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Repeated low-dose flubendazole treatment and its effect on glioblastoma cells
Namyslová, Marie ; Szotáková, Barbora (advisor) ; Králová, Věra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Marie Namyslová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Repeated low-dose flubendazole treatment and its effect on glioblastoma cells Glioblastoma (GBM) is one of the most aggressive and common primary brain tumours in the adults. GBM is resistant to current treatment and the median survival of treated patients with this disease is around 15 months. The very poor prognosis of this disease has prompted the research of new therapeutic strategies. A promising direction is represented by drugs already registered for other indications, such as the anthelmintic flubendazole (FLU) with potential antitumour activity. The aim of this work was to study the effect of FLU after repeated low-dose treatment of GBM cells. Two cell lines, U87MG and U87MG-IDH1mut, were used for this work and the effect of repeated doses of FLU on cell viability (using the WST-1) and on cell morphology (via phase contrast microscopy) was investigated. Furthermore, changes in the expression of cell cycle markers, proliferation, growth, and resistance markers after FLU treatment were evaluated at the mRNA level (RT-PCR) and protein level (Western blotting). Subsequently, the activity of initiator and...
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Testing of the efficacy and toxicity of new potential anthelmintics OMK3 AND OMK4
Žáková, Barbora ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Barbora Žáková Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Testing of the efficacy and toxicity of new potential anthelmintics OMK3 and OMK4 Haemonchus contortus is a parasite of smaller ruminants, mainly sheep and goats. It causes a disease called haemonchosis, the main symptom of which is anemia, whitch can lead to the death of the animal. Anthelmintics are used to treat this disease. However, these drugs have become ineffective due to resistance developing against them. Haemonchosis is thus becoming a significant global problem. Therefore, it is necessary to search for new anthelmintics with a new mechanism of action. In this study, the potential anthelmintic effects of new derivates of benzhydroxamic acide, OMK3 and OMK4, were investigated. The aim of this study was to determine whether these substances have an effect on eggs, larvae and adults of H. contortus, and to compare their effects on sensitive and resistant strains of H. contortus and to test their potential toxicity on sheep livers. For this purpose, eggs, larvae and adults of H. contortus were isolated, precision-cut liver slices from sheep were prepared, and the following tests were used: egg hatch...
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The expression analysis of aldo-keto reductases in Haemonchus contortus
Babičková, Markéta ; Matoušková, Petra (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Markéta Babičková Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of diploma theses: The expression analysis of aldo-keto reductases in Haemochus contortus Haemonchus contortus is a blood-sucking nematode found mainly in livestock. This nematode causes a disease called haemonchosis. During its evolution, it has developed emerging resistance to anthelmintics, a is growing global problem despite good strategies. Aldo-ketoreductases (AKRs) belong to the super family of NADPH-dependent oxidoreductases catalyzing oxidation-reduction reactions of aldehydes and ketones in various organisms from bacteria to mammals. In mammals, they are abundantly expressed in various tissues. Their essential role is mainly in the metabolism of hormones, xenobiotics, eobiotics, oxidative molecules or a role in drug resistance. Some species, such as aldosareduktasa (ADR), are also capable of damaging the organism. Unfortunately, very little has been described about these enzymes in H. contortus, and therefore this thesis deals with the investigation of putative AKR genes in this nematode. Genome sequencing identified 22 genes from the AKR superfamily. Expressions of all AKRs were detected in eggs, larvae...
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The effect of GP46 on function of HU protein of Francisella tularensis
Sokolová, Eliška ; Szotáková, Barbora (advisor) ; Skálová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Science Candidate: Bc. Eliška Sokolová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Supervisor - consultant: RNDr. Petra Špidlová, Ph.D. Title of diploma thesis: The effect of Gp46 on function of HU protein of Francisella tularensis Francisella tularensis is a highly virulent bacteria and the DNA binding HU protein is one of its important virulence factors. Recently, it was reported that the Gp46 protein inhibits the function of the Bacillus subtilis HU protein (Zhang et al., 2022). The main goal of the presented diploma thesis was to find out if the Gp46 protein of the B. subtilis bacteriophage SPO1 also works as an inhibitor of the F. tularensis HU protein. Given the high conservation of HU proteins, Gp46 might function as a universal inhibitor of all HU proteins. At the Department of Molecular Pathology and Biology (Military Faculty of Medicine, University of Defense) neither bacteriophage SPO1 nor its DNA were available, thus, it was initially necessary to acquire the gp46 gene, which encodes the Gp46 protein, using the overlap PCR method. Subsequently, the Gp46 protein was purified from the E. coli expression system using affinity chromatography. In both in vivo and in vitro conditions, we studied...
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Expression and activities of antioxidant enzymes in the in vivo models of non-alcoholic fatty liver disease
Sochová, Gabriela ; Boušová, Iva (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Gabriela Sochová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Advisor: Mgr. Gabriela Svobodová Title of diploma thesis: Expression and activities of antioxidant enzymes in the in vivo models of non-alcoholic fatty liver disease Non-alcoholic fatty liver disease is one of the most common liver diseases. This disease is caused by an imbalance between fat intake and fat degradation. Lipid accumulation can be caused by several factors (genetic predisposition, diet, metabolic diseases, etc.). If these factors act for a long enough period, the disease can lead to liver fibrosis or even cirrhosis. The aim of this diploma thesis was to compare the expression and activity of the antioxidant enzymes glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase in two models of NAFLD (dietary model (K) - high fat, fructose, and cholesterol diet (FFC); chemical model - induced by monosodium glutamate (MSG)). Furthermore, the effect of LiPR31 and liraglutide on the expression and activity of antioxidant enzymes were investigated in these models. Expression and activity were determined in 20,000×g supernatant of mouse liver. RT-qPCR was used...
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Determination of ganciclovir in lung transplant recipients
Hrach, Filip ; Szotáková, Barbora (advisor) ; Kotaška, Karel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Filip Hrach Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Consultant: doc. Ing. Eva Klapková, Ph.D. Title of diploma thesis: Determination of ganciclovir in lung transplant recipients Ganciclovir is a nucleoside analogue that is widely used for the treatment of infections caused by herpes viruses, including cytomegalovirus. Patients after lung transplantation are always given valganciclovir (ganciclovir prodrug) prophylactically to prevent cytomegalovirus infection. The aim of this thesis was to establish a reliable, sensitive and rapid analytical method for the determination of ganciclovir and to validate this method. Furthermore, to determine ganciclovir levels in patients after lung transplantation. The final objective was to evaluate the importance of therapeutic monitoring of ganciclovir. A liquid chromatography-tandem mass spectrometry method was established for the determination of ganciclovir. Validation parameters necessary for bioanalytical methods have been established.All validation parameters met the conditions for successful validation.Valganciclovir is used for oral administration of antiviral drugs and ganciclovir for intravenous administration. Patients after lung...
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Characterization of carbonyl-reducing enzymes in barber's pole worm
Kunzová, Lucie ; Szotáková, Barbora (advisor) ; Novotná, Eva (referee)
Charles University, Faculty of Pharmacy in Hradci Králové Department of Biochemical Sciences Candidate: Lucie Kunzová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Consultant: Mgr. Nikola Rychlá Title of Thesis: Characterization of carbonyl-reducing enzymes in barber's pole worm Barber's pole worm (Haemonchus contortus), one of the most pathogenic parasites of small ruminants as sheep and goats, has developed resistance against all used anthelmintic drugs. Detoxifying enzymes, which represent the main defense system against harmful xenobiotics, can also contribute to the development of drug resistance, and they are not fully explored in this organism. This research is focused on the study of carbonyl-reducing enzymes in barber's pole worm using specific substrates and inhibitors. Subcellular fractions (cytosol and microsomes) were prepared from adult worms for following in vitro experiments. Protein concentration and reductase activity towards different substrates were determined in each fraction. Substrates were chosen based on knowledge of human biotransformation enzymes. The highest activity in cytosol has been detected for glyceraldehyde (8,22 ± 0,82 [nmol/min/mg]) and menadione (4,63 ± 0,46 [nmol/min/mg]), in microsomal fraction for menadione (4,38 ± 0,36 [nmol/min/mg]) and metyrapone (1,92...
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Characterization of carbonyl-reducing enzymes in barber's pole worm
Kunzová, Lucie ; Szotáková, Barbora (advisor) ; Novotná, Eva (referee)
Charles University, Faculty of Pharmacy in Hradci Králové Department of Biochemical Sciences Candidate: Lucie Kunzová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Consultant: Mgr. Nikola Rychlá Title of Thesis: Characterization of carbonyl-reducing enzymes in barber's pole worm Barber's pole worm (Haemonchus contortus), one of the most pathogenic parasites of small ruminants as sheep and goats, has developed resistance against all used anthelmintic drugs. Detoxifying enzymes, which represent the main defense system against harmful xenobiotics, can also contribute to the development of drug resistance, and they are not fully explored in this organism. This research is focused on the study of carbonyl-reducing enzymes in barber's pole worm using specific substrates and inhibitors. Subcellular fractions (cytosol and microsomes) were prepared from adult worms for following in vitro experiments. Protein concentration and reductase activity towards different substrates were determined in each fraction. Substrates were chosen based on knowledge of human biotransformation enzymes. The highest activity in cytosol has been detected for glyceraldehyde (8,22 ± 0,82 [nmol/min/mg]) and menadione (4,63 ± 0,46 [nmol/min/mg]), in microsomal fraction for menadione (4,38 ± 0,36 [nmol/min/mg]) and metyrapone (1,92...
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