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Preparation of strategy for studying interaction of human constitutive androstane receptor with steroid receptor coactivator-1
Šachová, Ivana ; Martin, Jan (advisor) ; Mladěnka, Přemysl (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Ivana Šachová Supervisors: PharmDr. Jan Martin, PhD., Dipl. Ing. Mgr. Ferdinand Molnár, PhD. Title of diploma thesis: Preparation of strategy for studying interaction of human constitutive androstane receptor with steroid receptor coactivator-1 The aim of this study was to express wild type and mutated synthetic human SRC1 central domains in E. coli BL21 DE3 cells. Expressed recombinant proteins were purified afterwards and used for investigation of SRC1-CAR protein interaction using pull down experiments. The constitutive androstane receptor (CAR), a nuclear receptor (NR), is a transcriptional regulator, which influences the expression of various proteins, such as enzymes of biotransformation and transporters important for metabolism of both endogenous and exogenous compounds. CAR can be fully active only in presence of coactivators, such as steroid receptor coactivator-1 (SRC1). The most important fragment of SRC1 for binding to NRs is the central domain, made of three α-helical Leu-X-X-Leu-Leu (LXXLL) L1, L2, L3 motifs, in where L is a leucine and X represents any amino acid. hSRC1, as well as hCAR, can be successfully produced by expression of cDNA of interest in E. coli strains. In case of SRC1,...
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Determination of the stoichiometry of the copper complexes with dehydrosilybine A
Klimková, Kateřina ; Mladěnka, Přemysl (advisor) ; Hrdina, Radomír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Kateřina Klimková Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm. D., Ph.D. Consultant: Assoc. Prof. Kateřina Valentová, Ph.D. Title of Thesis: Determination of the stoichiometry of the copper complexes with dehydrosilybin A Silymarin, the standardized extract of the milk thistle (Silybum marianum), is a widely used approved over-the-counter drug that is recommended for a number of liver diseases. Silymarin contains as one of its components 2,3-dehydrosilybinA, which has an appropriate metal binding site in its structure. In general, flavonolignans, due to their structure, can interact with transition metals in the gastrointestinal tract by forming complexes. This property can be useful for the protection against excessive amounts of metals in the body. The aim of this in vitro study was to analyse the interaction of 2,3-dehydrosilybin A with copper, which plays a crucial role in the organism as a cofactor of many enzymes. Although being an essential element, it can, however, be toxic at elevated levels. Stoichiometry, as one of the most important characteristics of the complex, was determined by UV-Vis spectrophotometry in four (patho)physiological pH conditions (4.5; 5.5; 6.8; 7.5)...
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Copper chelating potential of iron chelators from the group of aroylhydrazones
Kříž, Jakub ; Mladěnka, Přemysl (advisor) ; Bárta, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Farmacology and toxicology Candidate Mgr. Jakub Kříž Consultant Doc. PharmDr. Přemysl Mladěnka, Ph.D. Title of Thesis Copper chelating potential of iron chelators from the group of aroylhydrazones Copper is one of the essential trace elements. It is present in two oxidation-reduction forms Cu+ and Cu2+ in the human body. The ability of an easy transformation between this two forms is the crucial feature for the activity of copper enzymes, like superoxide dismutase and cytochrome c oxidase. Copper kinetics is quite complicated. The key organ which affects copper kinetics is the liver, but the small intestine plays an important regulatory role too. There are disorders, which are associated with both copper deficiency and accumulation. The best known disorder caused by copper excess is Wilson's disease. It is caused by a mutation in the ATP7B gene. It codes a transporter, which is responsible for elimination of copper. Excessive accumulation of copper causes several symptoms, mainly hepatic and neurological problems. Wilson's disease is treated by chelators, which bind the excess of metal ions in the organism. The aim of my thesis was to verify the copper-chelation efficiency of three selected iron chelators. The...
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The influence of polyphenolic compounds and their metabolites on platelet activation
Applová, Lenka ; Mladěnka, Přemysl (advisor) ; Bultas, Jan (referee) ; Šimůnek, Tomáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Applová Lenka, MSc. Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of Doctoral Thesis: The influence of polyphenolic compounds and their metabolites on platelet activation Blood aggregation is a very difficult and complex process, in which platelets play crucial role. On the other hand, their activation is excessive and therefore undesirable under pathophysiological conditions. Cardiovascular diseases are the most common cause of mortality in developed countries and unfortunately, despite significant advances in treatment, the Czech Republic remains in mortality from these diseases behind most Western European countries. Epidemiological studies suggest, that higher intake of (iso)flavonoids is associated with reduced cardiovascular mortality. It is also worth mentioning, that the Czech Republic is among European countries with the lowest content of flavonoids in diet. The aim of this doctoral thesis was to find out, if polyphenolic compounds, especially (iso)flavonoids and/or their metabolites, are able to influence platelet aggregation and thus indirectly to support epidemiological findings, i.e. whether there may be a link between these substances and possible positive...
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Chelation of ferrous ions by derivatives of xanthene-3-one
Pohanová, Lucie ; Mladěnka, Přemysl (advisor) ; Hrdina, Radomír (referee)
v angličtině Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Pohanová Supervisor: Assoc. Prof.. Přemysl Mladěnka, Pharm.D., Ph.D. Title of Thesis: Chelation of ferrous ions by xanthen-3-one derivatives Iron is an essential element important for proper function of cells. Imbalance of iron levels can lead to serious diseases. Since there is no excretory mechanism, the homeostasis is regulated at the level of absorption in the intestine. The iron overload, which leads to tissue damage due to catalysis of the formation of free radicals, occur because of genetic disorders such as hemochromatosis or owing to frequent administration of transfusions. Rational therapy for iron overload is the administration of the chelators. The aim of this study was to evaluate the ability of derivatives of 2,6,7- trihydroxyxanthen-3-one (synthesized at the University of Sarajevo - Dr. Durić) to chelate iron in 4 (patho) physiologically relevant pH conditions. The ferrozine spectrophotometric method was used to determine the degree of chelation. Measurements showed dependency of the chelating effect on pH: lowering pH resulted in the decrease of the effect. At pH 7.5, most of the substances showed 100% ferrous ion chelation in the stoichiometric ratio of 1...
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Determination of the stoichiometry of the copper complexes with dehydrosilybine A
Klimková, Kateřina ; Mladěnka, Přemysl (advisor) ; Hrdina, Radomír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Kateřina Klimková Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm. D., Ph.D. Consultant: Assoc. Prof. Kateřina Valentová, Ph.D. Title of Thesis: Determination of the stoichiometry of the copper complexes with dehydrosilybin A Silymarin, the standardized extract of the milk thistle (Silybum marianum), is a widely used approved over-the-counter drug that is recommended for a number of liver diseases. Silymarin contains as one of its components 2,3-dehydrosilybinA, which has an appropriate metal binding site in its structure. In general, flavonolignans, due to their structure, can interact with transition metals in the gastrointestinal tract by forming complexes. This property can be useful for the protection against excessive amounts of metals in the body. The aim of this in vitro study was to analyse the interaction of 2,3-dehydrosilybin A with copper, which plays a crucial role in the organism as a cofactor of many enzymes. Although being an essential element, it can, however, be toxic at elevated levels. Stoichiometry, as one of the most important characteristics of the complex, was determined by UV-Vis spectrophotometry in four (patho)physiological pH conditions (4.5; 5.5; 6.8; 7.5)...
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Interference of reducing agents with the hematoxylin assay for determination of cupric chelation
Zsáková, Emily ; Mladěnka, Přemysl (advisor) ; Vorčáková, Katarína (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Emily Zsáková Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Interference of reducing agents with the hematoxylin assay for determination of cupric chelation Copper is an essential trace element of living organisms. Disorders of its homeostasis affect various pathological conditions. In excessive amounts, it is toxic due to its ability to form free radicals. Vitamin C, trolox (a water soluble form of vitamin E), glutathione and hydroxylamine possess reducing properties that protect our body from oxidative tissue damage. Oxidized glutathione (GSSG) was also tested for comparison. The object of study was to test the chelating ability of these substances. A simple and fast spectrophotometric method was used. The combination of reducing properties of copper and chelating agents could lead to an improvement in chelation therapy either in heavy metal poisoning, treatment of Wilson's disease, or other diseases. We found that all substances were chelators/copper-chelating agents in vitro in a slightly competitive environment, using hematoxylin as an indicator. However, by using the bathocuproine assays to verify chelating properties, none of the substances...
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Effect of cholinesterase inhibitors on monoaminergic system and energic metabolism
Kalinová, Tereza ; Mladěnka, Přemysl (advisor) ; Štěpánková, Šárka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Kalinová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Mentor: Assoc. Prof. Jana Hroudová, Pharm.D., Ph.D. Title of diploma thesis: Effect of cholinesterase inhibitors on monoaminergic system and energy metabolism Cholinesterase (ChE) inhibitors play an essential role in the treatment of Alzheimer's disease (AD). They effect positively cognitive, functional and behavior symptoms of AD. Up to date, donepezil, rivastigmine and galantamine represent the only ChE inhibitors approved for AD treatment. The first ChE inhibitor was tacrine, which was withdrawn from market due to its toxicity and adverse effects. Recently, novel tacrine and 7-methoxytacrine (7-MEOTA) derivatives were synthetized and extensively investigated to find less toxic compounds affecting pathological mechanisms associated with development of AD. There is less known about effects of these drugs on mitochondrial functions and cellular energy metabolism. The aim of this project is to examine in vitro effects of ChE inhibitors on energy metabolism and cellular respiration, specifically on mitochondrial electron transport chain complexes and an enzyme of the citric acid cycle - citrate synthase. Inhibitory effects...
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Ability of copper chelators to interact with iron and zinc
Hanuščinová, Lucia ; Mladěnka, Přemysl (advisor) ; Čonka, Patrik (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lucia Hanuščinová Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmDr., PhD. Title of diploma thesis: Ability of copper chelators to interact with iron and zinc Copper plays in the human organism a role of an element with indispensable significance, whose biological influence and effects depend on its quantity. With elevated concentrations in the human body, copper becomes toxic, resulting in pathological conditions. The most well-known diseases is the Wilson's disease, whose treatment consists of oral administration of chelators, i.e. chemical compounds, which are capable of binding copper ions in various proportions and eliminating them from the organism. Chelation therapy is currently the first choice after confirmation of the diagnosis. Chelation toxicity results from several factors, e.g. inhibition of copper dependent enzymes or low selectivity to metals. And precisely the selectivity of chelators is being discussed in this diploma thesis. An ideal chelator should not interact with any of the other physiological ions, that are necessary for the proper functioning of the organism. Five of the most frequently therapeutically or experimentally used substances /trientine, D-penicillamine,...
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Transcriptomic analysis of cutaneous inflammatory biomarkers in a mouse model of small fiber neuropathy
Benčová, Simona ; Mladěnka, Přemysl (advisor) ; Smutný, Tomáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Simona Benčová Supervisors: Dr. Claire Demiot, Dr. Aurore Danigo Assoc. Prof. Přemysl Mladěnka, Ph.D Title of diploma thesis: Transcriptomic analysis of cutaneous inflammatory biomarkers in a mouse model of small fiber neuropathy. Peripheral neuropathy is an expanding public health problem conditioned by various diseases and associated with several adverse effects such as the occurrence of chronic pain or increased risk of pressure ulcers (PUs). The aim of this study is to explore, whether the inflammatory state of the skin is modified during peripheral neuropathy and in the course of the formation of a pressure ulcer. The transcriptomic analysis was performed with two different models of mice: PU model and uninjured model, to determine genes that differ in expression and in particular, those involved in inflammation. Small fiber neuropathy was induced in young mice by intraperitoneal injection of resiniferatoxin (50 µg/kg, i.p.) - transient receptor potential vanilloid 1 (TRPV1) agonist. PUs were induced by applying two magnetic plates on the dorsal skin. Gene expression was obtained based on RNA microarray and the results were subsequently verified by qPCR. The transcriptomic analysis of PU...
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