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Characterization of barbiturates and benzodiazepines-human serum protein interactions by cappilary electrophoresis
Studničková, Kamila ; Wsól, Vladimír (advisor) ; Pospíšilová, Marie (referee)
An important topic in the drug discovery and development process is the role of drug binding to plasma proteins. In this diploma thesis the characterization of the interaction between benzodiazepines and barbiturates towards human serum albumin and α-1-acid glycoprotein under physiological conditions by capillary electrophoresis-frontal analysis is presented. Furthermore, the binding of these drugs to all plasma proteins is evaluated by using ultrafiltration and capillary electrophoresis. The results indicate that the hydrophobic character of compounds seems to be the key factor on the interaction between these drugs towards proteins. In fact, hydrophobic basic drugs (benzodiazepines) bind in great extension to HSA, while less hydrophobic acid drugs (barbiturates) present lower interactions with proteins and bind especially to AGP.
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Purification and characterization of membrane-bound reductase / / human liver tissue
Pelcová, Vendula ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Vendula Pelcová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of rigorous thesis: Purification and characterization of membrane-bound reductase from human liver. This study was focused on implementation of experimental techniques for purification of human liver microsomal fraction. There were chosen three basic steps of purification process. As a first step, ion-exchange chromatography was chosen followed by two purification steps on the base of gel filtration. Before purification the microsomal fraction had to be solubilized to release proteins from membrane bounds and conditions had to be optimized. Single fractions were subsequently incubated with specific substrate of reductases oracin and the quantity of metabolite 11-dihydroracin was assessed by achiral HPLC analysis. For the most active fractions the SDS-PAGE analysis was done to set size of unknown enzyme. By use of antibody against 11beta-HSD1 was axcleded the presence of this reductase in all analyses fractions. By use of described purification techniques we were successful to concentrate fraction with unknown human liver reductase.
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Study of resistance in cancer therapy - protein kinase inhibitors influence on activity of selected human reductases II.
Flaxová, Michaela ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Michaela Flaxová Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Study of resistance in cancer therapy - protein kinase inhibitors influence on activity of selected human reductases II. Nowadays cancerous diseases are significant problem, and the incidence is still increasing. Anthracycline antibiotics are important in therapy of cancerous diseases, unfortunately, they have serious side effects and drug resistance is often obstacle for the effective treatment. The origin of cardiotoxicity is still not clear, older theories were based on formation of reactive oxygen species (ROS). Nevertheless, newer theories confirm that anthracyclines or their metabolites influence complicated cell pathways. The enzymes, which metabolize anthracyclines, specifically daunorubicin, were the subject of this work. The carbonyl reducing enzymes are NADP(H)-dependent oxidoreductases, which are able to catalyse reduction of aldehydes and ketones to primary and secondary metabolites, daunorubicin is transformed to daunorubicinol directly by this way. Therefore we are most interested in enzymes from aldo-keto reductase family and short-chain dehydrogenases (SDR), namely AKR1C3 and CBR1. Many enzymes...
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Gene expression profiling after experimental perinatal asphyxia and the effect of complement-derived anaphylatoxin C3a
Šourková, Hana ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
Background: The complement system is involved in neuroprotection and brain repair after brain damage. To understand the molecular mechanisms of these processes, we performed gene expression profiling using quantitative real-time polymerase chain reaction (qPCR), which is the most accurate modern strategy for gene expression analysis. Project: Our project was directly aimed at expression profiling of selected genes potentially involved in loss and rescue of neural tissue during three weeks after hypoxic-ischemic brain injury, an experimental model of perinatal asphyxia. Recent experiments have shown that over-expression of C3a under the control of the GFAP promoter (C3a/GFAP) reduced hippocampal injury after left common carotid artery ligation in neonatal mice by 50%, compared to wild type mice. Here, we assessed how the local expression of C3a/GFAP transgene affects gene expression profiles. Gene expression was measured on samples from hippocampus ipsilateral and contralateral to the injury and ipsilateral part of cortex, taken at the time of injury, 6 and 24 hours; 3, 7 and 21 days after the injury. Results: Our data showed that the regulation of gene expression after hypoxic-ischemic injury differs in timing and intensity and may also be region dependent. The analysed genes belong to families...
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Zkoumání účinku CTA1R7K-ChrA-DD způsobujícího toleranci na BDC2.5 CD4 buňky vyvolávající diabetes
Kadová, Zuzana ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Zuzana Kadová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Investigations of tolerogenic effects of CTA1R7K-ChrA-DD on diabetogenic BDC2.5 CD4 cells This project was focused on the study of a novel tolerance inducing vaccine, CTA1R7K-ChrA-DD. It was investigated if this construct can inhibit autoimmune diabetes and if the CTA1R7K-ChrA-DD treatment can affect proliferation and cytokine production of BDC2.5 CD4 cells. The treatment with CTA1R7K-ChrA-DD was effective only one time of two repeated experiments (using the same protocol) when the mice received the dose of construct more times on the other hand when they were treated only once the treatment was without effect. After in vitro restimulation with the PS3 peptide it would be expected less INF gamma production and less proliferation in CTA1R7K-ChrA-DD treated mice but in most cases, we got the opposite result. This study incites hope for that CTA1R7K-ChrA-DD construct actually has ability to induce protection against diabetes and is a good start for further studies.
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Effect of selected cytostatics for the treatment of leukemia on the activity of human carbonyl reducing enzymes
Šmídlová, Monika ; Novotná, Eva (advisor) ; Wsól, Vladimír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Monika Šmídlová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Effect of selected cytostatics for the treatment of leukemia on the activity of human carbonyl reducing enzymes Key words: reductases, leukemia, cytostatics, inhibition Anthracycline antibiotics, especially daunorubicin, are widely used for the treatment of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Although the efficacy of these drugs is high, treatment is still limited due to cardiotoxicity and tumor cell resistance to anthracyclines. Mechanisms that contribute to the formation of anthracycline resistance include metabolic biotransformation (reduction) to less efficient secondary alcohols. The reduction is calatyzed by carbonyl reducing enzymes belonging to aldo-keto reductase (AKR) and short chain dehydrogenase/reductase (SDR) superfamilies. The discovery of AKR and SDR inhibitors could help to overcome anthracycline resistance and also reduce cardiotoxicity caused by these drugs. The aim of the diploma thesis was to find out whether all-trans-retinoic acid, cyclophosphamide, cytarabine, cladribine and prednisolone are able to inhibit anthracycline reductases AKR1A1, AKR1B10, AKR1C3, AKR7A2...
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Implementation of Experimental Techniques for Preparing of Recombinant Proteins on the Model of CBR3
Reissová, Helena ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Helena Reissová Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of rigorous thesis: Implementation of experimental techniques for preparing of recombinant proteins on the model of CBR3. This study was focused on implementation of experimental techniques for preparing of recombinant proteins. As a model protein was used the human carbonyl reductase 3 (CBR3). The recombinant CBR3 is used for numerous metabolic studies and its structure is already well described on both amino acids sequence and structure levels. Several of its substrates have been described, but its physiological functions and its role in metabolism of endogenous substances and xenobiotics are still not sufficiently described. The preparation of the recombinant protein started from isolation of the plasmid carrying its code sequence (vector pOTB7), followed by the polymerase chain reaction (PCR) with appropriate primers containing restriction sites for the choosen endonucleases. The PCR product was ligated into the amplification vector (pCR® 2.1-TOPO® ) and the vector was multiplied in transformed competent cells. The code sequence was removed from the vector by using restriction endonucleases and subsequently was...
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Cloning and expression of human carbonyl reductase CR-1
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
The purification of pOTB7 plasmid containing coding sequence of carbonyl reductase1 (CBR1) in cells of Escherichia coli (E. coli) was done by alkali hydrolysis. The sequence of CBR1 was multiplied by polymerase chain reaction (PCR) and synthesized primers with restriction sites were used. The left primer contained sequence for restrictive endonuclease NdeI and the right primer for restrictive endonuclease XhoI. Validation of the first step was confirmed by size measurement of the synthesized fragment with following restrictive analysis realized by restrictive endonuclease BamHI. Prepared sequence CBR1 was cloned into Topo vector, which was transformed into the competent E. coli cells. Topo vector was purified by alkali hydrolysis after innidation of E. coli cells. The size of cyclic Topo vector without CBR1 and Topo vector with CBR1 coding sequence was verified on agar gel by restrictive endonuclease BamHI. This was the validation of the second step. Subcloning of coding sequence CBR1 from Topo vector to express vector pET15b was the last step.
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Scheme " Heath city"
Valčíková, Šárka ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
1028 inhabitants of the town Kroměříž were investigated in the project "Healthy Town" in the years 2005, 2006 and 2007. Using acquired data, some epidemiological characteristics of region's inhabitants were evaluated and important experiences were put into preventive clinical practice. The experiences are following: 1. The project "Healthy Town" is not a pilot study. Contact centers are attended by citizens who pay attention to active interest of their health. In this population women and senior citizens dominate, many of them have health problems and the incidence of cardiovascular factors considerably exceeds "the average population". The study has shown that 37% of them are overweight and 13% are obese. 16% of inhabitants have hypertension of over 140/90 mm Hg. 57% suffer from lipid metabolism failure and 14% suffer from hyperglycemia. 2. Then we observed a relationship between BMI index and investigated high risk markers (systolic and diastolic pressure, serum concentration of total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and glucose). We have proved a significant relation between the degree of overweight and values of blood pressure and concentration of triglycerides, and on the other hand we have discovered a statistically insignificant relation between total cholesterol...
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