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Site-directed mutagenesis of the human histamin H4 receptor: The role of Arg-341 in the interaction with cyanoguanidine - type H4R agonists
Ládová, Kateřina ; Pávek, Petr (referee) ; Skálová, Lenka (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Ládová Consultant: Prof. Dr. Armin Buschauer Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Site-directed mutagenesis of the human histamine H4 receptor: The role of Arg-341 in the interaction with cyanoguanidine-type H4R agonists The human histamine H4 receptor (hH4R) was discovered in 2000. The H4R is supposed to be involved in immunological processes and is considered a potential drug target, e. g., for the treatment of inflammatory diseases. Cloning and expression of the hH4R inspired to the search for selective agonists and antagonists. Recently, UR-PI376 (2-cyano-1-[4-(1H- imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine) was identified within a series of cyanoguanidines as a highly potent and subtype-selective H4R agonist with pronounced preference for the human over the murine H4R (mH4R). According to molecular modelling studies, the cyanoguanidine moiety of UR-PI376 forms charge-assisted hydrogen bonds with Arg-341 of the hH4R, suggesting this amino acid brings about selectivity for the hH4R over the hH3R and is the reason for the preference of UR-PI376 for hH4R over mH4R as well. To elucidate the role of this amino acid in the interaction with...
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Doxorubicin cytotoxicity and metabolism in MCF7 cell line
Hanušová, Veronika ; Šimůnek, Tomáš (referee) ; Skálová, Lenka (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Bc. Veronika Hanušová Title, Name, Surname of tutor: Doc. RNDr. Lenka Skálová, Ph.D. Title of a diploma work: Doxorubicin cytotoxicity and metabolism in MCF-7 cell line. Doxorubicin (DOX) is ranked among anthracycline chemoterapeutics, significantly participant in the treatment of solid tumors, inclusive breast carcinoma and haematologic malignancies. DOX is metabolized to 13-OH-doxorubicinol (DOX-OL) produced by carbonyl reductase 1 (CBR1). DOX-OL is lower antineoplastic, but more cardiotoxic compared to the parent drug. Aim of this work was research of reduction DOX and testing possible increasing cytotoxicity of DOX through inhibition his reductase. For experiments has been selected human breast cancer cell line MCF-7. Initially has been elicit optimal composition of cultural medium and optimalized process on sample preparation for HPLC. In the study of DOX metabolism in cytosol the MCF-7 cells act oracin (potentional chemoterapeutic) as significantly kompetition's inhibitor of DOX reductases. In cytotoxicity tests acted DOX more toxic in cells with bovine serum in medium, oracin was again more toxic in cells without bovine serum. Combination DOX with oracin...
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Effect of flubendazole on activities of selected biotransformation enzymes in helminth parasite Haemonchus contortus
Babíková, Martina ; Lamka, Jiří (referee) ; Skálová, Lenka (advisor)
This diploma thesis treats of biotransformation enzymes issue in development of resistance of parasitic helminths on benzimidasole anthelmintics. The treatment of helminthic infections has become problematic because of frequent drug resistance of helminth parasites. The development of drug resistance can be facilitated by the action of xenobiotic metabolizing enzymes. Experimental model was represented by Haemochus contortus which is one of the most pathogenic parasites of domestic and wild ruminant species. Adults of H. contortus were isolated from infected sheep; these were treated by sub-therapeutic doses of anthelmintic flubendazole. And were also isolated from infected sheep but with no treatment applied. There were determined specific activities of selected biotransformation enzymes of the first and second phase in subcellular fractions of the hetminth homogenate. Comparing enzymatic activity values among examined groups of hetminths I was evaluating possible influence on enzymatic activities if there was flubendazole applied in the past.
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ZIP7, hlavní místo intracelulární signalizace zinku: studium prostřednictvím mutageneze
Manišová, Michaela ; Skálová, Lenka (referee) ; Pávek, Petr (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Michaela Manišová Supervisors: Dr. Kathryn Taylor, Ph.D. Doc. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: ZIP7, a hub for intracellular zinc signalling. Functional investigation by site-directed mutagenesis. Zinc is essential for many cellular processes. It is cofactor for enzymes, important for normall cell growth and can be classified as a second messenger. A significant role of zinc is ability to inhibit protein tyrosine phosphatases activity, resulting in activation of mitogen-activated protein kinases. Zinc is unable passively cross over the cell membranes and need special transporters. Zinc transporters have a main role in intracellular zinc homoeostasis, aberrations of which could lead to diseases such as cancer. The zinc transporter solute carrier family 39, member 7 (SLC39A7, commonly referred to as ZIP7) releases zinc from the endoplasmic reticulum into the cytoplasm and might be required for tyrosine kinases activation. ZIP7 is encoded by 1407 nucleotide pairs which fit into 469 amino acids. ZIP7 contains 8 transmembrane domains and two predicted phosphorylation residues one at serine 275 and one at serine 276. The presence of these phosphorylation sites is an...
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Transport and biotransformation of selected anthelmintics in lancet fluke
Dokoupilová, Ivana ; Skálová, Lenka (referee) ; Szotáková, Barbora (advisor)
(EN) Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Ivana Dokoupilová Consultant: Doc. Ing. Barbora Szotáková, Ph.D. Title: Transport and biotransformation of selected anthelmintics in lancet fluke Dicrocoeliosis is parasitic infection of small ruminants and mouflon is one of them. Lancet fluke (Dicrocoelium dendriticum) causes this disease. Energetic metabolism is inhibited by therapeutically significant group of pharmaceuticals - benzimidazoles. The aim of this project is to evaluate the transportation of albendazole (ABZ) - representative of benzimidazoles - and its metabolite albendazole sulfoxide (ABZSO) into the bodies of lancet flukes in ex vivo experiment and if lancet fluke biotransform these anthelmintics (ABZ to ABZSO and ABZSO2 (albendazole sulfone)). Lancet fluke (10 parasites) were incubated in medium with anthelmintics for 24 hours. We observed changes of anthelmintics concentration in medium and in parasite's bodies. After the incubation was finished, flukes and medium were separated. Concentration of ABZ and its metabolites was measured on HPLC. Results indicate that albendazole and albendazole sulfoxide are transported to the bodies of flukes. With growing concentration of anthelmintics in medium the concentration...
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The role of hDia1 and hDia2 in melanoma cell invasion
Šťastná, Jana ; Skálová, Lenka (advisor) ; Šimůnek, Tomáš (referee)
The aim of the diploma thesis was to elucidate the role of hDia1 and hDia2 proteins in A375-M2 melanoma cell invasion. Different methods, such as siRNA transfection, western blotting, fluorescence microscopy and confocal microscopy, were used. It was revealed that both proteins really play the role in invasion behavior of A375-M2 tumor cells. Furthermore, the hypothesis arose proposing the mechanism of hDia1 and hDia2 in A375-M2 tumor cell invasion.
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Flubendazole metabolism - effect of gender and selected inhibitors
Šťastná, Hana ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Flubendazole, a benzimidazole-like drug with strong effect on Nematoda and Cestoda (endoparasites), is routinely used in therapy of monogastric animals. Lately, it has been considered to use flubendazole also in treatment of ruminants. Hence, it is essential to obtain information about flubendazole metabolism in these species. The main aims of study presented here were to identify an enzyme participating in flubendazole metabolism (in sheep) (with use of specific enzyme inhibitors) and to examine whether male or female sex has a significant influence on rate of flubendazole metabolism in domestic sheep. Consequently, the contribution of phase I and mainly phase II enzymes (conjugation with glucuronic acid) to metabolism of flubendazole in rats was thoroughly studied. In the inhibition experiment, many specific enzyme inhibitors (menadione, naloxone, ketoprofen, coumarine, pyrazole, phenobarbital, quercitrin, α-methylcinnamic acid and estrone) were used to reveal a particular enzyme responsible for flubendazole biotransformation (reduction). Only menadion (specific inhibitor of cytosolic carbonyl reductase) was found to have strong inhibition effect on flubendazole reduction. Based on this result, cytosolic carbonyl reductase is assumed to be the main enzyme participating in flubendazole metabolism...
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Accumulation and biotransformation of nitramines in plant tissue
Šídlová, Petra ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Explosives are toxic, recalcitrant to degradation and contaminate large areas of land and ground water. Remediation of these xenobiotics is difficult and an enormous logistical task. Phytoremediation is a technique that offers an environment friendly, low-cost alternative to current remediation techniques. However this approach is hindered by the low metabolic abilities of plants towards these xenobiotic compounds and the phytotoxicity of these compounds. The fate of the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) on exposure to plant was investigated in hydroponic system of sunflower (Heliantus annuus L.) and hemp (Cannabis sativa L.) approximately for more than 10 days. Exposure levels were: 10mg/l and 50mg/l RDX, respectively HMX for sunflower; 25mg/l and 50mg/l RDX , 5mg/l and 50mg/l HMX for hemp. Their ecotoxicites were characterized by the test based on the inhibition of elongation of primary roots 3days-old mustard and hemp seedlings. Extracts of plants were analyzed by HPLC (liquid chromatography). These study showed that RDX and HMX was taken up by both plant species. The accumulation of these nitramines was observed more in the roots than in the aerial parts of the plants, except for RDX in hemp, where the...
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The assay of peroxidases activities and their comparison in various helminths
Sulanová, Lenka ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lenka Sulanová Supervisor: Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The assay of peroxidases activities and their comparison in various helminths The aim of diploma thesis was to search for simple methods for determination of peroxidases activities, to choose a suitable method, optimize it and compare it with the method used in the workplace. The 3,3′,5,5′-tetramethylbenzidine (TMB) has been used as a substrate in selected technique. I have optimized this method and compared it with the formely applied method using substrate O-phenylenediamine dihydrochloride (OPD). Method working with TMB is more precise, more comfortable but more expensive than method using OPD. I have applied both of methods for assay of peroxidases activities in the subcellular fractions of the two species of helminths: Dicrocoelicum dendriticum and Haemonchus contortus. I found out that activities of peroxidases are higher at Haemonchus contortus than at Dicrocoelicum dendriticum. Different activities of peroxidases were observed in the subcellular fractions of the Haemonchus contortus strains sensitive and resistant to anthelmintics. The ex vivo contact of Dicrocoelicum dendriticum with...
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