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The changes in expression of carbonyl reductases during life cycle of nematode
Ňorková, Kateřina ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Ňorková Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The changes in expression of carbonyl reductases during life cycle of nematode Barber 's pole worm (Haemonchus contortus, from the family Trichostrongylidae) is a nematode attacking the mucosa of the gastrointestinal tract of small ruminants. Infections caused by this nematode are manifested by anemia and edema. Anthelmintics, drugs against parasitic worms, are used for prophylaxis and treatment of these infections. However, a huge problem is the worldwide growing resistance to all available anthelmintics. Therefore, it is very important to study the possible mechanisms of resistance and factors that contribute to the development of resistance. In recent years, one of the mechanisms of resistance to anthelmintics has been found to be increased expression and activity of some enzymes of metabolizing drugs. This work dealt with the monitoring of changes in the expression of selected genes from the detrinth of Dehydrogenas/Reductas with a short chain (SDR). Expression of 20 SDR genes was determined using the quantitative PCR method in all developmental nematode stages (eggs, larvaes, adults) of...
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Preparation of STAT3-knockout glioblastoma multiforme cell line using CRISPR-Cas9 editing
Papinčáková, Dominika ; Skálová, Lenka (advisor) ; Laštovičková, Lenka (referee)
Charles University Fakulty of Pharmacy in Hradec Králove Department of Biochemical Sciences Candidate: Bc. Dominika Papinčáková Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Preparation of STAT3-knockout glioblastoma multiforme cell line using CRISPR-Cas9 editing Glioblastoma multiforme (GBM) is one of the most common primary brain tumours in adults and one of the most fatal cancers in humans A key oncogene in GBM development and progression is the signal transducer and activator of transcription 3 (STAT3). It is one of the mediators of tumorigenesis, plays a strong pro-survival role in GBM, and is permanently activated in GBM, leading to increased cell proliferation, invasiveness, and angiogenesis. The aim of this work was to establish a GBM STAT3-knockout (STAT3-KO) cell line. The U87MG cell line and the CRISPR-Cas9 gene knockout method were used for this purpose. In a first step, we investigated the possibility of silencing STAT3 expression by siRNA and the suitability of this cell type to pinpoint the mechanism for CRISPR gene editing into the cell. In the second step, we transfected the cells using the STAT3 gene knockout kit (CRISPR) and tested the knockout success. In the established STAT3-KO GBM cell line, we confirmed STAT3-KO at the DNA, RNA and protein levels. We...
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The changes in flubendazole metabolism during life cycle of nematode
Salvová, Martina ; Skálová, Lenka (advisor) ; Prchal, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Martina Salvová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The changes in flubendazole metabolism during life cycle of nematode The nematode barber's pole worm (Haemonchus contortus) is an infectious pathogen that causes gastrointestinal haemonchosis in ruminants. Its life cycle consists of egg, larval (L1, L2 and L3) and adult stages. Haemonchosis is a common and widespread disease of farm animals, which causes significant global economic losses. Whereas the treatment of haemonchosis is mainly based on pharmacotherapy using anthelmintics, the increasing drug resistance of barber's pole worm to these drugs is a big problem. Hence, the study of the mechanisms of the development of drug resistance in this parasite is in the foreground of scientific interest. One of the mechanisms of resistance may be an increased ability to deactivate the anthelmintics. In the case of the benzimidazole anthelmintic flubendazole, deactivation is based on a reduction of the carbonyl group by the enzymes aldo-ketoreductases and short-chain dehydrogenases/reductases. The metabolite with decreased anthelmintic efficacy is produced by the reduction of flubendazole, and in this way, nematodes...
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Optimization of a new analytical system for selected biochemical and immunochemical methods
Dvořáková, Kristýna ; Skálová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kristýna Dvořáková Thesis supervizor: prof. RNDr. Lenka Skálová, Ph.D. Consultant: Mgr. Martina Hrdinová Title of diploma thesis: Optimization of a new analytical system for selected biochemical and immunochemical methods Specific requirements for validation and verification of analytical methods according to the ČSN EN ISO 15189: 2013 standard must be met by every medical laboratory. The aim of the work was to meet the requirements of verification in the introduction of a new analytical system Cobas 8000 and also to compare the results with those obtained using older analyzer Cobas 6000 in the laboratory of clinical biochemistry. For the analysis of biochemical and immunochemical methods, we used diagnostic kits from the same supplier Roche s.r.o. We determined the selected clinic-biochemical markers by the basic methods using spectrophotometry, immunoturbidimetry and electrochemiluminiscence. We used certified calibration and control material in the process of method verification. The mixed patient serum was used for the evaluation of repeatability and comparability of the methods. We evaluated the verification and repeatability of methods using Microsoft Excel software. We compared the...
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The effect of fern extracts on cell line SW480
Zacharská, Andrea ; Matoušková, Petra (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Student: Andrea Zacharská Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of fern extracts on the SW480 cell line Cancer is one of the leading causes of death in the world. Although we have several drugs available for the treatment of cancer, none of them are completely effective and safe. The fundamental problem is toxicity, which experts try to eliminate by looking for other treatment alternatives like plants and their secondary metabolites with antiproliferative and antioxidant effects against free radicals, that can cause cancer. Our work is focused on the antioxidant, as well as the prooxidant effect of 4 species of ferns (Athyrium filix - femina, Dryopteris dilatata, Dryopteris borreri, Blechnum spicant), whose extracts of different concentrations were studied as potentially beneficial substances against oxidative stress on the SW480 cell line. We used the fluorescent probe H2DCF-DA to detect the production of reactive oxygen species (ROS) in cells. In the initial setup the potential prooxidant effect was monitored alongside with the terc- butyl hydroperoxide solution as a positive control. The results demonstrated that these test substances did not induce the ROS...
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The effect of thujone and piperitone on the expression of biotransformation enzymes in liver slices.
Štětková, Lenka ; Skálová, Lenka (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lenka Štětková Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The effect of thujone and piperitone on the expression of biotransformation enzymes in liver slices Monoterpenes are natural substances that are widely distributed in the essential oils of many plant species. Thujone and piperitone are found in certain plant species that can be part of dietary supplements, herbal preparations or used in foods as flavourings. The aim of this diploma thesis was to find out how thujone and piperitone affect the expression of selected biotransformation enzymes from the cytochrome P450 superfamily, the sulfotransferase and the UDP-glucuronosyltransferase superfamilies. Precision-cut liver slices obtained from the livers of 3 human donors were used for these experiments. Slices were incubated with thujone or piperitone for 24 hours at 10 μM and 50 μM concentrations. After incubation, the amount of mRNA and protein was quantified. The obtained results showed considerable interindividual variability. CYP1A2 gene expression was significantly increased in one donor sample after incubation with thujone (50 μM) and piperitone (10 μM, 50 μM). Piperitone (50 μM) increased the amount of mRNA of...
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The use of new ex vivo tests to find ferns with anthelmintic effect
Bednářová, Klára ; Skálová, Lenka (advisor) ; Szotáková, Barbora (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Bednářová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The use of new ex vivo tests to find ferns with anthelmintic effect Haemonchus contortus is one of the worldwide spread nematode parasitizing in the gastrointestinal tract of small ruminants. The nematodes cause a disease called haemonchosis. A typical symptom of the disease is anemia resulting from the sucking of blood by nematodes. The disease also reduces productivity and may result in the death of the host. Only a few drugs are used for the treatment of haemonchosis, the number of newly introduced drugs is low. Nowadays, the emerging resistance to anthelmintics is a growing problem due to the high H. contortus adaptivity as well as the incorrect use of synthetic drugs. In addition, the synthetic drugs pollute the environment, which is why there is an effort to find herbal products with an anthelmintic effect. Anthelmintic activity has been demonstrated in some Asian and African ferns, so we decided to test the European fern species and evaluate their anthelmintic activity. Anthelmintic activity against nematodes, specifically H. contortus, was tested using egg hatch test and ATP bioluminescence assay in...
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The effect of flubendazole on temozolomide treatment in glioblastoma cells
Dvořáková, Kateřina ; Skálová, Lenka (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Dvořáková Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The effect of flubendazole on temozolomide treatment in glioblastoma cells Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors in adults. Despite significant advances in treatment, GBM remains to have very poor prognosis and median survival of only 12 to 15 months after diagnosis. Standard treatment consists of maximal possible surgical resection followed by chemo-radiotherapy with antitumor drug temozolomide (TMZ), which use is problematic due to fast developing chemoresistance. The aim of this study was to examine the effect of potential anticancer drug, anthelmintic flubendazole (FLU) and effect of FLU in combination with TMZ on GBM cells. For this purpose, two GBM cell lines were used - A172 and T98G. In general FLU reduced cell proliferation more, especially in T98G cells. Moreover, the use of different combinations of TMZ + FLU showed even higher inhibitory effect on the viability of GBM cell lines. The combination of TMZ + FLU reduced the protein level of -tubulin and III-tubulin, simoultaneously interesting changes in STAT3 and EGFR expression, as well as lower...
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Advances in the discovery and testing of anthelmintics
Nguyen, Thuy Linh ; Skálová, Lenka (advisor) ; Lamka, Jiří (referee) ; Várady, Marián (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Training Workplace Department of Biochemical Sciences Doctoral Degree Program Xenobiochemistry and Pathobiochemistry Candidate Mgr. Thuy Linh Nguyen Supervisor Prof. RNDr. Lenka Skálová, Ph.D. Advisor Assoc. Prof. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis Advances in the discovery and testing of anthelmintics The parasitic nematodes cause a considerable problem in human and animal health worldwide. The group of gastrointestinal nematodes is responsible for economic losses in livestock production. The treatment relies on the use of a limited number of anthelmintics, but their efficacy is hampered due to widespread drug resistance. Given the need for new drugs, the present thesis focuses on discovering novel compounds with potential anthelmintic effects and on the development of testing methods. Based on the literature search, we presented the possible approaches in the current development of new anthelmintics and evaluated their advantages and disadvantages in a review article. For the experiments, we used a model organism Haemonchus contortus, which is one of the most important gastrointestinal pathogens of small ruminants. The primary phenotypic screening of a small compound library against larvae identified two 'hit' compounds...
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Advances in the discovery and testing of anthelmintics
Nguyen, Thuy Linh ; Skálová, Lenka (advisor) ; Lamka, Jiří (referee) ; Várady, Marián (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Training Workplace Department of Biochemical Sciences Doctoral Degree Program Xenobiochemistry and Pathobiochemistry Candidate Mgr. Thuy Linh Nguyen Supervisor Prof. RNDr. Lenka Skálová, Ph.D. Advisor Assoc. Prof. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis Advances in the discovery and testing of anthelmintics The parasitic nematodes cause a considerable problem in human and animal health worldwide. The group of gastrointestinal nematodes is responsible for economic losses in livestock production. The treatment relies on the use of a limited number of anthelmintics, but their efficacy is hampered due to widespread drug resistance. Given the need for new drugs, the present thesis focuses on discovering novel compounds with potential anthelmintic effects and on the development of testing methods. Based on the literature search, we presented the possible approaches in the current development of new anthelmintics and evaluated their advantages and disadvantages in a review article. For the experiments, we used a model organism Haemonchus contortus, which is one of the most important gastrointestinal pathogens of small ruminants. The primary phenotypic screening of a small compound library against larvae identified two 'hit' compounds...
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