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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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Inhibition Study of Human Membrane-bound Carbonyl Reductase
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Linda Laštovková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of thesis: Inhibition study of human membrane-bound carbonyl reductase. Carbonyl reductases are an important group of enzymes that participate in metabolism of both endogenous substances also xenobiotics. Potential anticancer drug oracin is one of such xenobiotics that is metabolised by various carbonyl reductases to two enantiomers of dihydrooracin. The new human microsomal carbonyl reductase also takes part in biotransformation of oracin. This enzyme was recently purified on Department of biochemical sciences (Faculty of Pharmacy in Hradec Králové). The aim of this study was to find some inhibitors of the new enzyme and distinguish it in terms of inhibitors from another microsomal carbonyl reductase 11β-hydroxysteroid dehydrogenase 1. Flavonoids are substances produced by plants, they have a different both positive and also negative effect on human organism. One of such effect is inhibition effect on diverse enzymes. Carbonyl reductases also fall in this group. It has been described inhibitory effect of different flavonoids on carbonyl reductases. Inhibition study of the new human micorosomal carbonylreductase was...
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Participation of Selected Carbonyl Reductases in Deactivation of Anticancer Drugs
Odiana, Romana ; Wsól, Vladimír (advisor) ; Szotáková, Barbora (referee) ; Heidingsfeld, Olga (referee)
Reduction is the reverse of oxidation and therefore it can involve loss of oxygen atom or the addition of two hydrogen atoms. The reduction of carbonyl groups in xenobiotics was the main topic of this thesis. We tried to identify and characterize human carbonyl reductases responsible for anticancer drugs deactivation. When cancer is among the most common death causes in the developed world, it is necessary to look for new and efficient ways of its treatment. Inhibition of enzymes, which may contribute to disease development or relapses and/or treatment efficacy decrease by drug inactivation, could be a possible way of treatment improvement and might also lead to decrease of drug doses and side effects of cytostatics. In the first part of our project, we focused on a soluble cytosolic reductase AKR1C3. This enzyme is involved in sex hormone metabolism and might play an important role in breast and prostate cancer development. We tested its ability to metabolize anticancer drugs by its incubation with oracin and doxorubicin with subsequent metabolite determination with use of HPLC. Our experiment proved that it can deactivate these two drugs with Km 355 μM for doxorubicin and 110 μM for oracin, respectively. AKR1C3 can therefore influence the anticancer therapy, expecially when overexpressed. The...
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Cloning, expression and purification of human AKR1C2
Tobolová, Veronika ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Veronika Tobolová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C2 The coding sequence of AKR1C2 inserted in vector pOTB7 was multiplied by PCR and purified by alkali hydrolysis. Then it was ligated together with TOPO 2.1 vector. Prepared sequence inserted in vector TOPO 2.1 was transformed into the competent E. coli cells and multiplied. To verify these steps we did incubation of cell culture with ampicilin and incubation of coding sequence with restriction endonucleases.The samples inserted in vector TOPO 2.1 were sent to do sequencing. The next step was the digestion of the coding sequence inserted in vector TOPO 2.1 and the opening of purchased expressed vector pET-15b. Then a several attempts to ligation were made. The control of these steps was done by transformation of vector pET-15b with coding sequence into Hb101 cells, by incubation of transformed cells with antibiotics and by restriction sequence. Finally, the vector with the coding sequence was transformed into competent BL-21 cells and the expression was done. The result of expression we could observe after sonication and digestion of the cells by...
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Cloning, expression and purification of human AKR1B1
Lundová, Tereza ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Lundová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1B1 Aldose reductase (EC 1.1.1.21) AKR1B1 is one of the 13 human enzymes of the AKR superfamily. All human AKRs are cytosolic and NADP(H) dependent. AKR1B1 plays an important role in metabolism of endogenous and exogenous substances. The main endogenous substrate is glucose. Its reduction to sorbitol is consistently linked to secondary diabetic complications. From xenobiotics metabolized by AKR1B1, daunorubicin, an anticancer drug from the group of anthracyclines, is reduced to daunorubicinol. This metabolite is less active than parent drug and is the cause of anthracycline related cardiotoxicity. At present, many projects are focused on AKR1B1 as a target enzyme and specific inhibitors of AKR1B1 are looking for. The recombinant protein of AKR1B1 was prepared in E. coli together with the pET expression system. First, cDNA for AKR1B1 in pOTB7 was isolated from E. coli. The coding sequence of AKR1B1 was amplified by a PCR. PCR was performed with Phusion Hot Start II polymerase and pair of forward and reverse primers, which contained NdeI and XhoI restriction...
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Characterization of human membrane-bound carbonyl reductase
Zvěřinová, Michaela ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Zvěřinová Supervisor: prof. Ing. Vladimír Wsól Ph.D. Title of diploma thesis: Characterization of a new human membrane-bound carbonyl reductase Reducing enzymes are involved in the metabolism of eobiotics and xenobiotics. Carbonyl reductases also belong to the group of reducting enzymes. These are grouped into three protein superfamilies, the SDR, AKR or MDR and they can be either soluble cytosolic or membrane-bound to endoplasmatic reticulum, mitochondria and peroxisomes. Recent research has focused mainly on characterization of microsomal carbonyl reducing enzymes. It has been decribed, only one microsomal carbonyl reductase in metabolism of xenobiotics, 11β-hydroxysteroid dehydrogenase 1. There is a discrepancy in metabolism of oracin between human liver microsomes and purified 11β-hydroxysteroid dehydrogenase 1 so an unknown microsomal carbonyl reductase participates in oracin biotransformation. This enzyme was purified. Carbonyl reductases have broad substrate specificity and also metabolize the strongest tobacco smoke carcinogen NNK. These enzymes reduce NNK to NNAL that can be excrected. The aim of this thesis is to optimize the method for determining metabolite NNAL...
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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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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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Cloning, expression and purification of human AKR1C3
Dudová, Radka ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Dudová Radka Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Cloning, expression and purification of human AKR1C3 The purification of the vector pOTB7 containing the coding sequence ( CDS ) of aldo-keto reductase 1C3 ( AKR1C3 ) in Escherichia coli ( E.coli ) cells was performed by an alkaline lysis method. The coding sequence was then amplified by polymerase chain reaction. Result accuracy of the demonstrated step was shown by verification of the size of the synthesized fragment and by a restrictive analysis with the restriction endonuclease PvuII. In the second step, the coding sequence was ligated into the Topo 2.1 vector which was transformed into competent E. coli cells. The cells were multiplied and Topo 2.1 vector with the inserted code sequence was obtained by the alkaline lysis method. The result was verified by comparing of the size of the Topo 2.1 vector without inserted CDS and the vector Topo 2.1 with the CDS on an agarose gel and the subsequent restrictive analysis with the restriction endonuclease HindIII. The coding sequence was verified by the sequenation. There was also carried out the subcloning of the CDS from the Topo 2.1 vector into the express...
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