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Identification of metabolites of anthelminthics in helminth parasites using mass spectrometry
Komrska, Jan ; Vokřál, Ivan (referee) ; Skálová, Lenka (advisor)
If we want be successful in the treatment of parasitic disease, it is important to get the utmost information on the fate of given drug. The discovery of the metabolism of anthelmintics and in cosequence of participating enzymes can help us fight the rising resistance to given drugs. The aim of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole (ABZ), flubendazole (FLU) and mebendazole (MEB) formed in ex vivo incubations by parasitic helminth Dicrocoelium dendriticum, using liquid chromatography-mass spectrometric techniques. In the ex vivo study, D. dendriticum adults were incubated in cultivating medium in presence of benzimidazole drug for 24 hours. After incubation of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid phase extraction. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS). We detected in phase I albendazole sulphoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methylderivatives of flubendazole, reduced flubendazole and reduced mebendazole were observed.
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Tumor markers
Karmazínová, Simona ; Skálová, Lenka (referee) ; Dršata, Jaroslav (advisor)
Tumorous diseases are as old as life itself. They pose a serious social problem and everybody has encountered them at some time. Due to the polluted environment and modern way of living, tumorous diseases are becoming increasingly responsible for human deaths. Tumorous diseases can appear in any multicellular organism in some form. Traces of them have been identified in prehistoric people as well as in Egyptian mummies. The widespread word CANCER has its origin in ancient Greece. The first reports of laboratory evidence of tumorous diseases were published in scientific publications as early as the mid-19th century. This was the time of the onset of clinical biochemistry as a branch of science and medicine. Laboratory techniques did not exist then and testing was based on simple physico-chemical reactions. It was only in the second half of the 20th century that the study and examination of tumour markers enjoyed rapid development. Currently, the most common tumour can be determined by nearly any biochemical laboratory. The subject of my paper, "Tumour markers" is very broad - actually, each chapter would deserve to be an independent subject - and still, my thesis is far from a detailed analysis of each individual marker and methods of its determination. I aimed at providing a brief overview of the...
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Effect of albendazole on activity of biotransformation enzymes in lancet fluke
Hradecká, Aneta ; Skálová, Lenka (referee) ; Szotáková, Barbora (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, name, surname of aspirant: Aneta Hradecká Title, name, surname of supervizor: Doc. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Effect of albendazole on activity of biotransformations enzymes in lancet fluke Disease caused by lancet flukes, dicrocoeliosis, can lead to dicrease of ruminants utility and consequently to economic loses. Albendazole (the benzimidazole anthelmentic) is one of the most important drugs being used in therapy of dicrocoeliosis. Because of the need to use high and repeated dosage of albendazole, a drug resistance can occure. The aim of present thesis was to determine the influence of albendazole on lancet fluke biotransformation enzyme activity. In this experiment, lancet flukes isolated from the liver of mouflons were used. One group of lancet flukes was incubated with albendazole, another group was used as a control sample. The enzyme activity of chosen enzymes (flavine-containing monooxygenases, carbonyl-reducing enzymes, glutathione S-transferases, UDP-glucuronosyl transferases, UDP-glucosyl transferases) was determined in cytosolic fraction, mitochondria and microsomes of both groups of parazites. In the majority of cases, albendazole did not affect the...
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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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