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Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines.
Kouklíková, Etela ; Novotná, Eva (advisor) ; Wsól, Vladimír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...
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Determination of the expression of enzymes DHRS7B and DHR7C in human tissues
Kozáková, Klára ; Wsól, Vladimír (advisor) ; Štambergová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Kozáková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Determination of the expression of enzymes DHRS7B and DHRS7C in human tissues Dehydrogenase/reductase (SDR family) member 7B (DHRS7B) and 7C (DHRS7C) are human enzymes that belong to the short chain dehydrogenase/reductase (SDR) superfamily. This very old and extensive superfamily includes members, which play an important role in different physiological and pathological processes. In spite of that the SDR enzymes are currently more studied, still about half of these enzymes are only poorly characterized, such as DHRS7B and DHRS7C. The aim of this study was the determination of the expression profiles of human enzymes DHRS7B a DHRS7C at mRNA and protein level and thus brings new knowledge of these enzymes. Expression of enzymes was tested in human tissues. RNA was isolated from the samples and used for transcription to cDNA. Obtained cDNA was utilized as a template for qPCR with fluorescence SYBR GREEN I detection. Determination of enzymes at protein level was done in homogenates of human tissues by using a Western blotting with chemiluminescence detection. The level of expression mRNA DHRS7B was strongest in testes...
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Interactions of muscarinic receptors and choline esterases: Functional examinations of esterase inhibitors in the rat.
Killi, Kumar Uday ; Wsól, Vladimír (advisor) ; Broďák, Miloš (referee) ; Delbro, Dick (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Uday Kumar Killi, M.Sc Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Co- Supervisor: Prof. Gunnar Tobin, D.D.S., Ph.D. Title of Doctoral Thesis: Interactions of muscarinic receptors and choline esterases: Functional examinations of esterase inhibitors in the rat. The parasympathetic nervous system regulates a number of vital body functions. The neurotransmission within the parasympathetic nervous system is exerted by acetylcholine, a phylogenetic old neurotransmitter, which acts on nicotinic and muscarinic receptors. Whilst the nicotinic receptors are located in the ganglia, the muscarinic receptors are located on the glands and smooth muscles. The muscarinic receptors belong to the large group of G glycoprotein- coupled receptors. Five subtypes exist of the muscarinic receptor (M1-M5), which can be either excitatory or inhibitory depending on subtype. Within the synaptic cleft, acetylcholinesterase hydrolyses acetylcholine and the inhibition of acetylcholinesterase, by for instance pesticides, causes accumulation of acetylcholine at the synaptic cleft, which in turn causes overstimulation of cholinoceptors. Muscarinic receptors and the acetylcholinesterase are important therapeutic...
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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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Molecular cloning of YinP gene from Leishmania major using two red fluorescent pXG-mCherry plasmids. Valuable tools for gene expression location.
Musilová, Kateřina ; Wsól, Vladimír (advisor) ; Štambergová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Musilová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Tutor: Prof. Paul Nguewa, Ph.D. Title of diploma thesis: Molecular cloning of YinP gene from Leishmania major using two red fluorescent pXG-mCherry plasmids. Valuable tools for gene expression location. In the 21st century, leishmaniasis remains a major health problem in numerous de- veloping countries. Around 2 million cases of leishmaniasis are reported every year and estimated mortality is over 20,000 deaths annually. Antileishmanial drugs are often unaf- fordable for affected people and display severe toxic side effects. Potent human vaccines are not available. This, together with increasing resistance, is a reason why new effective, safe, and affordable medicines are greatly needed. Leishmaniasis is caused by Leishmania species. These parasites are transmitted by phlebotomine sand flies, which also provide to the leishmania environment necessary for their development into infective forms. The process of transformation into a stage in- fective for vertebrate hosts is called metacyclogenesis. Nowadays, genes, enzymes, and proteins possibly exhibiting a function in the metacyclogenesis are extensively examined. One of the...
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Significance of genetic factors for prognosis and prediction of cancer therapy outcome.
Šůsová, Karolína ; Wsól, Vladimír (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Karolína Šůsová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Significance of genetic factors for prognosis and prediction of cancer therapy outcome Colorectal cancer (CRC) is one of the most common cancers in the world and also in the Czech Republic. There are many reasons for such a high prevalence and mortality. In general, we can say, that the level of awareness of risk factors and primary prevention is very low. For example, the inoperability of the tumor, caused by the diagnosis of CRC at a late stage, makes therapy of patients difficult and reduces survival. 5 - Fluorouracil and leucovorin are the gold standard in the treatment of CRC. The targeted therapy has a great success in the palliative therapy and prolongs survival. However, many patients do not respond to adjuvant and palliative therapy well. This failure is often caused by drug resistance. Efflux of cytotoxic drugs out of cells caused by transport proteins of cancer cells. ABC transporters remain among the many elements of drug resistance. Deregulation of gene expression of ABC transporters in tumor cells was discovered in connection with other types of cancer. The aim of this study is to find...
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Studies of the interaction between human AQP5 and PIP
Nešverová, Veronika ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Gothenburg Faculty of Science Department of Chemistry and Molecular Biology Candidate: Veronika Nešverová Supervisor: Vladimír Wsól Title of diploma thesis: Studies of the interaction between human AQP5 and PIP Aquaporin 5 is a membrane canal which maintains water balance mainly in lacrimal and salivary glands. A defect in this protein's trafficking from cytoplasm to cytoplasmic membrane is thought to play a role in development of Sjögren's syndrome - a chronic autoimmune disease. Prolactin-inducible protein is a cytoplasmic protein which was found to be contributing to this disease's pathogenesis. This project was aimed to study the proposed interaction between aquaporin 5 and prolactin-inducible protein. Both recombinant proteins were overexpressed in the yeast Pichia pastoris and purified using low-pressure column chromatography on an ÄKTA purification system. Glycosylation of prolactin-inducible protein was confirmed using specific glycoprotein staining. Two coelution tries were run on a HisTrap column. The fractions eluted from the second try were analysed by mass spectrometry. However results did not confirm any interaction. Further investigation is needed for the complete...
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Comparison of glomerular filtration methods
Dvořáková, Olga ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
9. ABSTRAKT Název práce: Srovnání metod vyšetření glomerulární filtrace Autor: Olga Dvořáková Vedoucí bakalářské práce: prof. Ing. Vladimír Wsól Ph.D. Cíl práce: Porovnání odhadů glomerulární filtrace ze sérových hladin cystatinu C a kreatininu stanoveného enzymaticky u vybraného souboru pacientů. Metody: Byl zpracován soubor 260 pacientů z laboratorního systému ENVIS biochemické laboratoře CEDELAB, u kterých byli k dispozici výsledky kreatininu stanoveného enzymově a cystatinu C z jednoho pacientského vzorku. Byl testován rozdíl mezi odhady GF podle rovnic MDRD a Leveyho. Ke statistickému hodnocení dat byla použita neparametrická regresní analýza Passing-Babloka . Výsledky: Výsledky ukázaly, že se odhady glomerulární filtrace z koncentrace kreatininu ze séra stanoveného enzymaticky (GFE) s odhady glomerulární filtrace z koncentrace cystatinu C ze séra (GFG) v souboru III (muži a ženy), IV (muži) a V souboru (ženy) dobře shodují, tj. nemají výraznou odchylku od linearity (P >0,05 a P >0,1). Závěry: odhady glomerulární filtrace vypočtené pomocí MDRD rovnice a pomocí Leveyho rovnice dobře shodují Klíčová slova: cystatin C, kreatinin, glomerulární filtrace
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Enzyme activity of recombinant enzymes. Influence of histidine tag.
Navrátil, Václav ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Václav Navrátil Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Enzymatic activity of recombinant enzymes. Evaluation of His- tag influence. AKR1B1, AKR1C1 and AKR1C3 are three representatives from 15 human enzymes of AKR superfamily, so far described. In the body they take part in a number of biochemical pathways and their substrates include both xenobiotics and endogenous compounds. Reduction of glucose to sorbitol catalyzed by AKR1B1 is associated with the development of secondary diabetic complications. AKR1B1 is also involved in cardiotoxic effect of the cytostatic drug daunorubicine. AKR1C1 is physiologically responsible for the conversion of progesterone to less efficient 20α- hydroxyprogesterone, thereby regulates progestational activity. Overexpression of AKR1C1 may be connected with resistance to certain cytostatic agents. AKR1C3 in human catalyzes a reaction in which testosterone and 5α-DHT is formed and it is also associated with development of several cancer types. Significant involvement of these enzymes in a number of pathological processes led to their closer study. The aim of this work was to evaluate influence of His-tag and chosen buffer on the...
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Interactions of Cyclin-dependent Kinase Inhibitors with ABC Drug Transporters in vitro and in situ
Hofman, Jakub ; Štaud, František (advisor) ; Wsól, Vladimír (referee) ; Kryštof, Vladimír (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Jakub Hofman Supervisor: Prof. PharmDr. František Štaud, Ph.D. Title of Doctoral Thesis: Interactions of cyclin-dependent kinase inhibitors with ABC drug transporters in vitro and in situ In the present work, we focused on the study of pharmacokinetic interactions of cyclin-dependent kinase inhibitors (CDKi) with drug efflux transporters breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). Using accumulation and transport methods with MDCKII-ABCG2 cells, we showed purvalanol A, olomoucine II, bohemine and roscovitine to inhibit BCRP. Employing accumulation method with MDCKII-ABCB1 cells, we further observed that purvalanol A potently inhibits P-gp and partial inhibition was recorded in the case of other studied CDKi (olomoucine II, roscovitine, flavopiridol, SNS-032). Transport method with the same cellular models was used for the study of substrate affinity of olomoucine II and purvalanol A when olomoucine II was determined to be a dual P-gp and BCRP substrate. Substrate affinity of purvalanol A toward any of the transporters tested was not observed. These findings were confirmed by the ATPase vesicular assay and using this experimental setup, we further...
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