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MicroRNA regulation of glutathion peroxidases
Boučková, Karolína ; Matoušková, Petra (advisor) ; Skarková, Veronika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Science Candidate: Bc. Karolína Boučková Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of diploma thesis: MicroRNA regulation of glutathion peroxidases Oxidative stress is an imbalance between the formation of free radicals and the organism's ability to remove those radicals naturally. There can be cell and DNA damage, premature aging, the emergence of various diseases if there is an excessive production of free radicals in the body and they are not scavenged. However, free radicals are also beneficial for the body, as it uses them to remove dead cells and bacteria. Normally, the organism has enough mechanisms to compensate for the activity of free radicals and prevent oxidative stress. Antioxidants can inhibit the activity of free radicals. They are divided into enzymatic and non-enzymatic antioxidants. The most important enzymatic antioxidants are glutathione peroxidases (GPx). Eight genes encoding GPx have been discovered in the human genome. GPx is a cytosol enzyme that catalyzes the reduction of hydrogen peroxide to water and oxygen, as well as it catalyzes the reduction of peroxide radicals to alcohols and oxygen. The reduction of expression or inactivation of enzymes may pose danger to the organism....
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Selection and validation of reference genes for relative mRNA quantification in human liver slices
Studenovská, Petra ; Boušová, Iva (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Petra Studenovská Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Title of diploma thesis: Selection and validation of reference genes for relative mRNA quantification in human liver slices The use of precision-cut liver slices is often employed in vitro system in the biochemical, pharmacological and toxicological studies. This is mainly due to the preservation of the cellular tissue architecture and therefore its functions. The aim of this diploma thesis was to select and validate reference genes for the relative quantification of mRNA in human liver slices. Liver tissues obtained from 3 patients of different age, diagnosis and pharmacotherapy were used as samples. Liver slices, prepared from samples of liver tissue, were exposed to dimethyl sulfoxide (control) or known inducers of cytochrome P450, rifampicin (10 µM) and β-naphthoflavone (10 µM). Subsequently, the stability of the expression of reference genes was verified using RefFinder program, which integrates the programs of the geNorm, BestKeeper, NormFinder and comparative ΔCT method. The optimal number of reference genes that should be used to evaluate the relative gene expression of target genes was determined using the geNorm program....
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Regulation of glutathione peroxidase 7 by microRNAs
Vávrová, Gabriela ; Matoušková, Petra (advisor) ; Boušová, Iva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Vávrová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Regulation of glutathione peroxidase 7 by microRNA Glutathione peroxidases (GPxs) are considered to be key antioxidant enzymes. A total of 8 human genes encoding GPxs are known. The main task of these enzymes is protecting biomolecules from oxidative damage due to oxidative stress, which arises from the imbalance between the generation and removal of free radicals. These enzymes catalyze the reduction of free hydrogen peroxide and organic hydroperoxides to water or corresponding alcohols. This work is focused on influencing gene expression of monomeric enzyme GPx7 by microRNA. GPx7 is involved in the correct composition of proteins in the endoplasmic reticulum, in the proper course of adipogenesis, and suppresses development of cancer. Short single-stranded non-coding microRNA molecules are involved in post-transcriptional regulation of gene expression. These molecules negatively influence the translation process, ie the translation of mRNA into the protein. MicroRNAs bind to the binding region predominantly inside of the 3'UTR mRNA of the target gene. The aim of this study was to experimentally...
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Examination of MPL in myeloproliferative diseases
Vitásková, Liana ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biochemical science Study program: Medical bioanalytics Author: Liana Vitásková, DiS. Supervisor: prof. PharmDr. Martin Beránek, PhD. Title: Examination of MPL in myeloproliferative diseases This bachelor thesis is focused on the myeloproliferative diseases related to mutations in the MPL gene. By identifying a specific mutation, it is possible to determine the diagnosis of patients with myeloproliferative diseases and thus set appropriate treatment. This thesis, therefore, aimed to introduce new diagnostic procedure at the Department of Molecular Genetics at the Regional Hospital Liberec. The aims of the work: The first objective was to get acquainted with the myeloproliferative diseases and their essential characteristics. The second objective was to study the role of mutations in the MPL gene in myeloproliferative diseases. The main aim was then to introduce a method for the determination of MPL gene mutations at the Department of Molecular Genetics at the Regional Hospital Liberec. Methods: The methodological part describes the protocol that I established for the diagnosis of mutations in the MPL gene: 1. Isolation of granulocytes from whole non-coagulated blood by gradient centrifugation 2. DNA column isolation 3....
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Effect of albendazol on the expression of selected enzymes in Haemonchus contortus
Božíková, Martina ; Matoušková, Petra (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martina Božíková Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of albendazol on the expression of selected enzymes in Haemonchus contortus Haemonchus contortus is the dominant gastrointestinal pathogen of small ruminants that can cause weakening or even death of the host organism. Losses are mainly recorded in young animals, which subsequently reflects on the farm economy itself. Therefore, prevention and therapy still play the most important role. On the other hand, the increased ability of worms to survive this treatment poses a serious problem. One of the possibilities that might be causing this problem is the increased activity of enzymes responsible for the metabolism of xenobiotics. The aim of this master's thesis was to investigate the effect of albendazole (ABZ) on the gene expression of selected biotransformation enzymes; UDP-glycosyltransferases (UGTs). For this purpose, two strains of H. contortus - anthelmintics-susceptible ISE (Inbred Susceptible Edinburgh) and anthelmintics-resistant WR (White River) were selected. One group of the parasites was cultivated for 12 hours in a media containing albendazole, the other group, which served as control, was...
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Constitutive expression of UDP-glucosyltransferases from Haemonchus contortus
Nekovová, Lucie ; Matoušková, Petra (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Nekovová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Constitutive expression of UDP-glucosyltransferases from Haemonchus contortus Haemonchosis is one of the most frequent parasitic disease on small ruminants caused by gastrointestinal nematode Haemonchus contortus. Attack of this blood sucking parasite does not mean only serious health damage but also lower productivity and economic losses. Anthelmintics are used as a medical treatment of haemonchosis however they are often insufficient because of worlwide developing resistence. Knowledge of mechanisms causing resistence is still not sufficient, therefore much effort is spent in order to find the factors which could be their source. It appears that changes of biotransformation enzymes and their expression might be one of the reason for resistence. At H. contortus more than 40 genes which code a different types of UDP- glucosyltransferases (UGTs) have been found. UGTs are the main enzymes in nematode which are connected with a deactivation of drugs (glycosylation). Therefore this thesis is focused especially on UGT family with the main goal to analyse changes in expression of these genes of susceptible (ISE) and...
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Epigenetic Regulation of Adhesive Molecules in High-grade Serous Ovarian Carcinoma
Baranová, Ivana ; Palička, Vladimír (advisor) ; Matoušková, Petra (referee)
5 Summary Title: Epigenetic Regulation of Adhesive Molecules in High-grade Serous Ovarian Carcinoma The lack of effective biomarkers for screening and early detection of ovarian cancer is currently considered as one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. Epigenetic mechanisms have been implicated also in regulation of adhesion molecules that play a major role in cancer progression. The main aim of this study was to investigate the methylation pattern of selected cadherin and catenin genes in ovarian cancer tissue by comparison with control tissue. The study group consisted of 68 patients with high-grade serous ovarian cancer (HGSOC) and 46 control patients. To determine the sites with the most significant methylation in selected genes next-generation sequencing was employed. For further confirmation of detected methylation of selected regions, methylation-sensitive high-resolution melting analysis and real-time methylation-specific polymerase chain reaction were used. In attempt to design potential biomarker panel for future screening of HGSOC as the secondary aim of our study, cadherins were evaluated together with transcription factors from our previous study....
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Epigenetic Regulation of Adhesive Molecules in High-grade Serous Ovarian Carcinoma
Baranová, Ivana ; Palička, Vladimír (advisor) ; Matoušková, Petra (referee) ; Friedecký, David (referee)
5 Summary Title: Epigenetic Regulation of Adhesive Molecules in High-grade Serous Ovarian Carcinoma The lack of effective biomarkers for screening and early detection of ovarian cancer is currently considered as one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. Epigenetic mechanisms have been implicated also in regulation of adhesion molecules that play a major role in cancer progression. The main aim of this study was to investigate the methylation pattern of selected cadherin and catenin genes in ovarian cancer tissue by comparison with control tissue. The study group consisted of 68 patients with high-grade serous ovarian cancer (HGSOC) and 46 control patients. To determine the sites with the most significant methylation in selected genes next-generation sequencing was employed. For further confirmation of detected methylation of selected regions, methylation-sensitive high-resolution melting analysis and real-time methylation-specific polymerase chain reaction were used. In attempt to design potential biomarker panel for future screening of HGSOC as the secondary aim of our study, cadherins were evaluated together with transcription factors from our previous study....
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Effect of imatinib on selected mice microRNAs
Heglasová, Silvia ; Matoušková, Petra (advisor) ; Skarková, Veronika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Silvia Heglasová Supervisor: Doc. Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of imatinib on selected mice microRNAs Imatinib (IMB) belongs to low molecular weight tyrosine kinase inhibitors (TKI). One of the undesirable effects of IMB is cardiotoxicity. Early diagnosis of heart damage is important for improving human health. MicroRNAs (miRNAs) are intensively studied as potential early biomarkers of cardiac damage. They are not only intracellular but also circulating freely in the bloodstream. Plasma miRNAs are stable, resistant to repeated freezing and methods of their detection are very sensitive. In my diploma thesis I have studied changes in the expression of miRNAs in plasma in the IMB induced heart damage. I have analyzed circulating plasma miRNAs and also miRNA levels in mice hearts. Selected miRNAs were measured by quantitative real-time PCR (qPCR). Plasma Troponin T levels were also measured as classical biomarker of cardiotoxicity. Troponin T levels in the IMB-treated plasma were very variable. This fact points to different responses of individuals to IMB. Changes in expression of observed miRNA in plasma affected by IMB were not significant. Statistically...
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Effect of doxorubicin on selected mice microRNAs
Dugasová, Lucia ; Matoušková, Petra (advisor) ; Jansová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lucia Dugasová Supervisor: doc. Ing. Petra Matošková Ph.D. Title of diploma thesis: Effect of doxorubicin on selected mice microRNAs Doxorubicin (DOX) belongs to the anthracycline antibiotics used by patients suffering from cancer. Its undesirable effect is cardiotoxicity. Prognostic of cardiotoxicity depends on its early detection, and therefore many studies examine the properties of microRNAs that could be appropriate biomarkers for heart failure in the future. MicroRNAs are involved in almost all processes in the human body, in development, differentiation, proliferation and apoptosis. They are also located in the extracellular space, they are stable and have many other positive properties. My diploma thesis is focused on monitoring changes in microRNA levels that can occur in heart damage by doxorubicin. The cardiac tissues and plasma of mice treated with doxorubicin were used as samples and compared with control samples. Samples were processed and evaluated by real-time quantitative PCR (qPCR). A statistically significant increase in expression in heart tissue occurred only in miR-34a and decrease in miR-205 level. Plasma mice samples showed a statistically significant increase in...
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