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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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Detection of oxidative DNA damage by comet assay
Kúdelová, Terézia ; Szotáková, Barbora (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Terézia Kúdelová Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Consultant: Prof. RNDr. Rudolf Štětina, CSc. Title of diploma thesis: Detection of the oxidative damage of DNA by the Comet Assay The reparative mechanisms have a very important role in organisms. Imbalance develops due to disruption of these mechanisms, which can lead to induction of many disorders. The aim of this diploma thesis was to study protection of DNA against oxidative damage. The ability of A549 cell line to cope with this damage was studied in vitro. Oxidative damage was induced by hydrogen peroxide (H2O2), which initiated induction of single strand breaks (SSBs) and at the same time oxidation of pyrimidine and purine bases. Mechanism of damage and reparation was detected by two methods. In the first case, cells were treated with antioxidant and then influenced by H2O2. In the second case, cells were influenced by H2O2 and then let to repair in the presence of antioxidants. Tested antioxidants were epigallocatechin gallate (EGCG), glutathione (GSH) and chemical modifications of GSH - S-allyldithioglutathione (GSH-A) and S-allyltrithioglutahione (GSH-B). The extent of damage was assessed using by alkaline version...
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Immunochemical determination of DHRS8 and DHRS12 enzymes in human tissues
Svobodová, Markéta ; Novotná, Eva (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Markéta Svobodová Supervisor: RNDr. Eva Novotná, Ph.D Title of diploma thesis: Expression of DHRS8 and DHRS12 enzymes in human tissues Dehydrogenase/reductase (SDR family) member 8 (DHRS8, SDR16C2) and dehydrogenase/reductase (SDR family) member 12 (DHRS12, SDR40C1) are human microsomal enzymes belonging to the superfamily of short-chain dehydrogenases/reductases (SDR). This superfamily represents one of the largest protein groups. SDR enzymes participate in the metabolism of various xenobiotic and endogenous compounds and are involved in physiological and pathological processes. However, there are still many enzymes which are only poorly characterised. To this date, the expression on mRNA level and catalytic activity toward 5α- androstane-3α,17β-diol are the only available information about DHRS8. Moreover, there is still no published information (apart from the prediction) regarding DHRS12. The aim of this study was to examine the protein expression of DHRS8 and DHRS12 enzymes in various human tissues. The tissue samples were collected from four middle aged male subjects after the sudden death without apparent disease. Proteins of interest were detected using western blotting and...
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Marriage and divorce in the Czech Republic in light of present morality
Zemanová, Lydie ; Ovečka, Libor (advisor) ; Sládek, Karel (referee)
The work draws a comparison between different views on marriage and divorce (e.g. from the perspective of the Catholic Church, profane view, the legal system of the Czech Republic). The aim of the study is to compare convictions and development of views on marriage and divorce in contemporary society. The work observes the release of morals under the influence of television and commercials. A question, whether art reflects the reality of people's lives, or determines them, also needs to be asked. Further, it examines if current people are able to think critically and looks at people's motivation. Other question is, how hard it may be to do a right thing when major society became reconciled to wrong. The issues will be answered in questionnaires. It is necessary to note though that the work is not binding as the research does not include a sufficient amount of questionnaires. In the work, the released sexuality and reasons for being "single" will be investigated. Using statistics we will try to predict the future course and to analyse natural and sacramental values of marriage, double indissolubility and unity, the relationship of marriage and society. Keywords: marriage, divorce, sexuality, theology, morals, canon law, family
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Studies of the interaction between human AQP5 and PIP
Nešverová, Veronika ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Gothenburg Faculty of Science Department of Chemistry and Molecular Biology Candidate: Veronika Nešverová Supervisor: Vladimír Wsól Title of diploma thesis: Studies of the interaction between human AQP5 and PIP Aquaporin 5 is a membrane canal which maintains water balance mainly in lacrimal and salivary glands. A defect in this protein's trafficking from cytoplasm to cytoplasmic membrane is thought to play a role in development of Sjögren's syndrome - a chronic autoimmune disease. Prolactin-inducible protein is a cytoplasmic protein which was found to be contributing to this disease's pathogenesis. This project was aimed to study the proposed interaction between aquaporin 5 and prolactin-inducible protein. Both recombinant proteins were overexpressed in the yeast Pichia pastoris and purified using low-pressure column chromatography on an ÄKTA purification system. Glycosylation of prolactin-inducible protein was confirmed using specific glycoprotein staining. Two coelution tries were run on a HisTrap column. The fractions eluted from the second try were analysed by mass spectrometry. However results did not confirm any interaction. Further investigation is needed for the complete...
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Optimization of transfection of eukaryotic cell lines
Šplíchalová, Iva ; Zemanová, Lucie (advisor) ; Štambergová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Iva Šplíchalová Supervisor: RNDr. Lucie Škarydová, Ph.D. External supervisor: Mgr. Vojtěch Vejvoda, Ph.D. Title of Master thesis: Optimization of Transfection of Eukaryotic Cells An optimization of a transfection is a process of searching for the best conditions for the transfection. Expression systems corresponding to concrete requirements are chosen first of all. A speed, an economy and a functionality of prepared recombinant protein that can be influenced by posttranslational modifications must be considered. Expression vectors are used to insert genes of the interest into expression systems. The goal of this diploma thesis was to optimize conditions for transfection of eukaryotic cell lines CHO PF a HEK293 using of three commercially available kits. It was followed by a comparison of these transfection efficiency with standard procedures that are used in the company Generi Botech s.r.o. A plasmid pMaxGFP was used for the optimization. A cell counting in Neubaurer's chamber and a quantification of GFP in cell lysate were used to evaluate the efficiency of the transfection. Commercially available kits ...
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Effect of the subchronic and early-life permethrin exposures on rat liver: oxidative stress and endogenous antioxidant responses
Palan, Matyáš ; Zemanová, Lucie (advisor) ; Dršata, Jaroslav (referee)
Charles University in Prague University of Camerino Faculty of Pharmacy in Hradec Králové Faculty of Pharmacy Department of Biochemical Sciences Department of Molecular Biology Candidate: Matyáš Palan Supervisor: Associate Prof. of Biochemistry Rosita Gabbianelli, Ph.D. RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: Effect of the subchronic and early-life permethrin exposures on rat liver: oxidative stress and endogenous antioxidant responses Permethrin is a widely used insecticide with a low acute toxicity to mammals; however, there are concerns about consequences of a long term and an early life exposure to this compound. Two different animal models were chosen in order to study effects of permethrin on liver. In the model of subchronic treatment, adolescent rats treated for 60 days with permethrin (150 mg/kg body weight/day) were used and sacrificed immediately after the treatment. As far as the model of early life is concerned, the treatment with permethrin (34.05 mg/kg body weight/day) lasted for 15 consecutive days in early life and sacrifice took place after the interval of 300 days since the beginning of the treatment. To evaluate the impact of permethrin treatment on liver tissue, markers of oxidative stress (lipid peroxidation, content of carbonyl groups), amount of superoxide...
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HPLC method for determination of testesterone and its use for chatacterization of carbonyl reductases
Plucha, Tomáš ; Zemanová, Lucie (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tomáš Plucha Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: HPLC method for determination of testesterone and its use for chatacterization of carbonyl reductases Prostate cancer belong to hormone-dependent tumors, the proliferation of transformed cells is dependent on activation of the androgen receptor by testosterone. Androgen deprivatin therapy is complicated by productin of active androgens directly in prostate cells. It was proved that among other enzymes also carbonyl-reducing enzymes participate in androgen production, especially enzyme aldo-keto reductase 1C3 (AKR1C3). The aim was to develop a HPLC method for the determination of testosterone and use this method for in vitro screening of activity of carbonyl-reducing enzymes in formation of potent androgen testosterone from its inactive precursor androstenedione. Enzyme samples were incubated with androstenedione c = 20-120 µmol/l and after incubation were extracted by ethyl acetate. Testosterone formed during the incubation was determined by HPLC analysis (column BDS Hypersil C18, 5µm, 250×4.6 mm, mobile phase metanol:voda, 70:30, detection: DAD při 240 nm). Samples of human liver subcelular fractions,...
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