reverse transcription, using...
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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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Effect of monepantel on the expression of selected enzymes in Haemonchus contortus
Jelenková, Klára ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Jelenková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of monepantel on the expression of selected enzymes in Haemonchus Contortus Barber's pole worm (Haemonchus contortus) is parasitic nematode which is the cause of haemonchosis, worldwide distributed disease of small ruminants leading to decrease in productivity and in many cases even to animals death. For prevention and treatment of this disease are used drugs for a long time - anthelmintics. Due to their frequent use and lack of innovation in this group of drugs parasites have gradually built an extensive resistance to these drugs. New hope in therapy of haemonchoses is monepantel, drug belonging to aminoacetonitrile derivatives which have unique mechanism of action. This drug was launched more than 25 years after registration of the last drug from the group of anthelmintics. Despite the initial great success information about occurrence of resistance often appear in recent years. Current knowledge about the mechanisms of resistance development and the factors that influence them is as well as information about drug action at the molecular level still insufficient. To limit the development of...
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The effect of alpha-humulene and carophyllene oxide in ovarian cancer cells
Žáková, Kateřina ; Matoušková, Petra (advisor) ; Svobodová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Žáková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: The effect of alpha-humulene and caryophyllene oxide in ovarian cancer cells. Sesquiterpenes α-humulene and β-caryophyllene oxide are substances with antiproliferative activity against number of tumor cell lines. In this study, the antiproliferative efect of α-humulene and β-caryophyllene oxide was tested alone and in combination with doxorubicin, using neutral red uptake assay. Doxorubicin- sensitive cell line A2780 was more sensitive to the action of doxorubicin and sesquiterpenes than SKOV3 cell line, which is naturally doxorubicin-resistant. To determine the interactions between sesquiterpenes and doxorubicin, software program CalcuSyn which is based on Chou-Talay method was used. The combination indexes ofsesquiterpenes with doxorubicin were determined. Both sesquiterpenes had antagonistic effect on doxorubicin in A2780 cell line and synergistic effect on SKOV3 cell line. The mechanism of the antiproliferative effect of all three tested substances is explained by the production of ROS. For the determination of pro-oxidant effects of the tested substances the DCF assay was used. Our results showed good...
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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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