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Effect of ivermectin on the adults of Haemonchus contortus
Mertová, Ivana ; Matoušková, Petra (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Ivana Mertová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of ivermectin on the adults of Haemonchus contortus Helminthiasis is a disease caused by helminth parasites including barber's pole worm (Haemonchus contortus - H. contortus) that causes haemonchosis. Haemonchosis is a serious disease harming small ruminant breeding and causing loss of productivity of animals, breeding weakening and ultimately death of the animals. Anthelmintics are widely used in struggle against the disease but frequent use in the past and present has led to developing drug resistance of the parasites. Increasing resistance against compounds with anthelmintic activity is becoming a worldwide problem. The effort is to know the resistance mechanisms and contributing factors which would prevent formation and expansion of the resistance. One of the most widely used substances of veterinary therapy of helminthiasis remains ivermectin (IVE). It is a macrocyclic lactone whose mechanism of action is likely damaging the parasite's chloride channels of glutaminergic synapses. This project studies the effect of IVE on adults of two strains of H. contortus: the sensitive strain, called ISE...
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Gene expression analysis of selected UDP-glucosyltransferases from Haemonchus contortus
Janura, Michal ; Matoušková, Petra (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Michal Janura Supervisor: Ing. Petra Matoušková, PhD. Title of thesis: Gene expression analysis of selected UDP-glucosyltransferases from Haemonchus contortus Haemonchosis is one of the most important parasitical diseases in small ruminants caused by Haemonchus contortus. Resistance to anthelmintics causes several problems in therapy of the disease and it increases costs of the therapy. With an effective therapy of resistant parasites the economical situation in agriculture would improve. Resistance mechanisms in H. contortus are still insufficiently understood. The resistant H. contortus isolate forms significantly more glucose conjugates of benzimidazole anthelmintic albendazole (ABZ) than the susceptible one. More than 40 UDP-glucosyl transferases (UGT) genes were identified in the genome of H. contortus, which displays a great variety in this family of enzymes. This thesis is a part of a systematic research focused on studying UGT enzymes, by which ABZ is detoxified. To investigate, which one of the UGTs is responsible for the increased metabolism of ABZ, mRNA levels of eight selected genes were analyzed by real-time PCR in adults of resistant isolate and compared with...
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Reference gene selection for mRNA quantification in Heamonchus contortus
Růžičková, Michaela ; Matoušková, Petra (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Michaela Růžičková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Reference gene selection for mRNA quantification in Haemonchus contortus Haemonchus contortus is a parasitic nematode, whose multiresistance to ant- helmintics means global problem, threatening mostly small ruminants farming. Genom and transcriptom have been published in 2013, allowing gene expression studies to be conducted. Reference genes for this studies have not been validated yet. Use of suitable reference genes is essential for accurate normalization of gene expression levels. Aim of this work was to identify and validate potential reference genes for gene expression studies in H. contortus adults. Eleven genes were chosen, stability of their expression was assessed in males and females of two H. contortus strains, one drug-susceptible (ISE) and one multi-drug-resistant (WR). Total RNA was extracted and reverse trancribed to cDNA. cDNA was diluted and analyzed using quantitative real-time PCR with SYBR Green I detection. Expression stability was evaluated by computer programs BestKeeper, geNorm, NormFinder and the comparative CT method. Ncbp, ama, sodc, gapdh and farb were found to be most stable...
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Construction of promoter-luciferase constructs for DHRS3 gene
Voborová, Lucie ; Malátková, Petra (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Voborová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Construction of promoter-luciferase constructs for DHRS3 gene Membrane-bound reductases, which include DHRS3, are still under-researched area. It is assumed that DHRS3 plays a role in the metabolism of retinoids, which contributes to maintaining the balance of all-trans-retinoic acid (ATRA) signaling and thereby regulates the formation of body axis. Gene regulation of DHRS3 by retinoids has not been described yet. Therefore, we decided to examine the gene regulation of DHRS3. The clarification of gene regulation may tell more about DHRS3 function. In order to perform the analysis of the promoter it was necessary to prepare luciferase constructs containing varying lengths of the promoter region of DHRS3. The preparation of the constructs involved amplification, restriction, ligation, transformation, colony PCR, restriction analysis and sequencing. The individual steps were optimized for specific constructs so as to achieve their correct assembly. Thanks to appropriate procedures and optimization constructs were created in sufficient quantity and quality, and can thus be used in the analysis of the promoter...
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Constuction of plasmids for human GPX7 gene reporter assay
Hanousková, Barbora ; Matoušková, Petra (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Barbora Hanousková Supervisor: Ing. Petra Matoušková, Ph.D Title of diploma thesis: Construction of plasmids for human GPx7 gene reporter assay Glutathione peroxidases (GPx) catalyze the reduction of H2O2 and organic hydroperoxides to H2O or corresponding alcohols. GPx remove compounds dangerous for the cell and protect cells against oxidative stress. Until now, in human genome in total 8 GPx coding genes were nidentified. Thesis deals with GPx7, a monomeric enzyme with antioxidant effect and that participates in protein folding. Lower expression of GPx7 was observed in breast cancer, Barrett's dysplasia and esophageal cancer. GPx7 might be a classic tumor marker and its inactivation can lead to cancer development. Possibly, microRNAs (miRNAs) cause a low expression of the GPx7 gene. MiRNAs are small non-coding single stranded RNA molecules that are significantly involved in regulation of gene expression. Their main function is inhibition of gene expression at post-transcriptional level, thus prevention translation of mRNA into protein. It is caused by specific binding miRNA to binding sites in 3'UTR region of mRNA. The aim of this thesis is the construction of plasmids with ligated...
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The effect of green tea polyphenols on antioxidant enzymes in mice in vivo
Kvita, Vojtěch ; Svobodová, Hana (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Vojtěch Kvita Supervisor: PharmDr. Hana Bártíková, Ph.D. Title of diploma thesis: The effect of green tea polyphenols on antioxidant enzymes in mice in vivo Antioxidant enzymes belong to compounds with proven effects on human organism. Their main purpose is to defend cells against oxidative stress. In order to support this process, we are taking substances called antioxidants either in regular diet or as dietary supplements. Recently there are a lot of companies on the market, offering the supplement, promising wight loss or defence against cancer. Green tea contains many natural substances, such as polyphenolic compounds which include catechins, which have proven effects on our bodies, but the specific way they act on selected antioxidant enzymes is still matter of study. The aim of this work was to determine the effect of green tea polyphenols on antioxidant enzymes in mice. We studied 8 groups (4 normal, obese 4) wherein the two groups were left as a control which means without administration of Polyphenon. Three groups were given a Polyphenon enriched diet and for various period of time (28 days, 3 days) and at different concentrations (0.1%, 0.01%). Activity was studied in liver...
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The effect of green tea polyphenols on antioxidant enzymes in mice in vivo
Kvita, Vojtěch ; Svobodová, Hana (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Vojtěch Kvita Supervisor: PharmDr. Hana Bártíková, Ph.D. Title of diploma thesis: The effect of green tea polyphenols on antioxidant enzymes in mice in vivo Antioxidant enzymes belong to compounds with proven effects on human organism. Their main purpose is to defend cells against oxidative stress. In order to support this process, we are taking substances called antioxidants either in regular diet or as dietary supplements. Recently there are a lot of companies on the market, offering the supplement, promising wight loss or defence against cancer. Green tea contains many natural substances, such as polyphenolic compounds which include catechins, which have proven effects on our bodies, but the specific way they act on selected antioxidant enzymes is still matter of study. The aim of this work was to determine the effect of green tea polyphenols on antioxidant enzymes in mice. We studied 8 groups (4 normal, obese 4) wherein the two groups were left as a control which means without administration of Polyphenon. Three groups were given a Polyphenon enriched diet and for various period of time (28 days, 3 days) and at different concentrations (0.1%, 0.01%). Activity was studied in liver...
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The impact of selected isoflavonoids on the effectiveness of anticancer therapy in MDA-MB-231 cell line
Lokvencová, Kateřina ; Svobodová, Hana (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Kateřina Lokvencová Title, Name, Surname of tutor: PharmDr. Hana Bártíková, Ph.D. Title of a diploma work: The impact of selected isoflavonoids on the effectiveness of anticancer therapy in MDA-MB-231 cell line. Breast cancer is the most common cancer disease in women. There is an extensive amount of therapeutic regimens for the treatment of this disease, but it is often limited by lack of efficiency or serious side effects. Isoflavonoids genistein (GEN), daidzein (DAID) and formononetin (FORM) belong among the natural substances with bioprotective effects on the human body. Some epidemiological studies have confirmed the protective effect of isoflavonoids against certain types of cancer, including breast cancer. The aim of this work was to study the effect of isoflavonoids on cancer cells and assess their ability to support the effect of cytostatics, namely anthracycline antineoplastic agents doxorubicin (Dox). For the experiments was chosen the breast cancer cell line MDA-MB-231. To study the effect of isoflavonoids on tumor cells we chose concentrations of 1 and 10 µM which is according to the bioavailability of isoflavonoids. We have shown that isoflavonoids...
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In vitro and in silico evaluation of biological activity of new compounds
Želazková, Jana ; Pávek, Petr (advisor) ; Matoušková, Petra (referee)
The cytotoxicity against HepG2 cell line has been determined in the MTS and LDH assay for the six coumarins, some of which have been determined in previous study to have platelet antiaggregatory activities and to act as cyklooxygenase I inhibitors. Three of the coumarins did not decrease HepG2 cells viability at 30μM concentration after 48h of exposure. None of the other three coumarins caused decrease in viability under 79% under identical conditions. Further, for all the coumarins, the important physicochemical properties determining the behaviour in biological systems have been calculated using the ADMET Predictor. The software has predicted four coumarins to have suitable drug-like properties (zero global risk) and the other two with the lowest risk in the ADMET Risk scale range. These two coumarins were identical with the coumarins showing certain cytotoxicity in the in vitro assays. Cytotoxicity in vitro has been also determined for the representative of the flavones' derivatives group the 3-hydroxyflavone. The 3-hydroxyflavone led to the decrease of HepG2 cells viability observed in MTS assay up to 80%, whereas no cytotoxicity has been observed in LDH assay. This may be explain by the cytostatic and not cytotoxic effect of 3-hydroxyflavone. Thus, the flavone derivative shall be further investigated....
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In vitro effect of flavonoids on activity of selected biotransformation enzymes in intestinal cell lines
Pokorná, Petra ; Szotáková, Barbora (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Pokorná Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: In vitro effect of flavonoids on activity of selected biotransformation enzymes in intestinal cell lines Anthocyanins belong to a class of flavonoids which are part of major natural pigments in plants. They are considered to be very beneficial for the human organism. Anthocyanins are widely consumed as fruits, vegetables and red wine and they represent significant constituents of dietary supplements. The popularity of dietary supplements with high concentrations of flavonoids is still increasing, but in addition to positive effects on human health it can also have side effects influence drug metabolizing enzymes and thus affect either desirable or undesirable effects of many drugs. In this thesis in vitro effect of some anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin) on the proliferation of the intestinal cell line Caco-2 was investigated using the assay of metabolic activity (MTT) and determination of the integrity of the cytoplasmic membrane of cells (neutral red). Then was studied their effect on the activity of selected biotransformation enzymes: glutathione S-transferase (GST),...
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