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Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Jana Pavlosková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Consultant: Ing. Marek Link, Ph.D. Title of diploma thesis: Analysis of MHC-II bound peptides of dendritic cells Antigen presentation is an important process for the immune system, which is mediated by antigen presenting cells. These cells can capture and process antigen material and present antigenic peptides to T lymphocytes. The immunopeptidome represents a collection of peptides displayed by MHC molecules on the surface of antigen presenting cells. Analysis of immunopeptidome plays a crucial role in identifying peptides to which the organism responds. This knowledge is important for developing new vaccines, medication, and disease diagnostics. This thesis focuses on the analysis of MHC-II bound peptides of dendritic cells, with the aim of verifying and demonstrating the suitability of methodological procedures for immunopeptidome analysis. The study was conducted on the mouse dendritic cells. Additionally, the cells were pulsed with the protein antigen ovalbumin to model foreign antigen binding and presentation. An immunoaffinity resin protein G agarose with immobilized antibody targeting I-Ab was successfully created for...

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...

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...

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Mikešková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Study of the effect of albendazole in the environment on the antioxidant system of red clover (Trifolium pratense) Today, anthelmintics are among the essential drugs for the therapy and prophylaxis of infectious diseases caused by parasitic worms. However, these substances may be further released into the environment by the excrements of treated animals and affect non-target organisms, such as plants, which have the ability to absorb and accumulate various substances. In this study, we focused on the effect of the often used anthelmintic albendazole on the antioxidant system of red clover (Trifolium pratense), which is commonly found in meadows and pastures. The effect of low concentrations of albendazole available in the environment on the antioxidant enzymes catalase, superoxide dismutase, ascorbate peroxidase, peroxidase, glutathione peroxidase, glutathione reductase and glutathione S-transferase was studied. In addition to antioxidant enzymes, we also monitored the concentration of plant photosynthetic pigments chlorophyll a, b and carotenoids. The results were evaluated in control and...

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...

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Magdalena Mičundová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The use of new ex vivo tests to find synthetic compounds with anthelmintic effect Haemonchus contortus is a parasitic nematode causing disease called haemonchosis in sheep and goats. This worldwide spread parasite feeds on the host's bloodstream, causing anemia and edema that can lead to death of the animal. Anthelmintic drugs are used for haemonchosis treatment, but recently their efficacy has decreased due to development of drug resistance in nematodes. Therefore, it is necessary to look for new drugs that will be effective in resistant nematode strains. In this work the anthelmintic effect of 13 new benzyloxyamide derivates (BLK) was studied in H. contortus eggs and adults. An egg hatch test, in which BLK8 was the most effective compound, was used first. A new method of bioluminiscence determination of adenosine triphosphate (ATP) was used in adults of H. contortus to monitor the effect of BLK analogues on viability of adult nematodes. The efficacy of BLK analogues was compared with the effect of classical anthelmintic levamisole. It is obvious from the results that several BLK analogues (especially...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Samanta Rysová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Consultant: RNDr. Radka Podlipná, Ph.D. Title of diploma thesis: The effect of selected herbicides on UGTs expression and activity of antioxidant enzymes in Arabidopsis thaliana Herbicides are widely used to control weeds in agriculture. Although their use is essential to ensure crop production, their effect occurs to be reduced due to the development of herbicide resistence. Therefore it is important to clarify the mechanism of herbicide resistence, so that herbicides can be used effectively and new substances can be developed if necessary. At the same time, herbicides acts as an abiotic stress factor on the plant, thus affecting the antioxidant apparatus of plant cells. The aim of this work was to investigate the effect of selected herbicides - tribenuron-methyl (TBM), pinoxaden (PNX) and sulcotrione (SLC), on the expression of selected UGTs that could play a role in herbicide resistence. Secondary, we want to find out the effect of TBM on the activity of selected antioxidant enzymes - ascorbate peroxidase (APX), catalase (CAT) and superooxide dismutase (SOD). Arabidopsis thaliana was used as a model plant and the experiment was...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Tereza Vojtěchová Supervisor: assoc. prof. PharmDr. Iva Boušová, Ph.D. Advisor: PharmDr. Martin Ambrož, Ph.D. Title of diploma thesis: Optimization of liver spheroids preparation Various in vitro liver models play an irreplaceable role in preclinical drug evaluation. One of them is multicellular hepatic spheroids. These are 3D cell aggregates that are formed by the process of cellular aggregation. They are used in drug development and represent a promising tool for identifying new targets and drug screening for various liver diseases. The aim of this thesis was to introduce and optimize the preparation of liver spheroids by the "forced-floating" method. The liver of male and female Wistar rats was used for hepatocyte isolation and spheroid preparation. Due to the low viability of isolated hepatocytes, we tried to optimize the isolation procedure. Nevertheless, we managed to obtain only a small number of hepatocytes with sufficient viability for subsequent formation of spheroids. The spheroids were incubated with various lipogenic reagents for 7 and 14 days and their viability was monitored by measuring adenosine triphosphate (ATP) production. Incubation of spheroids with lipogenic substrates...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Andrea Wagnerová Supervisor: prof. Ing. Barbora Szotáková, Ph.D., Mgr. Kateřina Černá Pilátová, Ph.D. Title of diploma thesis: Single nucleotide polymorphism in the patogenesis of mature B-cell neoplasms Chronic lymphocytic leukemia (CLL) is a disease characterized by clonal proliferation of B-lymphocytes. CLL is a lymphoproliferative disorder with a number of defined risk factors, but its etiopathogenesis remains unresolved to some degree. Over the past 10-15 years, there have been several studies that primarily suggest a genetic predisposition to the disease. The influence of external factors on the etiology of CLL has not been demonstrated. There is also a defined asymptomatic condition associated with CLL called monoclonal B-lymphocytosis, which is often referred to as a precursor of CLL. Immunophenotyping of lymphocytes by flow cytometry was performed in subjects of the control group (100 subjects) and the CLL/MBL group (patients diagnosed with CLL or MBL, 50 subjects). All subjects in the control and CLL/MBL groups were screened for selected genetic polymorphisms (rs17483466, rs735665, rs7176508, rs872071, rs13397985) that have been associated with the occurrence of CLL and MBL in other...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Samanta Rysová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Consultant: RNDr. Radka Podlipná, Ph.D. Title of diploma thesis: The effect of selected herbicides on UGTs expression and activity of antioxidant enzymes in Arabidopsis thaliana Herbicides are widely used to control weeds in agriculture. Although their use is essential to ensure crop production, their effect occurs to be reduced due to the development of herbicide resistence. Therefore it is important to clarify the mechanism of herbicide resistence, so that herbicides can be used effectively and new substances can be developed if necessary. At the same time, herbicides acts as an abiotic stress factor on the plant, thus affecting the antioxidant apparatus of plant cells. The aim of this work was to investigate the effect of selected herbicides - tribenuron-methyl (TBM), pinoxaden (PNX) and sulcotrione (SLC), on the expression of selected UGTs that could play a role in herbicide resistence. Secondary, we want to find out the effect of TBM on the activity of selected antioxidant enzymes - ascorbate peroxidase (APX), catalase (CAT) and superooxide dismutase (SOD). Arabidopsis thaliana was used as a model plant and the experiment was...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Monika Lisoňková Supervisor: prof. Ing. Barbora Szotáková, PhD. Title of diploma thesis: The effect of albendazole on the antioxidant system of alfalfa in real conditions Anthelmintics are drugs used to treat diseases caused by parasitics worms. In the Czech republic they are mainly used in veterinary medicine. Benzimidazole anthelmintic albendazole is used in cattle and sheep, during its biotransformation metabolites are formed, which are excreted together with the parent compound in faeces. Application of faeces from treated cattle to the field may affect non-target organisms, occurring in the field. An example of such organisms are agricultural crops, including alfalfa (Medicago sativa). Plants do not have the ability to eliminate the absorbed drugs and its metabolites, therefore they are deposited in plants and can affect the antioxidant system of plants. The aim of this study was to monitor the effect of albendazole on the antioxidant system of alfalfa (M. sativa) in real conditions. Plants were affected by faeces of sheep treated with albendazole. Subcellular fractions were prepared from harvested plants in the years 2019 and 2020. The concentrations of photosynthetic pigments...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Josef Krátký Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Introduction of the MST method for the study of interactions between biomolecules MicroScale thermophoresis (MST) is currently a recognized method for studying interactions between biomolecules; the method is using directed movement of biomolecules in a temperature gradient, which allows to analyze practically all interactions where changes in analyte properties are possible. This thesis was focused on introduction of the MST method for research of protein interactions with low molecular weight substances focusing on verifying the possibility of using MST in monitoring the interaction of glutathione S-transferase (GST) with selected terpenes ((-)-fenchone, (-)-isopulegole, (+)-isomenthone, (-)-carvone, (+)-carvone, piperitone, and thujone). Another aim was to determinate the properties of terpenes by classical spectrofluorimetric methods and MST. Using spectrofluorimetric methods it was found that terpenes affect the fluorescence of human serum albumin, and they do not affect the fluorescence spectrum of the dye NHS, which is used as a dye for samples for MST. Next part of experimental work was to determine...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Mikešková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Study of the effect of albendazole in the environment on the antioxidant system of red clover (Trifolium pratense) Today, anthelmintics are among the essential drugs for the therapy and prophylaxis of infectious diseases caused by parasitic worms. However, these substances may be further released into the environment by the excrements of treated animals and affect non-target organisms, such as plants, which have the ability to absorb and accumulate various substances. In this study, we focused on the effect of the often used anthelmintic albendazole on the antioxidant system of red clover (Trifolium pratense), which is commonly found in meadows and pastures. The effect of low concentrations of albendazole available in the environment on the antioxidant enzymes catalase, superoxide dismutase, ascorbate peroxidase, peroxidase, glutathione peroxidase, glutathione reductase and glutathione S-transferase was studied. In addition to antioxidant enzymes, we also monitored the concentration of plant photosynthetic pigments chlorophyll a, b and carotenoids. The results were evaluated in control and...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Bc. Helena Vondráčková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Specialist consultant: MUDr. Pavel Pick Title of diploma thesis: Relationship between compensatory status and renal function in patients with diabetes mellitus type II Diabetes mellitus is a chronic disease accompanied by a number of complications. Among the most common and at the same time the most serious are kidney diseases, which often lead to their failure. These patients then rely on lifelong dialysis treatment or a possible kidney transplant. By compensating for diabetes mellitus effectively, the development of complications can be reduced or at least delayed. Fasting and postprandial blood glucose level and glycated hemoglobin are used to assess the state of compensation. The renal function is assessed by the examination of albumin and proteins in urine, serum creatinine concentration and estimation of glomerular filtration. The aim of the thesis was to determine whether there is a relation between the level of compensation for diabetes mellitus and the renal function. The data from 70 patients diagnosed with diabetes mellitus type II regularly checked in a diabetological clinic were evaluated. Samples were...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Patrícia Graňáková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Consultant: RNDr. Radka Podlipná, Ph.D. Title of diploma thesis: Modulation of expression and activity of selected plant detoxifying enzymes by anthelmintic Anthelmintics represent risk to environment as they may impact non-target organisms including plants, which come into contact with these pharmaceuticals in fields by fertilization with dung from treated animals or in pastures by excrements of treated animals. After uptake, these substances can increase the production of reactive oxygen species in plants, with the risk of oxidative stress and plant damage, and also affect the antioxidant enzymes. The aim of this work was to investigate the effect of two widely used anthelmintics ivermectin and fenbendazole on the activity and expression of selected antioxidant enzymes in soybean (Glycine max). Soybean was cultivated in a greenhouse and watered with a 10 µM solution of the selected drug. The changes of activity and gene expression of antioxidant enzymes were measured in root, leaf, pod and seed samples. Results showed that both anthelmintics caused significant decrease of superoxide dismutase, ascorbate peroxidase, glutathione...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Tereza CYRUSOVÁ Supervisor: prof. Ing. Barbora SZOTÁKOVÁ, Ph.D. Consultant: RNDr. Radka PODLIPNÁ, Ph.D. Title of Doctoral Thesis: Effect of Nanoparticles on Plant Metabolism Along with the expansion of nanotechnologies, which are used in many disciplines, the field of nanotoxicology has recently been developed. It deals with the negative effects of engineered nanoparticles on the environment and biota. Considerable attention has already been paid to the toxicity of nanoparticles to plants, however, the mechanisms responsible for their effects are not fully understood yet. At the same time, the possible positive effect of nanoparticles on plants has been investigated in recent years. The aim of our work was to study and evaluate the effect of nanoparticles on the growth of selected plant species and to evaluate their phytotoxicity. We focused on the study of nanoparticles in the form of metal oxides. The content of accumulated metals in germinating plants of mustard (Sinapis alba L.), in in vitro cultures of true-fox sedge (Carex vulpina L.) and the concentration of metal ions released into the culture media were measured by atomic absorption spectrometry (Cu, Fe, Mn, Zn). and inductively...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Anna Křížová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of diploma thesis: Effect of selected bicyclic monoterpenes on the activity and expression of antioxidant enzymes in human liver Monoterpenes are plant secondary metabolites, which are used as flavourings or aromas in the food industry due to their smell and flavour. They also play an irreplaceable role in the cosmetic and pharmaceutical industry. Monoterpenes are substances with a wide spectrum of biological activities. They exert, for example, antimicrobial, anti- inflammatory, antioxidant, hypotensive, and analgesic effects. In addition to the positive effects of these substances, some cases of their toxic effects in various organs were also observed, mostly in the liver. Monoterpenes also affect the activity and expression of antioxidant enzymes. The aim of this thesis was to determine the effect of five selected bicyclic monoterpenes, (+)-camphor, (-)-camphor, (-)-fenchone, camphene, and (-)-α-thujone, on the activity and expression of antioxidant enzymes. Of those enzymes, glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) were studied. At...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Marie Elstnerová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Pistacia lentiscus essential oil: in vitro testing of cytotoxicity and anti-inflammatory activity This project, which has been initiated in cooperation with University of Sassari (Italy), is aimed to find out more details about biological activity of essential oil from Pistacia lentiscus. We want to contribute to the decision, if this essential oil can be used as a part of oral health products. The essential oil was prepared from leaves of wild occurring plants growing in North Sardinia. The cytotoxicity and anti-inflammatory activity of not only the pure essential oil but also of the lecithin nanoemulsion with P. lentiscus oil and Tween 80 nanoemulsion with P. lentiscus oil and moreover, the two most abundant terpenes in the essential oil, (−)-α-pinene and (−)-terpinen-4-ol, were tested. The cytotoxicity was measured by the colorimetric assay using tetrazolium salt WST-1 on four oral cell lines (gingival fibroblasts, periodontal ligament fibroblasts, dysplastic oral keratinocytes and primary gingival keratinocytes). The viability of human oral cells was the most diminished by lecithin nanoemulsion, pure...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Eliška SYSLOVÁ Supervisor prof. Ing. Barbora SZOTÁKOVÁ, Ph.D. Consultant RNDr. Radka PODLIPNÁ, Ph.D. Title of Doctoral Thesis: Anthelmintics in Plants - Uptake, Biotransformation and Transcriptional Response The use of anthelmintics, drugs against parasitic worms, is almost necessary in veterinary medicine today. However, anthelmintics may enter the environment with the excrements of treated animals, and influence non-target organisms there. Plants which can receive, biotransform or accumulate these substances, are an integral part of the environment. Biotransformation of anthelmintics may affect the capacity of the antioxidant system, interfere with endogenous plant metabolism or polyphenol biosynthesis where plants use the same detoxifying enzymes. The aim of this thesis is to study the influence of the most commonly used anthelmintics albendazole (ABZ), fenbendazole (FBZ), flubendazole (FLU) and ivermectin (IVM) on plants. To study their uptake by plants, their biotransformation in plants and transcriptional response of plant cells to their presence. The results of the dissertation include the finding that model systems of cell suspensions and in vitro regenerants of Plantago lanceolata...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Tereza Švábová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Consultant: Mgr. Michaela Šadibolová Title of diploma thesis: Effect of monoterpene citral on the expression of detoxification enzymes in HepaRG cells Citral is an acyclic monoterpene, which is mainly produced by higher plants. It is one of the many secondary metabolites, which serve the plant as detoxifying factors, morphoregulators and attractants for pollinators or defense substances. Besides other things, it has numerou positive effects such as antibacterial, anti-inflammatory or antitumor, but it may be toxic, especially for the liver, if used incorrectly. The main goal of this thesis was to find out the effect of citral on selected biotransformation and antioxidant enzymes in HepaRG cells. Differentiated HepaRG cells were incubated with 10 µM rifampicin, 10 µM β-naphthoflavone, 30 µM oltipraz, 10 µM citral, 30 µM citral and 100 µM citral for mRNA expression detection for 12 and 24 hours and for protein expression evaluation for 24 and 48 hours. At the mRNA level, rifampicin induced CYP3A4 at 12- and 24-h incubation, β-naphthoflavone induced CYP1A2 at 12- and 24-h incubation and aldo-ketoreductase (AKR1C) at 24-h incubation,...

ASBSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Jana Štěpánková Supervisor: Prof. Ing. Barbora Szotáková, Ph.D. Title of Thesis: Characterization of flubendazol reduction in human in vitro Flubendazole (FLU) is an anthelmintic of the benzimidazole type used as a treatment or prophylaxis of helminthoses in livestock and wild animals. FLU is also registered for the treatment of parasitic diseases caused by intestinal nematodes in humans. Its mechanism of action lies in its specific binding to tubulin, which disrupts the structure and function of microtubules for the therapy of parasitosis in humans. The advantage of this drug is also the low systemic toxicity. That is why the anticancer activity of FLU is now under study. The aim of this rigorous work was to characterize the reduction of FLU in human liver subcellular fractions as this information is still lacking in available literature. Stereospecificity, subcellular localization, preference of coenzymes, enzyme kinetics of FLU reduction, and activity of enzymes involved in FLU reduction were studied in this project. Measurement results have shown that FLU is predominantly reduced in cytosol and nicotinamide adenine dinucleotide phosphate reduced (NADPH) coenzyme is preferred. FLU...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: MSc. Lucie Zemčíková Supervisor: Assoc. Prof. PharmDr. František Trejtnar, CSc. Title of doctoral thesis: The effect of natural compounds on transport by OATP drug transporters OATP membrane transporters belong to carrier proteins responsible for transporting certain drugs (e.g. hypolipidemics from the group of statins) and other xenobiotics across the biological membranes and tissue barriers within the body. These transport proteins play an important role in pharmacokinetic processes such as absorption, distribution and elimination. The potential modulation of their transport function by natural compounds commonly present in plant food or food supplements may result in the changes of the concentration of their substrate (drug) in the cells and body fluids, that may affect the effect and toxicity of these drugs. The aim of this study was to obtain data on the interactions of selected natural compounds with human transporters OATP2B1 and OATP1A2 and their ability to affect drug transport mediated by these transporters. These two OATP transporters are involved in the drug uptake especially in organ barriers important for pharmacokinetics. For the study natural compounds from the group of...

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Kateřina Duchačová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of rigorous thesis: Influence of glutamate-induced obesity on detoxification enzymes contains glutathione Obesity and related comorbidities occur more frequently in the population and belong to the most studied contemporary health topics. Aging of the organism is much discussed topic as well. Various animal models are used to observe changes in the organism at the organ, tissue, cellular and molecular levels. Comparison of specific activities and protein expressions of glutathione peroxidase (GPx) and glutathione reductase (GR), and specific activity of glutathione-S-transferase (GST) in the liver of obese mice and healthy controls according to the pathological state, aging and sex was the aim of this study. Mice with monosodium glutamate (MSG)-induced obesity, osmolal controls (NaCl) and mice without treatment of both sexes and at 8 and 20 weeks of age were used. Specific enzyme activities were determined spectrophotometrically. Protein expressions were determined using polyacrylamide gel electrophoresis followed by immunoblotting. The biggest obesity-related changes in enzyme activity were observed in GST, which activity...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Veronika Mazurková Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of rigorous thesis: Effect of selected sesquiterpenes on carbonyl reductase 1 activity in vitro Sesquiterpenes, the main components of plant essential oils, are integral parts of spices, traditional food and beverages. They are used in folk medicines, pharmaceutical industry as well as in cosmetics. Several sesquiterpenes possess interesting biological activities but they could interact with concurrently administered drugs via the modulation of activity and/or expression of drug-metabolizing enzymes. Enzyme carbonyl reductase 1 (CBR1) is one of the enzymes participating in the metabolism of a number of endogenous and xenobiotic compounds. One of the CBR1 substrates is chemotherapeutic agent doxorubicin, which is metabolized by this enzyme to the less effective metabolite doxorubicinol, which is responsible for the doxorubicin cardiotoxicity. The aim of this rigorous thesis was to find out the ability of five selected sesquiterpenes (i.e. valencene, β-caryophyllene oxide, α-humulene, cis-nerolidol and trans-nerolidol) to affect the activity of CBR1 in vitro. The effect of studied substances on the activity of this enzyme was...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bibiána Lancošová Supervisor: doc. PharmDr. Boušová Iva, Ph.D Title of diploma thesis: Toxicity of pulegone and menthofuran and their effect on miRNA expression in human liver slices Monoterpenes are secondary plant metabolites, characterised by their odour, which have a positive impact on human body and are used not only in cosmetic and food industry, but also in medication. R-pulegone (PUL) and its metabolite R-menthofuran (MF) are the most represented in plants of genus Mentha (Mint), with higher doses of these substances exerting certain risk of hepatotoxicity. The aim of this thesis was to assess the concentration, at which the viability of liver cells decreases and also to observe the changes in individual microRNA expression as a result of influence of PUL, MF and paracetamol, as the reference compound. Five samples of human liver obtained from patients of both genders and at age of 57 to 73 years represented biological material for hepatotoxicity testing. The samples were stored in Euro-Collins solution and subsequently cut into precision-cut liver slices with thickness of 150- 170 µm, placed to 12-well plate and put into incubation medium with the tested substances for 24 hours. The...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Nikoleta Dojčanská Supervisor: prof. Ing. Barbora Szotáková, Ph.D Title of diploma thesis: Environmental risks of anthelmintics Anthelmintics are drugs used in human and veterinary medicine for the treatment and prophylaxis of infectious diseases caused by parasites- helminths. They are admittedly beneficial to the treated organisms. However, their overall effect on the ecosystem has not yet been sufficiently explored. Anthelmintics, together with their metabolites, enter the environment in multiple ways. One possibility is directly through urine and excrements of treated livestock or indirectly by leakage of contaminated manure into surface and groundwater. Such pollution has an effect on various animal organisms and plants that have the ability to uptake xenobiotics and remove them through metabolic pathways. The aim of this work was to investigate the effect of antihelmintics ivermectin and fenbendazole on soybean plants (Glycine max). We investigated the concentration of total phenolic compounds spectrophotometrically using the Folin-Ciocalteu method. Using the colorimetric method of aluminum chloride, we found the concentration of flavonoids in soybean plants. The concentration of isoflavons-...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jaroslav Bobek Supervisor: Doc. PharmDr. Iva Boušová, Ph.D. Supervisor specialist: RNDr. Veronika Skarková, Ph.D. Title od diploma thesis: The effect of trabectedin on the cells derivated from colorectal carcinoma Trabectedin is alkaloid, which occurs naturally in sea tunicate Ecteinascidia turbinata. Its mechanism of action consist of creation complexes with DNA, transcription factors and nuclears proteins. This cause blocade of transcription and defect of repair mechanism of DNA in cell. We can use it in therapy of some types of cancer, in this particular work we was researching influence on colorectal carcinoma. We tested this substance on two cancerous cell lines SW 480 - derivated from primary deposit of adenokarcinoma and SW 620 - metastatic line from lymph nodes from same patient. Prime cell cultures marked as 36B and 39B were secured from patients in University Hospital in Hradec Králové and their following cultivation. Both samples 36B and 39B were derived from the primary colon tumor deposit. On all cell lines was performed cytotoxicity assay and then was individualy calculated IC50. These values were used in migration assay for cells 36B and 39B. After treatment with trabectedin both...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lenka Matějková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Consultant: RNDr. Radka Podlipná, Ph.D. Title of diploma thesis: Effect of nanoparticles on plant proteome The environment is contaminated by increasing amounts of different types of xenobiotics. Nanomaterials are relatively "new" group of pollutants. Effective method used for decontamination of the environment can be phytoremediation which uses higher plants for biotransformation and accumulation of pollutants. The plant is suitable for phytoremediation when it can absorb, metabolize and store contaminants without significant impact on its metabolism. Influence on the function and construction of the plant can be detected at the proteome level. In this work, we observe the effect of copper oxide nanoparticles on plant proteome of Arabidopsis thaliana. The effect of standard copper oxide particles and copper sulfate ions was also studied in one-day and four-day period for comparison. The proteomes were compared and changes were analyzed by software PDQuest. Changes in intensity of protein spots comparing to the control sample correspond to modulation of the amount of different types of plant proteins. This modulation depends on the...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Kateřina LNĚNIČKOVÁ Supervisor: prof. Ing. Barbora SZOTÁKOVÁ, Ph.D. Title of Doctoral Thesis: MODULATION OF BIOTRANSFORMATION AND ANTIOXIDANT ENZYMES BY SELECTED NATURAL COMPOUNDS Public interest in various dietary supplements containing herbs, herbal extracts or isolated active compounds has increased significantly over past decades. Consumption of these supplements increases worldwide and they are often consumed in unreasonably high doses, as they are generally considered as safe. Upon the intake to organism, these compounds are, as other xenobiotics, modified mostly by xenobiotic-metabolizing enzymes and they could influence these enzymes at the same time. Potential modulation of xenobiotic-metabolizing enzymes' activity (induction or inhibition) can seriously affect pharmacokinetics of concomitantly administered drugs. Knowledge of the possible impact of natural compounds on the xenobiotic-metabolizing enzymes is essential for their safe use. The aim of this doctoral thesis was to study the effects of selected herbal extracts and their active chemical constituents on the activity and expression of xenobiotic-metabolizing and antioxidant enzymes. We have focused on the study of effects of...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Martina Plšková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of rigorous thesis: The systemic inflammatory response of the organism to Clostridium difficile infection Clostridium difficile is currently considered to be the major etiological agent of healthcare-associated diarrhoea in the developed countries. CDI (Clostridium difficile infection) is a potentially life threatening disease of increasing incidence. Despite worldwide research of new highly potent drugs, CDI treatment remains a major problem with regard to the high probability of recurrence and treatment resistance. The aim of this thesis is to find out the number of patients with CDI in a given time period and how CDI affects the C-reactive protein (CRP), leukocyte and neutrophil levels. Rapid membrane enzyme immunoassay for the simultaneous detection of C. difficile glutamate dehydrogenase (GHD) antigen and toxins A and B in a single reaction was used. The RT-PCR method was applied to confirm where a positive result for antigen but negative for toxin appeared. The biochemical determination of CRP was performed turbidimetrically. Complete blood count including a differential count was performed using electrical impedance...