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Study of genetic factors modifying the risk of onset and progression of colorectal and pancreatic cancer
Mohelníková Duchoňová, Beatrice ; Souček, Pavel (advisor) ; Skálová, Lenka (referee) ; Macek, Milan (referee)
Introduction: The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal and pancreatic cancer. The first part deals with the etiological factors and the importance of polymorphisms in biotransformation enzymes and genetic alterations in the gene CHEK2 in the origin of these malignancies. In the second part, the ABC transporter genes were analyzed as potential prognostic and predictive markers of a treatment's outcome. Materials and methods: The polymorphisms and other genetic alterations were detected using real-time PCR, allelespecific PCR and PCR-RFLP methods in DNA which was extracted from the blood of patients. The frequency of polymorphisms was evaluated and their importance was assessed with regard to the available epidemiological data. Gene expressions were determined by qPCR in paired samples of tumor tissue and adjacent non-tumorous parenchyma. Results: A majority of the observed polymorphisms failed to show a relationship between their presence and the risk of any of these malignancies. CYP2A13 variant allele*7 coding inactive enzyme was found in 7 of 265 controls and in none of 235 pancreatic carcinoma patients. In contrast, GSTP1-codon 105 Val variant allele and GSTT1-null genotype were associated with an elevated...
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New analogs of anorexigenic neuropeptides involved in food intake regulation
Pražienková, Veronika ; Maletínská, Lenka (advisor) ; Novotný, Jiří (referee) ; Skálová, Lenka (referee)
This work focuses on anorexigenic neuropeptides, cocaine- and amphetamine-regulated transcript (CART) and prolactin-releasing peptide (PrRP), which decrease food intake and body weight. CART peptide is an anorexigenic neuropeptide and, despite many efforts, its receptor has not yet been identified. We found CART peptide specific binding sites in pheochromocytoma PC12 cells. Cells differentiated to neurons increased significantly the number of binding sites. On the other hand, after differentiation to chromaffin cells the number of binding sites was so low that it was impossible to determine their density. To clarify the importance of each of the three disulfide bridges in the CART molecule, analogs with one or two disulfide bridges were synthetized. The biological activity was maintained in analog with two disulfide bridges in positions 74-94 and 88-101. Moreover, we demonstrated the stimulation of JNK and subsequently c-Jun activation in PC12 cells. Neuropeptide PrRP belongs to the RF-amide peptide family and has anorexigenic properties. PrPR has a high affinity to GPR10 and neuropeptide FF (NPFF2) receptor. In our laboratory lipidized analogs of PrRP were synthesized, which are able to decrease food intake after peripheral administration and may cross the blood-brain barrier. We tested biological...
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Cytotoxicity of new taxol derivatives in selected tumor cell lines
Némethová, Ingrid ; Skálová, Lenka (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Ingrid Némethová Supervisor: Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Cytotoxicity of new taxol derivatives in selected tumor cell lines. Paclitaxel (Taxol, PTX) is a natural product with antitumor activity, used in the treatment of various types of solid tumors, especially breast and ovarian cancer. It belongs to the so-called mitotic poisons, which cause cell cycle arrest and apoptosis of cells by interference with microtubule dynamics. The aim of this study was to compare the antiproliferative effect of PTX and its derivatives, prepared in the ÚEB AVČR by conjugation of peptide receptor agonists for gonadotropin-releasing hormone (GnRH) within a targeted cancer therapy. For the experiment was chosen cell line MCF-7, which is derived from breast adenocarcinoma and has receptors for GnRH. The results were evaluated by two different methods (NRU, MTT). Of the six tested substances showed derivative MP 265 the most significant antiproliferative effect. When using the other substances occurred more pronounced decrease in cell-life at higher concentrations compared to PTX.
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Effect of monepantel on expression of biotransformation enzymes in sheep
Štolcová, Tereza ; Skálová, Lenka (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Štolcová Supervisor:prof. RNDr. Lenka Skálová Ph.D. Title of diploma thesis: Effect of monepantel on expression of biotransformation enzymes in sheep Monepantel (MOP) represents a new anthelmintic drug from the amino-acetonitrile derivatives group with the different mode of action in comparison with commonly used broad-spectrum drugs. It is used for the treatment and control of gastrointestinal nematode infection in sheep and cattle. This study focuses on the effect of MOP on expression of selected cytochromes P450 (CYP) in sheep in vivo and in vitro. The sheep treated with the recommended therapeutic dose of MOP was used for the in vivo experiment. Non-treated animals represented the control group. Changes of mRNA expression of selected CYPs in primary cultures of ovine hepatocytes were observed after 24 h incubation with MOP (10 μM) in the in vitro experiment. The qPCR method (real time quantitative PCR) was used for the quantification of transcription levels of mRNA. Both in vivo as well as in vitro results showed a significant up-regulation in CYP3A24 expression. The induction effect of MOP was even stronger than induction effect of well-known CYP3A inducer rifampicin....
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METABOLISM AND EFFECTS OF NEW ANTHELMINTIC DRUG IN HELMINTHS AND ITS HOSTS
Stuchlíková, Lucie ; Skálová, Lenka (advisor) ; Kučera, Radim (referee) ; Machala, Miroslav (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Lucie STUCHLÍKOVÁ Supervisor prof. RNDr. Lenka SKÁLOVÁ, Ph.D. Title of Doctoral Thesis METABOLISM AND EFFECTS OF NEW ANTHELMINTIC DRUG IN HELMINTHS AND ITS HOSTS Diseases caused by helminths, called helminthoses, are one of the most serious problems in veterinary and human medicine. These diseases are treated by anthelmintics but their effectiveness is often not sufficient due to resistance of helminths against common anthelmintics. The global anthelmintic resistance encouraged the developing of new drugs with a different mode of action. Monepantel (MOP) belongs to a new class of anthelmintic drugs known as amino- acetonitrile derivatives (AADs) that act on the nicotinic acetylcholine receptors. In 2011, MOP was approved for the treatment of gastrointestinal diseases caused by nematodes in the veterinary practice in the Czech Republic. It is produced commercially under the trade name Zolvix® (Novartis AG). The aim of this doctoral thesis was to study metabolism and effects of MOP and other AADs in order to contribute to the global research in this field. For this purpose, adults nematodes Haemonchus contortus (the sensitive strain ISE and the resistant strain WR) and its hosts Ovis...
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Identification and quantification of anthelmintics and their metabolites in tapeworms by LC/MS
Briestenský, David ; Szotáková, Barbora (advisor) ; Skálová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: David Briestenský Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Identification and quantification of anthelmintics and their metabolites in tapeworms by LC/MS Biotransformation of anthelmintics, as well as the activity of biotransformation enzymes, represents areas that are not yet so much explored at the tapeworms. However especially biotransformation enzymes can to a certain extent protect organism of the parasite against effects of anthelmintics (and xenobiotics in general). The ability of the parasite to metabolize administered anthelmintic drug to an inactive metabolite can represent a convenient defence mechanism that may contribute to the formation of resistance. The aim of my thesis was to evaluate the metabolism of selected anthelmintics (albendazole sulphoxide, flubendazole, mebendazole) in Hymenolepis diminuta. Results of metabolism show that the rat tapeworm is unable to oxidize or reduce albendazole sulphoxide. Tapeworm is able to reduce mebendazole and flubendazole. For mebendazole and flubendazole reduction the kinetic parameters were determined. It was shown that the rate of conversion to reduced metabolites of flubendazole is twenty times slower...
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Biotransformation of flubendazole and albendazole in plant cells
Seidlová, Helena ; Skálová, Lenka (advisor) ; Karlíčková, Jana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Helena Seidlová Supervisor: Prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Biotransformation of flubendazole and albendazole in plant cells Benzimidazole anthelmintics, the drugs against parasitic worms, are widely used in human as well as in veterinary medicine. Following excretion, these substances may persist in the environment and impact non-target organisms. Phytoremediation might be a suitable method for elimination of anthelmintics from the environment. In order to test detoxification abilities of plants, biotransformation pathways of albendazole (ABZ) and flubendazole (FLU) were studied in reed (Phragmites australis) in vitro. It was found that reed cells were able to uptake and biotransform both anthelmintics. These drugs did not significantly affect viability of the reed cells. Using HPLC/MS 10 ABZ metabolites and 5 FLU metabolites were found. Some atypical biotransformation metabolites (glucosylglucosides, acetylglucosides and xylosylglucosides), which have not been previously reported were identified. Based on the obtained results, the schemes of metabolic pathways of ABZ and FLU in reed were proposed. The amount of main metabolites (reduced FLU, ABZ-sulfoxide,...
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Effect of inositol hexaphosphate on adhesion and migration of cell lines SW480 and SW620
Peterková, Alena ; Szotáková, Barbora (advisor) ; Skálová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Alena Ježková Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Supervisor specialist: RNDr. Veronika Hanušová, Ph.D. Title of diploma thesis: Effect of inositol hexaphosphate on adhesion and migration of cell lines SW480 and SW620 Inositol hexaphosphate (IP6) is molecule, which is usually present in nature, especially in grain and legumes. It has been described several processes, which are influenced in human body by IP6, such as decrease of platelet aggregation, promoting effect on insulin secretion, hypocholesterolemic, as well as anticancer effect. Aim of this work was to study adhesion and migration properties, which are the most important processes in metastasis of cancer cells. For experiments, colorectal adenocarcinoma cell line SW480 and metastatic lymph node cancer cell line SW620, both isolated from identical patient, have been selected. The ability of migration in both cell lines was tested. The influence of IP6 in different concentrations and several time intervals on mRNA (RT-PCR) and protein level (western blot, immunofluorescence) expressions of adhesion and invasion molecules has been compared as well. Considerable ability of migration was found in metastatic cell line...
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Differences in expression of selected microRNA in sensitive and resistant nematodes
Hegrová, Lenka ; Skálová, Lenka (advisor) ; Jirkovská, Anna (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Hegrová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Differences in expression of selected microRNA in sensitive and resistant nematodes Anthelmintic resistance has become a worldwide growing problem in veterinary medicine due to excessive and inappropriate drug use. Understanding the mechanisms of resistance development could help prevent the spread of resistance and effectively fight resistant individuals. Haemonchus contortus is the most extensively studied parasitic nematode in this regard, because it has the ability to develop resistance to all major classes of anthelmintic drugs and at the same time it has good biological and physiological aspects. Because several recent studies have shown that a change in the expression or activity of so-called microRNAs (miRNAs), small non-coding ribonucleic acid molecules, is also involved in the drug resistance, were monitored in this diploma thesis the differences in the expression of nine selected miRNAs between susceptible (ISE) and multidrug- resistant (WR) strains of H. contortus using the qPCR method. Several significant differences in the expression of selected miRNAs were found between ISE and WR adults....
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The effects and metabolism of anthelmintics in fodder plants
Nováková, Anežka ; Skálová, Lenka (advisor) ; Karlíčková, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Anežka Nováková Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Consultant: Ing. Lenka Langhansová, Ph.D. Title of diploma thesis: The effects and metabolism of anthelmintics in fodder plants Drugs are potentially dangerous contaminants of the environment. The entry of anthelmintics into the environment is mainly via excrement of treated animals. Anthelmintics can negatively affect the ecosystem by acting on non-target organisms such as plants and soil microorganisms. The accumulation of medicines in plants is also at risk. The consumption of these plants may promote the development of resistance in helminths. This thesis looked at the study of the influence and biotransformation of frequently used anthelmintics of albendazole (ABZ), ivermectin (IVM) and monepantel (MOP) in the forage plants of alfalfa seté (Medicago sativa) and the meadow clover (Trifolium pratense). Plants cultivated in in vitro conditions and plants grown in a greenhouse were used for evaluation. Phytotoxicity was monitored on seeds and whole plants by measuring the length of the roots, by determining the content of proline, light-collecting pigments and the total protein content. Mild phytotoxicity was observed after ivm...
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