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The Study of the Cell and Molecular Mechanisms of B Cell and Intracellular Pathogen Francisella Tularensis Interaction
Bramborová, Lucie ; Wsól, Vladimír (advisor) ; Krejsek, Jan (referee) ; Šinkorová, Zuzana (referee)
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...
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Cloning, expression and purification of human 17beta-HSD1
Popovská, Lenka ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
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...
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Effect of vitamin E on oxidative stress and the basement membrane in lungs in vitamin A-defficient rats
Fabšíková, Katarína ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
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...
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Cloning, expression and purification of human AKR1C4
Kosánová, Kateřina ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
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...
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Cloning, expression and purification of human AKR1C1
Vomočilová, Iva ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
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...
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The Role of Membrane Bound Enzymes in Metabolism of Xenobiotics in Human
Skarka, Adam ; Wsól, Vladimír (advisor) ; Bílková, Zuzana (referee) ; Hodek, Petr (referee)
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...
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Regulation of Human Carbonyl Reductase 3 (CBR3) Expression
Malátková, Petra ; Wsól, Vladimír (advisor) ; Pávek, Petr (referee) ; Machala, Miroslav (referee)
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...
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Role of systemic inflammation in Parkinson's disease
Veselá, Karolína ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
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...
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Nitric oxide mediated functional effects in the normal and inflamed urinary bladder of the rat
Cabalková, Jana ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
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...
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On Purinoceptors in the Rat Urinary Bladder
Veselá, Renata ; Wsól, Vladimír (advisor) ; Študent, Vladimír (referee) ; Broďák, Miloš (referee)
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...
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