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The use of differential scanning fluorimetry in characterization of selected carbonyl reductase
Tomanová, Radana ; Zemanová, Lucie (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Radana Tomanová Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: The use of differential scanning fluorimetry in characterization of selected carbonyl reductase Differential scanning fluorimetry (DSF) is simple, rapid method that enables to determine optimal conditions for stabilization of proteins and discover their ligands. DSF monitors thermal unfolding of a protein in the presence of fluorescent dye (e.g. SYPRO Orange, 2,6-ANS). The dye is highly fluorescent in non-polar environment such as hydrophobic sites of unfolded protein that appear on the surface in gradual increase of temperature. Melting temperature (Tm) of a protein expresses its stability. Ligand screening relies upon the fact that protein stability is enhanced upon ligand binding (ΔTm 0). The aim of this study was to introduce the DSF method on our department, use it for characterization of carbonyl reductase 1 (CBR1) and evaluate the obtained results with an independent methods and literature. First, the functionality of method was verified with citrate synthase and the conditions were optimized for CBR1. The group of several buffers pH 3-10 without additives or with NaCl or glycerol were tested...
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Role of systemic inflammation in Parkinson's disease
Veselá, Karolína ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Seville, Faculty of Pharmacy Department of Biochemistry and Molecular Biology Candidate: Karolína Veselá Supervisors: Prof. Dr. José Luis Venero Recio, Prof. Ing. Vladimír Wsól, Ph.D Title of diploma thesis: Role of Systemic Inflammation in Parkinson's Disease. Parkinson's disease (PD) is the second most common ageing-related neurodegenerative disorder after Alzheimer's disease and the prevalence in population is increasing. The characteristic movement disorder is caused by selective dopaminergic neurons loss, while the mechanism of this neurodegeneration is not well understood. Increasing evidence points out the key role of vicious cycle of microglial overactivation and oxidative stress, while the questions "where it begins" and "how to stop it" remain without clear answers. This thesis investigates implication of peripheral inflammation as a deteriorating circumstance and possible inductor of brain inflammation and progressive dopaminergic neurodegeneration. We use mice model of Parkinson's disease employing single intraperitoneal injection of toxin N-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) that induces specific degeneration of DA neurons and in contrast to other animal...
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Evaluation of biological activity of resveratrol and its derivatives in cell cultures
Miler, Jaroslav ; Novotná, Eva (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague University Roma TRE Faculty of Pharmacy in Hradec Králové Department of Biology Department of Biochemical Sciences Candidate: Jaroslav Miler Supervisors: Prof. Sandra Incerpi Mgr. Eva Novotná, Ph.D. Title of diploma thesis: Evaluation of biological activity of resveratrol and its Derivatives in cell cultures The aim of this thesis was to research especially antioxidative and partly cytostatic features of resveratrol, its four derivatives (trimethoxy-resveratrol, pterostilbene, oxyresveratrol and AVD 163 - compound which has different position of one methoxy group than trimethoxy-resveratrol) and 2,2',5'-trihydroxychalcone. Experiments were carried out with two different cell cultures (L6 myoblasts from skelatal muscles of rats and human THP-1 monocytes from peripheral blood of patients with acute monocytic leukaemia). Four varied methods were used - fluorimetric ROS determination with DCF-DA (dichlorofluorescein diacetate) as a probe; MTT assay for evaluation of the cytotoxicity (just with L6 myoblasts); cell counting to evaluate the (anti)proliferative activity; electron paramagnetic resonance spectroscopy (EPR) was carried out only with the most promising compounds (2,2',5'-trihydroxychalcone, resveratrol, oxyresveratrol and pterostilbene. Main method was ROS...
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Apoptosis and its role in selected human diseases
Černá, Hana ; Kvasničková, Eva (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Hana âerná Supervisor: Prof. RNDr. Eva Kvasniãková, CSc. Title of diploma thesis: Apoptosis and its role in selected human diseases Apoptosis, which represents a specific form of programmed cell death, is an essential physiological process. It is very important for the organism's developement and for the maintenance of tissue homeostasis. It is a highly regulated process that is always triggered by a definite signal. This signal activates specific apoptotic pathways, a series of mechanisms that subsequently lead to cellular decline. The dysregulation of apoptosis is associated with a variety of pathological conditions such as cancer, some disorders of the immunty system or of the cardiovascular system, or neurological diseases. To the last mentioned belong neurodegenerative disorders that are characteristic of the decline of selective neuronal populations. Examples of these are Alzheimer's and Parkinson's disease. Although its pathogenesis is still not fully elucidated, many studies indicate that the loss of neurons could be tightly related to excessive apoptotic activity. In spite of some discrepancies apoptosis can therefore be expected to an important part of the pathogenesis of these...
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Cloning, expression and purification of human AKR1B1
Lundová, Tereza ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Lundová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1B1 Aldose reductase (EC 1.1.1.21) AKR1B1 is one of the 13 human enzymes of the AKR superfamily. All human AKRs are cytosolic and NADP(H) dependent. AKR1B1 plays an important role in metabolism of endogenous and exogenous substances. The main endogenous substrate is glucose. Its reduction to sorbitol is consistently linked to secondary diabetic complications. From xenobiotics metabolized by AKR1B1, daunorubicin, an anticancer drug from the group of anthracyclines, is reduced to daunorubicinol. This metabolite is less active than parent drug and is the cause of anthracycline related cardiotoxicity. At present, many projects are focused on AKR1B1 as a target enzyme and specific inhibitors of AKR1B1 are looking for. The recombinant protein of AKR1B1 was prepared in E. coli together with the pET expression system. First, cDNA for AKR1B1 in pOTB7 was isolated from E. coli. The coding sequence of AKR1B1 was amplified by a PCR. PCR was performed with Phusion Hot Start II polymerase and pair of forward and reverse primers, which contained NdeI and XhoI restriction...
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