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Preparation of expression vectors for the production of ZIKA and Dengue NS3 helicase
Daňhelová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
Zika and Dengue viruses have spread, due to globalisation, to all continents which lie at least in part in the subtropic and tropic climatic zones. This spread of these viruses is a reason of an increasing number of severe diseases caused by them. New drugs, which would be effective against these infections, could be an answer to this challenge. Various viral proteins, among them also viral helicase, which is the topic of this bachelor thesis, can be a suitable drug target. The task was to prepare expression constructs for production of recombinant helicases of Zika and Dengue viruses via the suitable bacterial strain Escherichia coli. Several constructs derived from plasmid pET-16b were prepared with inserted helicase of Zika and Dengue viruses. One of them was used for the preparation of recombinant purified helicase of Zika virus, that will be used for further research. [IN CZECH] Keywords: Flavivirus, Zika virus, Dengue virus, helicase, expression, purification, enzyme activity [IN CZECH]
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Optimization of flavin monooxygenase activity assay
Hovorková, Adéla ; Dračínská, Helena (advisor) ; Heidingsfeld, Olga (referee)
Flavin monooxygenases, biotransformation enzymes catalyzing the oxidation of broad spectrum of xenobiotics, have long been overlooked compared to cytochromes P450, a larger group of biotransformation enzymes. Liver microsomes containing both flavin monooxygenases and cytochrome P450 are often used to study metabolism of xenobiotics. The catalytic aktivity of both enzymes may overlap due to the need of identical cofactors and it is therefore necessary to differentiate them appropriately. This bachelor thesis deals with the optimization of the method for the determination of enzyme activity of flavin monooxygenases in rat liver microsomes. Model reaction for optimization of the method was the oxidation of methyl p-tolyl sulfide to methyl p-tolyl sulfoxide. To determine the activity, the most suitable conditions were set: sample buffer with pH 9.5, 2 mM methyl p-tolyl sulfide and a 10 minute incubation time. It has been found that in the oxidation of methyl p-tolyl sulfide cytochromes P450 are also involved, mostly isoform 1A1. Various inhibitors of both of the above mentioned biotransformation enzymes (lipoic acid, methimazole, Brij 35 and Triton X-100) have also been tested. Brij 35 was selected as a suitable inhibitor of the catalytic action of cytochromes P450, because it had no effect on the rate...
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Monitoring of myrosinase enzymatic activity by capillary electrophoresis in off-line and on-line setting
Orgoníková, Andrea ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
4 Abstract Glucosinolates are secondary products of plants, that are hydrolysed by the enzyme myrosinase. Hydrolysis of glucosinolates leads to a production of toxic compounds, such as isothiocyanates. These degradation products are involved in the defense system of the plant against external pathogens. This bachelor thesis deals with monitoring of myrosinase enzymatic activity by capillary electrophoresis. As a substrate, 3-indolylmethylglucosinolate known by trivial name glucobrassicin, was selected. The enzymatic reaction was tested in both off-line and on-line setting by capillary electrophoresis. As the background eletrolyte a solution containing 9 mM sodium tetraborate, 15 mM phosphoric acid and 10 mM hexadecyltrimethylammonium chloride (pH = 7.02) was used. The sample was injected hydrodynamically by pressure (5 kPa, 3 s). A voltage of -20 kV was applied to the capillary and solution in the capillary was simultaneously mobilized by a pressure 5 kPa. The optimized method was evaluated by measuring calibration curve, limit of detection, limit of quantification, repeatability of injection of glucobrassicin in off-line and on-line setting and also repeatability of on-line enzymatic reaction. The limit of detection was 0.011 mg ml-1 and the limit of quantification was 0.035 mg ml-1 . In both off-line and...
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Hydrogels with incorporated enzymes
Geistová, Karolína ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of incorporation of enzymes into phase separated hydrogels. The aim of this work is to determine the enzyme activity in phase separated gels. Gels were prepared by the dry-way based on the interaction of negatively charged polyelectrolyte (hyaluronan) with positively charged surfactant (Septonex). Two enzymes, bromelain and collagenase, were incorporated into the hydrogels. To determine enzyme activity, the modified albumin protein with bound sulfanilamide group (azoalbumin) was used as a substrate. The enzyme activity of the enzyme itself, the enzyme activity affected by one of the two components of the system as well as the activity of the enzyme directly in the hydrogel was determined on UV-VIS spectrophotometry. The enzyme was found to be incorporated in the hydrogel. Furthermore, a significant effect of the positively charged surfactant on the enzyme activity was detected in phase-separated hydrogels.
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Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta
Lukačiková, Karolína ; Vokřál, Ivan (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Karolína Lukačiková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta The resistance of parasitic helminths to anthelmintic drugs is a growing worldwide phenomenon and a concerning issue. Xenobiotic metabolizing enzymes play an important role in drug resistance development as they can lower the concentration of the anthelmintics in the parasite's body and therefore protect the parasite from the anthelmintic effect. The role of drug metabolizing enzymes in drug resistance development has been already described in the group of roundworms and flukes. Limited information is available about this topic in tapeworms. In our study we decided to test the possibility of the anthelmintic mebendazole to affect the activity of these enzymes and possibly to influence the drug resistance development in rat tapeworm (Hymenolepis diminuta). Our first goal was the isolation of adult tapeworms from the definitive host (rat, Rattus norvegicus). We used mealworm beetle (Tenebrio molitor) as an intermediate host. After the successful isolation, adult tapeworms were incubated with the mebendazole (1 and 10µM) in...
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Effect of albendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta
Krejzová, Andrea ; Vokřál, Ivan (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Andrea Krejzová Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Effect of albendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta The efficacy of anthelmintics used to treat diseases caused by helminths is not always sufficient, and in some cases, we are directly facing resistance to these drugs. Helminths, including tapeworms, are able to defend against the toxic effect of anthelmintics using several mechanisms. Xenobiotic metabolizing enzymes and transport proteins belong to these mechanisms. When xenobiotic metabolizing enzymes are induced, the efficacy of therapy may be significantly reduced. The effect of xenobiotic metabolizing enzymes on the drug resistance development has been already described in number of helminths. In tapeworms this information is still missing. Main aim of this study was to determine effect of drug albendazole on the activity of selected xenobiotic metabolizing enzymes in rat tapeworm (Hymenolepis diminuta). Tapeworms were incubated with albendazole (1 μM and 10 μM) for 24 hours. Then activities of selected enzymes in cytosol-like, microsome-like and mitochondria-like fractions were determined. This study is focused on...
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Expression and activity of rat cytochromes P450 3A after exposure to benzo[a]pyrene and Sudan I
Ličko, Vojtech ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
The aim of this Bachelor thesis is the study of the effect of two carcinogenic compounds, benzo[a]pyrene and Sudan I, co-administered to rats individually or in combination, on the expression and the activity of important biotransformation enzymes cytochromes P450 of subfamily 3A in liver - a main organ of xenobiotic metabolism, in which the amount of CYP3A is especially high. Using the quantitative PCR method, the decrease of the gene expression of CYP3A1/2 in the livers of rats exposed to benzo[a]pyrene and Sudan I individually or in combination, was observed. Using the Western Blot method with a consecutive immunodetection, we found the decrease of the protein expression of CYP3A in the livers of rats treated with benzo[a]pyrene and Sudan I alone. Specific activity of CYP3A, determined by marker reaction of CYP3A, which is 6β-hydroxylation of testosterone, did endorse the previous results only in some of the premedicated groups of rats. It can be concluded that the exposure of rats to both studied compounds with carcinogenic potential resulted in a decrease in the expression of hepatic CYP3A in vivo. (In Czech) Keywords: cytochromes P450, benzo[a]pyrene, Sudan I, expression, enzyme activity
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Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta
Lukačiková, Karolína ; Vokřál, Ivan (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Karolína Lukačiková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta The resistance of parasitic helminths to anthelmintic drugs is a growing worldwide phenomenon and a concerning issue. Xenobiotic metabolizing enzymes play an important role in drug resistance development as they can lower the concentration of the anthelmintics in the parasite's body and therefore protect the parasite from the anthelmintic effect. The role of drug metabolizing enzymes in drug resistance development has been already described in the group of roundworms and flukes. Limited information is available about this topic in tapeworms. In our study we decided to test the possibility of the anthelmintic mebendazole to affect the activity of these enzymes and possibly to influence the drug resistance development in rat tapeworm (Hymenolepis diminuta). Our first goal was the isolation of adult tapeworms from the definitive host (rat, Rattus norvegicus). We used mealworm beetle (Tenebrio molitor) as an intermediate host. After the successful isolation, adult tapeworms were incubated with the mebendazole (1 and 10µM) in...
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Effect of albendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta
Krejzová, Andrea ; Vokřál, Ivan (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Andrea Krejzová Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Effect of albendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta The efficacy of anthelmintics used to treat diseases caused by helminths is not always sufficient, and in some cases, we are directly facing resistance to these drugs. Helminths, including tapeworms, are able to defend against the toxic effect of anthelmintics using several mechanisms. Xenobiotic metabolizing enzymes and transport proteins belong to these mechanisms. When xenobiotic metabolizing enzymes are induced, the efficacy of therapy may be significantly reduced. The effect of xenobiotic metabolizing enzymes on the drug resistance development has been already described in number of helminths. In tapeworms this information is still missing. Main aim of this study was to determine effect of drug albendazole on the activity of selected xenobiotic metabolizing enzymes in rat tapeworm (Hymenolepis diminuta). Tapeworms were incubated with albendazole (1 μM and 10 μM) for 24 hours. Then activities of selected enzymes in cytosol-like, microsome-like and mitochondria-like fractions were determined. This study is focused on...
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Metabolism adaptation to excercise
Nencini, Ricky ; Šulc, Miroslav (advisor) ; Stiborová, Marie (referee)
Skeletal muscles are remodeled in response to chronic exercise training. Training induces adaptations, which can be reflected by changes in contractile protein functi- ons, mitochondrial amount and functions and also in specific enzyme activity. Due to extensive training muscle proteins and enzymes permeate through sarcoplasm and can be detected at blood plasma. Six male rowers, 19,6 ˘ 2,1 years old (trained), six sedentary men, 20,2 ˘ 1,8 years old (untrained) were included within this report. Except mentioned groups, two individual clinical cases: one male user of androsten, 28 years old, and one male user of erythropoetin (EPO), 32 years old, were also included at this theses. To report any species influence, the groups (five females per group) of two domestic animal species (cow, pig) with different treatment (housed, outdoor) were also included. Blood concentration of erythrocytes, blood plasma concentration of proteins, acti- vity of lactatedehydrogenase (LDH), malatedehydrogenase (MDH) and enoyl-CoA- hydratase were measured as well as quantitative pattern of LDH izoenzymes. The only one difference for man was observed: a significantly higher concentration of plasma protein for untrained group. On the other hand, the outdoors cows had a significantly higher concentration of erythrocytes and activity...
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