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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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Crime on the open drug scene in Prague
Matoušková, Petra ; Fidesová, Hana (advisor) ; Janíková, Barbara (referee)
The main topic of this theses is the relationship between crime and open drug scene in Prague. The use of illegal drugs alone and crime related to drug use, fundamentally threatens, not only individuals, but the entire society. Therefore it is necessary to address this problem as best as possible and consequently seek the most effective ways of problem solving. The theoretical part of the study is trying to explain the character of the drug scene during the previous political regime as well as the present. It deals with the general relationship between the crime and the drug use. It shows an overview of the model connections between those phenomena. The qualitive part of the research has the goal to describe the lifestyle of proble drug users, a way of raising money for drugs and livelihood. It focuses on detection of criminal offenses for which respondents were prosecuted, convicted and sentenced. It is also asking for so-called tertiary crime. Statement here counts whether respondents were also sometimes the victims of crime. The aim of this theses is to verify the following hypotheses and try to define what kind of crime associates with the open drug scene. The research results were obtained by semistructured interview between clients outreach program. The study involved 33 clients from the...
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