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Olaparib is a potent poly (ADP-ribose) polymerase inhibitor currently used in targeted therapy for treating cancer cells with BRCA mutations. Here we investigate the possible interference of olaparib with daunorubicin (Daun) metabolism, mediated by carbonyl-reducing enzymes (CREs), which play a significant role in the resistance of cancer cells to anthracyclines. Incubation experiments with the most active recombinant CREs showed that olaparib is a potent inhibitor of the aldo&ndash, keto reductase 1C3 (AKR1C3) enzyme. Subsequent inhibitory assays in the AKR1C3-overexpressing cellular model transfected human colorectal carcinoma HCT116 cells, demonstrating that olaparib significantly inhibits AKR1C3 at the intracellular level. Consequently, molecular docking studies have supported these findings and identified the possible molecular background of the interaction. Drug combination experiments in HCT116, human liver carcinoma HepG2, and leukemic KG1&alpha, cell lines showed that this observed interaction can be exploited for the synergistic enhancement of Daun&rsquo, s antiproliferative effect. Finally, we showed that olaparib had no significant effect on the mRNA expression of AKR1C3 in HepG2 and KG1&alpha, cells. In conclusion, our data demonstrate that olaparib interferes with anthracycline metabolism, and suggest that this phenomenon might be utilized for combating anthracycline resistance.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Andro Haddad Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Consultant: Anselm Morell García, Ph.D. Title of diploma thesis: ISOCITRATE DEHYDROGENASE 2 INHIBITOR ENASIDENIB SYNERGIZES DAUNORUBICIN CYTOTOXICITY BY TARGETING ALDO-KETO REDUCTASE 1C3 Treatment with anthracyclines is crucial in treating several oncologic disorders. However, several molecular mechanisms hinder the effectivity of anthracyclines, which is a significant obstacle in cancer therapy. Carbonyl reducing enzymes (CREs), a type of NAD(P)H-dependent oxidoreductase, contribute to anthracycline resistance by reducing these drugs to fewer active alcohols. These enzymes also play a role in the proliferation and differentiation of cancer cells, leading to increased tumour aggressiveness. Therefore, targeting these enzymes is essential for effective anticancer therapy. This study aimed to uncover the potential off-targets of the isocitrate dehydrogenase (IDH) inhibitor enasidenib (ENA) that could counteract the resistance to anthracycline, specifically in relation to the detoxification role of CREs. For this, we screened the ability of ENA to inhibit different recombinant CREs that can reduce daunorubicin (Daun) to daunorubicinol...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Tučková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Consultant: Mgr. Lenka Laštovičková, Ph.D. Title of diploma thesis: The use of selected inhibitors to overcome anthracycline resistance in cancer therapy (in vitro study) Resistance to chemotherapy is a severe problem in treating cancer patients, significantly reducing their chances of recovery. The increased expression of carbonyl reductases in tumour cells is one of the leading causes of the resistance. These enzymes can reduce anthracycline-based chemotherapy drugs to their less effective derivatives and thus significantly reduce their efficiency. Therefore, the inhibitors of carbonyl reductases could increase the effectiveness of anthracycline-based chemotherapy. The goal is to find an inhibitor with high inhibitory activity and low side effects. This thesis tested inhibitors asciminib, tucatinib, sotorasib, and umbralisib hydrochloride against carbonyl reductases from the aldo-keto reductase superfamily (AKR1C3, 1A1, 1B1, 1B10) and short-chain dehydrogenases/reductases superfamily (CBR1), using chemotherapy drug daunorubicin. The most significant inhibitory potential was observed between asciminib and the enzyme AKR1C3,...

5 Abstract Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Havlíčková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The effect of zanubrutinib on tumor cell resistance to daunorubicin due to carbonyl reducing enzymes Anthracycline antibiotics (ANT) are considered the first-line medication for oncologic diseases, including acute myeloid leukemia. Daunorubicin (DAUN) with a carbonyl group in position 13 is often used in chemotherapeutic treatments. Nicotinamidadenindinucleotid-phosphate dependent carbonyl reducing enzymes (CRE) create its hydroxyl metabolite daunorubicinol (DAUN-ol), which demonstrates higher toxicity on myocardial tissue and lower cytotoxicity on cancer cells, which acquire tolerance towards its effects. Overexpression of enzymes catalyzing the conversion of effective antineoplastic DAUN causes the gradual development of resistance to administered ANT. For this reason, the new pharmacotherapeutic options for preventing this process are being searched for to maintain the drug in the target cell as long as possible. One option is to use inhibitors, which lower enzymatic activity CRE and prevent unfavorable DAUN metabolism on cardiotoxic and ineffective DAUN-ol. This diploma thesis aimed to study the...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kamila Krtilová Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Inhibitory effect of tepotinib, entrectinib, and sapanisertib on an activity of selected reductases from AKR superfamily. The lung carcinoma has an increasing trend in the Czech Republic. These findings correspond to the fact that lung carcinoma is the most common type of cancer worldwide. Carbonyl reducing enzymes occur in different types of tissues, and they are responsible for the development of inflammation, cancer, and cancer resistance. These NADPH-dependent oxidoreductase cause the reduction of carbonyl groups to alcohol compound and decrease the toxicity of drug for tumor cells. Last but not least, these enzymes are responsible for tumor cell proliferation, differentiation, and increased tumoral aggressivity. This work aimed to study the inhibition effect of chosen cyclin-dependent kinase inhibitors (CDKi) on the activity of Aldo-keto reductases. Besides inhibition of CDK, the ability to inhibit efflux transporters and carbonyl reducing enzymes was proved at CDK inhibitors. The inhibition effect of tepotinib, entrectinib and sapanisertib was determined by UHPLC analysis. The most significant inhibition...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Alexandra Pěčková Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The influence of enasidenib, quizartinib and glasdegib inhibition on the activity of selected reductases from AKR and SDR superfamilies Acute myeloid leukemia is the most common cancerous disease among the adult population. The treatment is dependent on many factors, where the effectiveness of anthracycline antibiotic cytostatic treatment plays a significant role. Therapy is often complicated by resistance to anthracyclines. This resistance can be caused by carbonyl reducing enzymes which also may aid in tumor growth. Carbonyl reducing enzymes are NAD(P)H-dependent oxidoreductases, reducing anthracyclines to respective alcohols, which not only have lower toxicity towards the cancerous cells, but can also damage the cardiac tissue. These enzymes also aid in the differentiation and proliferation of cancerous cells and increase the tumor aggressiveness. The topic of this thesis was to study the inhibitors of carbonyl-reducing enzymes from aldo-keto reductase and short chain dehydrogenase/reductase superfamilies, reducing daunorubicin to less effective metabolite daunorubicinol. The three selected inhibitors were:...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Adéla Slámová Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The influence of belinostat inhibition on the activity of selected reductases from AKR and SDR superfamilies Anthracycline antibiotics (ANTs) are important antineoplastic agents. One of them, daunorubicin (DAUN), is used for the treatment of acute leukaemia and other malignancies in children and adults. Factors limiting its clinical use include mainly resistance and cardiotoxicity. Enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies mediated the reduction of DAUN to its C-13 alcohol metabolite daunorubicinol (DAUNOL). The metabolite is more cardiotoxic, less antineoplastic, and is causing anthracycline resistance. This diploma thesis aimed to examine the inhibitory effect of belinostat on the activity of AKR1A1, 1B1, 1B10, 1C3, and CBR1. The specific enzyme activity and inhibitory potential were estimated in vitro using recombinant enzymes, and the enzymatic production of DAUNOL was evaluated by the liquid chromatography (UHPLC) system. The inhibition was decreased in order AKR1C3  AKR1B10  AKR1A1  AKR1B1  CBR1. The most inhibited enzyme, AKR1C3, expressed 50,7%...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Katharina Zenkerová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Effect of evobrutinib on cancer cell resistance to daunorubicin caused by carbonyl reducing enzymes Anthracyclines (ANT) have been among the first line treatments for many types of cancer, including acute myeloid leukemia, for decades. These chemotherapeutic agents target topoisomerase II, interfere with DNA and RNA synthesis by intercalation, and induce apoptosis by forming reactive oxygen species. As with many other chemotherapeutics, administration of ANT is associated with a range of adverse effects, particularly cardiotoxicity and resistance. The culprit responsible for this cardiotoxicity is the hydroxy metabolite of ANT, formed by reduction of the carbonyl group at position 13. This metabolite is also considerably less cytotoxic; cancer cells excessively metabolize ANT, thus developing resistance to their effects. Enzymes involved in the ANT reductions are NADPH dependant carbonyl reducing enzymes, primarily from aldo-keto reductase and short-chain dehydrogenase/reductase superfamilies. These enzymes are frequently over-expressed in cancer cells and they might be an attractive target of novel...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Alžběta Lekešová Supervisor: prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The effect of olaparib on tumor cell resistance to daunorubicin due to carbonyl reducing enzymes Daunorubicin (DAUN) belongs to the group of anthracycline antibiotics, belonging to the group of cytostatics used in a wide range of different carcinomas. However, even with this group of drugs, the resistance to chemotherapy, followed by treatment failure is becoming more and more common. This problem of resistance is often dealt with by combination therapy, which uses the addition of drugs that affect the cytostatic by different mechanisms. One possibility is to keep the cytostatic in tumor cells in their active form for as long as possible. This may be affected by the blockade of efflux transporters or inhibition of enzymes responsible for the inactivation of cytostatics. The possible inhibition of enzymes by the low molecular weight drug olaparib (OLA) was studied in this diploma thesis. OLA is a drug with a cytostatic effect on its own and has been used in certain cancer therapies for several years. We tested its inhibitory potential to selected carbonyl reducing enzymes, which are involved in the metabolism of...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Hudáčová Supervisor: Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The influence of bosutinib, neratinib and ibrutinib inhibition on the activity of selected reductases from AKR and SDR superfamilies Anthracycline antibiotics (ANTs) are antineoplastic drugs. Daunorubicin (DAUN) is used in the treatment of acute leukaemia. Enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies mediate the reduction of DAUN to its C-13 alcohol metabolite daunorubicinol (DAUNOL), which is more cardiotoxic, less antineoplastic and is causing anthracycline resistance. In my diploma thesis, I examined the inhibitory effect of bosutnib, neratinib and imatinib on the activity of enzymes from the SDR and AKR superfamilies. The specific enzyme activity and inhibitory effect were estimated on the base of in vitro enzymatic production of DAUNOL by the ultra-high-performance liquid chromatography (UHPLC) system. MTT assay was used to measure DAUN and ibrutinib cytotoxicity effect on HCT116 cell culture. In vitro enzymatic activities for recombinant enzymes were decreased in order CBR1 > AKR1C3 > AKR1B1 > AKR1A1 > AKR7A2 > AKR1B10 > AKR1C1 > AKR1C2 > AKR1C4 > CBR3. The...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Jaroslav Milan Supervisor: prof. Ing. Vladimír Wsól, PhD. Consultant: RNDr. Eva Novotná, PhD. Title of diploma thesis: The influence of midostaurin, vistusertib and talazoparib inhibition on the activity of selected reductases from AKR and SDR superfamilies. Key words: reductase, AKR, inhibitors, midostaurin, vistusertib, talazoparib, anthracyclines, KG1a Multi drug resistance is for a lot of years still a big problem in therapy of cancer. Anthracycline antibiotics are highly efficient for treating cancers but multi drug resistance and severe side effects sometimes restrain the use of them and lead therapy to fail. One of the worst adverse effect is a cardiotoxicity. By older studies, the mechanism of a cardiotoxicity was because of formation of reactive oxygen species (ROS). Many times, the negative effects of ROS on cardiac muscle cells was confirmed but nowadays the evidence opens some other and more complex mechanisms of its damage. The main point of this work was examination of enzymes which metabolize anthracyclines, mainly daunorubicin. Metabolites which are formed are less potent than parent drug and they have bigger toxicity. This can have an impact on therapy and can cause a...

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Dagmar Louvarová 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 I. The anthracycline antibiotics are a significant group of drugs for treatment of various types of cancer. Difficulties related to their usage are cardiotoxicity and multiple drug resistance. Many factors are involved in the development of resistance; one of them is inactivation of anthracyclines by the activity of enzymes, human reductases, which are involved in biotransformation of anthracyclines by reducing their carbonyl group. Daunorubicin was selected from the group of anthracycline antibiotics, in which the potential involvement of selected carbonyl reducing enzymes in its metabolism (AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, CBR1 and CBR3) was subject of verification. The specific activity of selected reductases was determined by UHPLC analysis of the major metabolite, daunorubicinol. The most active reductases were AKR1B10, AKR1C3, AKR1A1 and CBR1. There is a great potential use of cyclin-dependent kinase inhibitors (CDKi) in cancer therapy. Expected effect is primarily...

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...

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...

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...

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...

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...

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...

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

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...

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...

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...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Hana ŠTAMBERGOVÁ Supervisor prof. Ing. Vladimír WSÓL, Ph.D. Title of Doctoral Thesis Human membrane-bound carbonyl reductases Human membrane bound enzymes involved in the metabolism of carbonyl containing compounds consitute a highly interesting group of enzymes. Despite the great effort of scientists over the world most of them still remain uncharacterized, mainly those from the large short-chain dehydrogenases/reductases superfamily (SDR). At present, 75 SDR members have been indentified in the human genome whereas only 20 % of them is considered to be well characterized. On the other hand 30 % SDRs remain completly uncharacterized. SDR enzymes are involved in the metabolism of steroids, saccharides, retinoids or prostaglandins and therefore play a crucial role in a number of physiological pathways as well as several serious diseases (e.g. hormon dependent cancer, metabolic syndrome, diabetes mellitus). Moreover, SDR proteins contribute to the biotransformation of some xenobiotic compounds. Several SDR enzymes have been identified to be involved in the phase I metabolism of drugs such as doxorubicin, dolasetron, benfluron, metyrapone or ketoprofen, but so far only one...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Kořínková Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning and expression of human AKR1A1 The aldo-keto reductase 1A1 (AKR1A1) is monomeric cytosolic enzyme from aldo- keto reductase superfamily (AKR) with carbonyl reducing activity. The members of AKR superfamily have an important role in reductive reactions in metabolism of some endogenous substrates and in the first phase of biotransformation of xenobiotics. Fifteen members of this large family have been found in the human body. These proteins are expressed in different tissues within all our organism. The highest concentrations of proteins have been detected in the hepatocytes and in the renal cells. The function of AKR1A1 is to catalyse reductive reactions of aromatic and aliphatic aldehydes to the respective alcohols. Among its important substrates belong mevalonate (endogenous substance) and some drugs like anthracykline antibiotics - doxorubicin and daunorubicin. AKR1A1 also participates in biotransformations of sorbitol and in the development of diabetes complications. Preparation of recombinant protein was performed in E. coli with using of pET28b(+) as an expression vector. Isolated cDNA was...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Adam Skarka Supervisor Prof. Ing. Vladimír Wsól, Ph.D. Title of Doctoral Thesis The role of membrane bound enzymes in metabolism of xenobiotics in human Heterogeneous substances named xenobiotics are commonly present in wide surroundings of man. Some of them are actively or passively acquired into organism and undergo metabolic transformation, which lead to their excretion. In this case, important role is played by different enzymatic systems. The most known system is superfamily of cytochrome P450s catalyzing oxidation reactions. However, the importance of reducing systems like superfamilies of short chain dehydrogenases/reductases (SDR), middle chain dehydrogenases/reductases (MDR) and aldo keto reductases (AKR) have grown recently. Carbonyl group, the target of reduction catalyzed by SDR, MDR and AKR superfamilies, is also present in various groups of drugs, e.g. antineoplastics and non-steroidal anti-inflammatory drugs. Conversion of this group can lead to the loss of therapeutic effect and/or to the increase of toxicity for organism. Some xenobiotics contain so called prochiral functional group in their structure, e.g. carbonyl group. Conversion of this group can lead to the...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Renata Veselá Supervisor Prof. Ing. Vladimír Wsól, Ph.D. Title of Doctoral Thesis On Purinoceptors in the Rat Urinary Bladder Both divisions of autonomic nervous system regulate the urinary bladder function. In addition to the classical autonomic neurotransmitters, noradrenaline and acetylcholine, other autonomic transmitters and signalling molecules play important roles in the physiology and pathophysiology of the lower urinary tract. Interstitial cystitis (IC) is a chronic inflammation of the bladder of non-infectious origin and unclear etiology and pathophysiology, characterized by urinary frequency, urgency and sharp pelvic pain that fade away while urinating. Non-adrenergic, non-cholinergic (NANC) systems influence functional responses in the inflamed bladder. The aim of this study was to investigate purinergic and nitrergic mechanisms involved in the IC pathogeneses and in urodynamic dysfunction. Methods: A single dose of i.p. injection of cyclophosphamide caused CYP-induced cystitis; a condition very similar to IC and for that property was used as a model of the inflamed bladder throughout our experiments. The organ bath set was employed in investigation of functional...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Popovská Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human 17β-HSD1 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1; SDR28C1; EC 1.1.1.62) belongs to the SDR superfamily and catalyzes the NAD(P)(H) dependent oxidoreduction of hydroxyl/keto groups at position C17 of androgens and estrogens and in this manner regulate intracellular availability of steroid hormone ligands to their nuclear receptors. This pathway constitutes a pre-receptor control mechanism. Estradiol is the most potent female sex steroid and the only one responsible for estrogen action in women. The ovary is the primary source of estradiol present in the circulation of premenopausal women, but circulating estrone and androgens originating from the adrenal gland are also converted to estradiol in peripheral tissues such as adipose tissue. After menopause, estrogen biosynthesis in peripheral tissues has a major role in estrogen action. For production of recombinant protein 17β-HSD1 bacterial strain E. coli DH10B with inserted vector pOTB7, which contained coding sequence of the respective enzyme, was used. The sequence of the enzyme was isolated from...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Petra Malátková Supervisor Prof. Ing. Vladimír Wsól, Ph.D. Title of Doctoral Thesis Regulation of Human Carbonyl Reductase 3 (CBR3) Expression The regulation of human carbonyl reductase 3 (CBR3) expression has been complete mystery until recently and is still not well understood. Because the transcriptional regulation of a gene is closely related to the function of encoded protein, the elucidation of the regulation of CBR3 might help to understand its physiological role which has not been elucidated up to the present. The promoter of CBR3 has been described in 2009. The CBR3 promoter contains several putative binding sites for various transcription factors. In 2010, we have shown that CBR3 is regulated via the Nrf2/ARE signaling pathway. This was the first study about the transcriptional regulation of CBR3. The involvement of Nrf2 in the regulation of CBR3 has been recently confirmed by another research group. The functional antioxidant response element (ARE) is located at 2698 bp upstream of the translation initiation codon of CBR3 (−2698ARE). However, the analysis of CBR3 promoter encompassing 2500 bp indicated the presence of cis regulatory upstream element in sequence between...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Katarína Fabšíková Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Effect of vitamin E on oxidative stress and the basement membrane in lungs in vitamin A-deficient rats In the experiment described in this thesis there was being observed the effect of long-term deficiency of vitamin A on the basement membrane in rat lungs. Rats were divided during the experiment into several groups differing in composition of feed and the changes of pulmonary basement membrane were being observed. Consequently, the effect of vitamin E and retinoic acid on membrane modifications was watched, too. During the experiment there was evaluated the rate of oxidative stress by the medium of glutathione and malondialdehyde levels, which act as its markers. These levels in all experimental animals were specified by HPLC chromatography. Also, the oxidative stress was assessed using other markers such as thickness of the basement membrane, deposits of collagen fibers and levels of retinol and α-tocopherol in plasma and tissues. This experiment should confirm the important role of vitamins acting as antioxidants in prevention and treatment of pathological changes caused by increased oxidative...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jana Cabalková Supervisors: Prof. Ing. Vladimír Wsól, Ph.D (Charles University in Prague, Faculty of Pharmacy) Prof. Gunnar Tobin, O.D., Ph.D. (University of Göteborg in Sweden, Sahlgrenska academy) Title of diploma thesis: Nitric oxide mediated functional effects in the normal and inflamed urinary bladder of the rat. Interstitial cystitis (IC) is a condition characterized by a diffuse inflammation. It affects mainly women and the symptoms include beside pain, which is the most significant one, urge and frequency. The etiology and the pathogenesis of IC are, in spite of many years of research, largely unknown. So far, the research has mainly been focused on IC-induced alterations of sensory mechanisms in the micturition reflex arch. The reason to this is that the levels of the sensory nerve activator ATP are significantly increased in patients suffering from IC. However, two other pivotal molecules in IC are nitric oxide (NO) and acetylcholine, which do not only affect the afferent side in the micturition reflex, but also the efferent side. This project focused on the mechanism of the NO effects in the normal and the inflamed rat urinary bladder. The influence of arginine and sodium...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Iva Vomočilová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C1 This work is focused on synthesis of human AKR1C1. AKR1C1 coding sequence (cDNA), incorporated into plasmid pOTB7, was purchased and delivered in Escherichia coli cells. These cells with modified plasmid were multiplied in LB medium. After the multiplication plasmid was isolated and purified by the alkaline lysis process. Coding sequence for AKR1C1 was amplified by PCR method. The primers were designed in advance and contained restriction sites for XhoI and NdeI endonucleases. The results of PCR were validated by gel electrophoresis. Then the PCR product was purified on ultra-pure agarose gel. In the next step plasmid pET-28b(+) was used to insert prepared coding sequence. Plasmid was multiplied in competent cells E. coli HB101 and purified by the alkaline lysis process. Purified PCR fragment and plasmid were double digested by a pair of restriction endonucleases mentioned above. These digested fragments were purified and PCR fragment was put into the open vector pET-28b(+) by T4 DNA ligase enzyme. This modified plasmid was transferred into the...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Kosánová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C4. AKR1C4 is one of four enzymes in men belonging to subfamily of aldo-keto reductases AKR1C. It is monomeric cytosolic protein with length of 323 amino acids expressed in liver. It plays an important role both in metabolism of endogenous and exogenous substances. It is involved in the metabolism of steroid hormones and bile acids and many drugs, for example tibolone and naltrexone. It also plays a role in activation of some cancerogenic substances, e.g. PAHs. cDNA of enzyme was delivered in cells of E. coli DH10B, in pDNR-LIB vector. After lysis of cells and isolation of plasmid, the coding sequence was amplified by PCR. Afterwards it was ligated into vector pET-28b, thanks to added restriction sites for Nde I and Xho I endonucleases in designed PCR primers. The recombinant plasmid prepared by this way was transformed by heat shock to cells E. coli HB101. After amplification of ligated plasmid it was transformed to E. coli BL21. Adjusted cells BL21 were used for expression of the protein. IPTG was used as induction reagent for overexpression. Pure...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Seville, Faculty of Pharmacy Department of Biochemistry and Molecular Biology Candidate: Karolína Veselá Supervisors: Prof. Dr. José Luis Venero Recio, Prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Role of Systemic Inflammation in Parkinson's Disease. Parkinson's disease (PD) is the second most common ageing-related neurodegenerative disorder after Alzheimer's disease and the prevalence in population is increasing. The characteristic movement disorder is caused by selective dopaminergic neurons loss, while the mechanism of this neurodegeneration is not well understood. Increasing evidence points out the key role of vicious cycle of microglial overactivation and oxidative stress, while the questions "where it begins" and "how to stop it" remain without clear answers. This thesis investigates implication of peripheral inflammation as a deteriorating circumstance and possible inductor of brain inflammation and progressive dopaminergic neurodegeneration. We use mice model of Parkinson's disease employing single intraperitoneal injection of toxin N-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) that induces specific degeneration of DA neurons and in contrast to other animal...

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...

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...

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...

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...

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...

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...

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...

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...

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.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Lucie Bramborová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Supervisor specialist: RNDr. Zuzana Kročová, Ph.D. Title of Doctoral Thesis: Study of cellular and molecular mechanisms of interactions between B-cells and intracellular pathogen Francisella tularensis F. tularensis is Gram-negative, intracellular pathogen responsible for the zoonotic disease tularemia. Immunity to F. tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes during early stage of infection was previously uncovered. The target host cells for F. tularensis replication are macrophages. After the entry into macrophages, F. tularensis resides in phagosomes and then escapes into the cytosol. It replicates within the cytosol and leads macrophages to apoptosis. Recently, important role of B lymphocytes in innate immunity against F. tularensis was demonstrated. Elkins et al. demonstrated strong early protection of mice that was highly dependent on B cells. Direct interaction of B lymphocytes with F. tularensis has not been described. We detected adhesion of F. tularensis to mice and human B lymphocytes, later the entry of bacteria into cells and morphological changes on mitochondria of host...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Hartmanová Supervisor: Dr. Claudia Staab, Dr. Hans-Jörg Martin, Prof. Ing. Vladimír Wsól, Ph.D. Title of the diploma thesis: Studies on carbonyl reductases 1 and 3 variants with emphasis on S-nitrosoglutathione as substrate and inactivator Human CBR1 and CBR3 (carbonyl reductases 1 and 3) are monomeric, NADPH-dependent enzymes belonging to the short chain dehydrogenase/reductase superfamily. Although they are highly similar at the amino acid level (72 % identity) the enzymes exhibit considerable differences in substrate specificity. The CBR1 substrate spectrum is well described, including a variety of compounds e.g. the endogenous indol isatin, S-nitrosoglutathione (GSNO), prostaglandins, quinones, and many carbonyl group bearing xenobiotics. In contrast, CBR3 shows a distinct and much narrower range of substrates and its role is still not fully clarified. Nevertheless, the dissimilar substrate spectra strongly indicate that CBR1 and CBR3 play different metabolic roles. In the present study, the catalytic properties of CBR1 towards the latest CBR1 substrate described, GSNO, were investigated. CBR3 was assessed for potential GSNO-reducing activity, but no in vitro activity was...

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...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Anita Székelyová Supervisor: Prof. Gunnar Tobin, O.D., Ph.D. Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: The impact of inflammation on muscarinic receptor subtypes expression - Immunohistochemical examination of lacrimal and salivary glands of rat and human This project was focused on the characterization of muscarinic receptor subtype expression in lacrimal and salivary glands of the rat. Immunohistochemical methods were used to reveal the presence of the M1, M3 and M5 receptors and the expression of these subtypes was further studied under experimentally induced inflammatory conditions. The expression of the above mentioned muscarinic receptor subtypes was also studied in human labial glands, both healthy and with autoimmune sialadenitis compatible with Sjögren's syndrome. M1, M3 and M5 muscarinic receptors were found in the lacrimal and submandibular glands of the rat using immunoenzyme and immunofluorescent methods. The M5 receptor in the lacrimal gland was also detected on intracellular, possibly nuclear localization. The tissue with induced inflammation didn't show any significant differences in the muscarinic receptor expression. The immunofluorescent labeling of the...