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Preparation and characterization of neutral trehalase for structural studies
Šmídová, Aneta ; Obšilová, Veronika (advisor) ; Jiráček, Jiří (referee)
This study is part of a project which aim is solving the structure of the catalytic domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is the preparation of new constructs of yeast Nth1 and optimization of their purification protocols, the selection of the ideal buffer for crystallization trials using the method of differential scanning fluorimetry (DSF) and at last the protein crystallization. Another part of the thesis is the measurement of the enzymatic activity of pNth1 WT in the presence of Bmh1 protein, verification of trehalose binding to the selected constructs of Nth1 using differential scanning fluorimetry (DSF), thermoforesis (MST) and further crystallization with trehalose. Neutral trehalase is highly conserved trehalase that has been found in a wide variety of organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and hydrolytically cleave trehalose into two glucose molecules. Trehalose is a naturally occurring non-reducing disaccharide serving in yeast cells a source of carbon and energy as well as protection against stress conditions such as a thermal shock. Trehalose hydrolysis is essential for flying insects, because it is present as the main sugar component of insect haemolymph, therefore trehalase inhibitors...
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High-throughput screening for the discovery of small molecules modulating cell fate
Ribeiro Pombinho, António José ; Bartůněk, Petr (advisor) ; Bařinka, Cyril (referee) ; Jiráček, Jiří (referee)
The discovery of chemical compounds able to modify the way cells proliferate, differentiate or die can lead not only to the formulation of new drugs for disease treatment or prevention but also to their use as biological probes in the study of the molecular pathways involved in these processes. In order to test thousands of these small molecules in cellular assays, instrument automation and assay miniaturization are necessary. In this thesis, applications of High-Throughput Screening campaigns are described. The Hypoxia and Wnt pathways involved in stem and cancer cell proliferation; the differentiation of hematopoietic, neural and mesenchymal stem cells; and the TRAIL pathway leading to selective cancer cells death were the main subjects chosen. With this approach, it was possible to test the effect of small molecules in eukaryotic cells and in unicellular organisms as exemplified by the search of compounds leading to the death of the protozoan parasite Leishmania. Several chemical compounds were identified as active in modulating cell fate. Of remark were: Monensin that inhibits the Wnt pathway and prevents the growth of tumors in a mouse model of colorectal cancer; Homoharringtonine that, only in combination with TRAIL, induces the death of cancer cells implanted in immunodeficient mice; and...
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The Specific Features of Collectivization in Svitavy District between 1949 and 1960
Jiráček, Jiří ; Jančík, Drahomír (advisor) ; Jakubec, Ivan (referee)
The thesis deals with the process of collectivization in the area which was the called Svitavy National District Committee. The thesis represents economic and social transformation after the expulsion of Germans from Czechoslovakia. First chapter serves general characteristic of Svitavy District and Svitavy National District Committee which was the intermediary among the government and individual vil- lages. The next chapter focuses on the process of collectivization in Svitavy District divided into three phases - first phase (1949-1953), second phase (1953-1954) and third phase (1954-1960). Regional development is associated with nationwide con- texts which were connected with the process of collectivization. The following chap- ter devotes to individual process of collectivization in selected villages. The author attempts to show the peculiarities of realization of collectivization on the base of micro-historical insight. The aim of the last chapter is to outline the mechanism of judicial repression against private farmers. The main attention is focused on emi- nent local private farmer František Blažek. The trial of František Blažek became de- terrent tool which should accelerate the liquidation of "kulaks". The thesis describes the distinctions among the newly settled villages (by Czech people)...
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Synthesis and characterization of the new insulin analogues with the aim to clarify the interaction of insulin with its receptor
Kletvíková, Emília ; Jiráček, Jiří (advisor) ; Obšil, Tomáš (referee) ; Hrabal, Richard (referee)
The objective of this thesis is to characterize insulin analogues modified at the C-terminus of the B-chain with the aim to observe the impact of the inserted modifications on the insulin-insulin receptor (IR) interaction and the ability of the analogues to dimerize. Therefore, a series of analogues with modifications at B24-B26 positions was prepared. Using the synthetic and semisynthetic methods we inserted coded and non-coded amino acids to this part of B-chain. We studied full-length analogues and analogues truncated by three to four amino acids. Binding affinity of all analogues to the insulin receptor was determined by competition of analogue with radioactive (125I) human insulin. Dissociation constant in the dimer dissociation process of selected analogues (especially of those with N- methylation of B23-B24, B24-B25 and B25-B26 peptide bonds) was determined by isothermal titration microcalorimetry. The crystal structures of several analogues were resolved by X-ray crystallography and nuclear magnetic resonance. The structural results showed the consequences of inserted modifications to the insulin molecule. We characterized analogues with higher, equipotent and lower binding affinity to the IR. The results...
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The metabolism and signaling of hydrogen sulfide: the role of CBS-related proteins in Caenorhabditis elegans
Vozdek, Roman ; Kožich, Viktor (advisor) ; Macůrková, Marie (referee) ; Jiráček, Jiří (referee)
Hydrogen sulfide (H2S) is a toxic gas that causes respiratory failure and death at high concentrations, but at low concentrations, it functions as a signaling molecule in vasodilation and neuromodulation, and it protects cells and tissues from reperfusion injury, hypoxia, hyperglycemia and endothelial dysfunction. Several model organisms have been used to study the physiological roles and signaling pathways of H2S. The roundworm Caenorhabditis elegans is a remarkable model for studying the physiology, developmental biology and signaling of H2S; however, the metabolism of H2S in this animal is largely unknown. Cystathionine beta-synthase (CBS) is one of three H2S-producing enzymes in mammals. Notably, C. elegans possesses 6 genes that encode proteins homologous to CBS, namely cbs- 1, cbs-2, cysl-1, cysl-2, cysl-3 and cysl-4. In this thesis we studied the roles of these genes in H2S metabolism and signaling. First, we identified cbs-1 as the gene encoding CBS in C. elegans; the recombinant purified CBS-1 protein exhibited canonical CBS activity, and RNA interference-mediated silencing of cbs-1 resulted in decreased CBS activity and increased homocysteine levels in worm extracts, recapitulating the phenotypes of CBS deficiency in mammals. Notably, the nematode and human enzymes differ in their domain...
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Study of physiological functions of betaine homocysteine S-methyltransferase and betaine homocysteine S-methyltransferase 2
Mládková, Jana ; Jiráček, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kožich, Viktor (referee)
Betaine homocysteine S-methyltransferase (BHMT) and betaine homocysteine S-methyltransferase 2 (BHMT-2) are mammalian cytosolic metalloenzymes. They both participate in the metabolism of homocysteine (Hcy), specifically Hcy remethylation, mainly in liver and kidney cells. BHMT catalyzes the transfer of a methyl group from betaine to L-Hcy, yielding L-methionine and dimethylglycine (DMG). BHMT-2 catalyzes the transfer of a methyl group from S-methylmethionine (SMM) to L-Hcy as well, yielding two molecules of L-methionine. Disorders in Hcy metabolism could lead to the so called hyper- homocysteinemia and homocystinuria, which can be connected with several pathological conditions. BHMT is already relatively well characterized enzyme. Its crystal structure and reaction mechanism have been described and a series of BHMT inhibitors have been prepared. The specific inhibitors enabled further in vivo studies and, recently, Bhmt-/- mice model has been successfully developed. In contrast, the research of BHMT-2 is still at the beginning and physiological functions of the enzyme are unknown so far. The reason is that BHMT-2 is a highly unstable enzyme and also there is a lack of selective BHMT-2 inhibitors. BHMT and BHMT-2 are very similar enzymes which have 73% amino acid identity. This thesis provides new...
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Study of physiological functions of betaine homocysteine S-methyltransferase and betaine homocysteine S-methyltransferase 2
Mládková, Jana ; Jiráček, Jiří (advisor)
Betaine homocysteine S-methyltransferase (BHMT) and betaine homocysteine S-methyltransferase 2 (BHMT-2) are mammalian cytosolic metalloenzymes. They both participate in the metabolism of homocysteine (Hcy), specifically Hcy remethylation, mainly in liver and kidney cells. BHMT catalyzes the transfer of a methyl group from betaine to L-Hcy, yielding L-methionine and dimethylglycine (DMG). BHMT-2 catalyzes the transfer of a methyl group from S-methylmethionine (SMM) to L-Hcy as well, yielding two molecules of L-methionine. Disorders in Hcy metabolism could lead to the so called hyper- homocysteinemia and homocystinuria, which can be connected with several pathological conditions. BHMT is already relatively well characterized enzyme. Its crystal structure and reaction mechanism have been described and a series of BHMT inhibitors have been prepared. The specific inhibitors enabled further in vivo studies and, recently, Bhmt-/- mice model has been successfully developed. In contrast, the research of BHMT-2 is still at the beginning and physiological functions of the enzyme are unknown so far. The reason is that BHMT-2 is a highly unstable enzyme and also there is a lack of selective BHMT-2 inhibitors. BHMT and BHMT-2 are very similar enzymes which have 73% amino acid identity. This thesis provides new...
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Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
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Chiral analysis of beta-alanyl-D,L-tyrosine and its derivatives by capillary electrophoresis with 2-hydroxypropyl-beta-cyclodextrin stereoselector
Sázelová, Petra ; Šolínová, Veronika ; Schimperková, Tereza ; Mášová, Alice ; Jiráček, Jiří ; Kašička, Václav
A new capillary electrophoretic method was developed for separation of enantiomers of antimicrobial dipeptide betha-Ala-D,L-Tyr and its derivatives using 50 mM Tris-phosphate, pH 2.50, as background electrolyte and 2-hydroxypropyl-betha-cyclodextrin at concentrations 20 – 60 mg/mL as chiral selector. In addition, association constants of complexes of the enatiomers of dipeptide betha-Ala-D,L-Tyr and its derivatives with 2-hydroxypropyl-betha-cyclodextrin chiral selector were determined.
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Capillary electrophoresis applied to analysis and characterization of mono-N-acyl-2,6-diaminopimelic acid derivatives
Vítovcová, M. ; Hlaváček, Jan ; Pícha, Jan ; Vaněk, Václav ; Jiráček, Jiří ; Kašička, Václav
Capillary zone electrophoresis and micellar electrokinetic chromatography have been employed for determination of electrophoretic purity degree, limit of detection and limit of quantification of twelve mono-Nacylated derivatives of 2,6-diaminopimelic acid (DAP). In addition, the DAP derivatives were characterized by effective electrophoretic mobilities of their cationic and anionic forms in several classical and isoelectric buffer-based background electrolytes within a broad pH range 2.18 – 8.64.
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