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Preparation of neprosin I - unique aspartic protease with potential in structural biology, proteomics and treatment of gluten intolerance.
Pelechová, Kamila ; Man, Petr (advisor) ; Šmídová, Aneta (referee)
Proteases are being found in all realms from viruses and microorganisms to plants and animals, including humans. They are necessary for living organisms. They degrade proteins to peptides and amino acids, allowing organisms to better absorb nutrients. Proteases also have a regulatory function - they control the cell cycle, they are responsible for converting precursors to biologically active substances, they are part of signaling cascades. Thanks to their properties, proteases are used for therapeutic purposes but found their used also in industry and research. This work deals with the production of recombinantly prepared neprosin - a newly discovered prolyl endopeptidase isolated from the carnivorous plant Nepenthes ventrata. It is an acidic protease with potential for use in structural biology, proteomics and medicine. Due to its ability to cleave primarily C-terminal proline residues, neprosin become a promising option for celiac enzyme therapy. (In Czech)
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Structural studies of 14-3-3 protein complexes
Šmídová, Aneta
The 14-3-3 proteins are small regulatory biomolecules that are important for many important metabolic or signaling pathways. These conserved proteins were identified in all types of eukaryotic cells and they affect, for example, activity and stability of binding partners, subcellular localization and interactions with a number of proteins. The 14-3-3 proteins are the subject of research of neurodegenerative diseases and cancer. This thesis reveals the study of 14-3-3 proteins and several binding partners, which are important for various metabolic processes for humans or yeasts. Due to the number of verified 14-3-3 interaction partners the structural characteristic i s crucial for understanding of the mechanism of their action in a variety of cellular processes. In this work, the machanims of regulation of yeast neutral trehalase 1 is studied, as well as inhibition of human variant of caspase-2 and the influence of 14-3-3 protein binding on regulation of yeast cytoplasmic antiporter Na+ K+ /H+ are investigated. A wide range of biological, dominantly fluorescence and protein crystallography methods, have been used. Based on the experimental data, the crystal structure of the fully active neutral trehalase 1 (Nth1) in complex with the 14-3-3 protein was resolved, the structure revealed a unique...
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Preparation of neprosin I - unique aspartic protease with potential in structural biology, proteomics and treatment of gluten intolerance.
Pelechová, Kamila ; Man, Petr (advisor) ; Šmídová, Aneta (referee)
Proteases are being found in all realms from viruses and microorganisms to plants and animals, including humans. They are necessary for living organisms. They degrade proteins to peptides and amino acids, allowing organisms to better absorb nutrients. Proteases also have a regulatory function - they control the cell cycle, they are responsible for converting precursors to biologically active substances, they are part of signaling cascades. Thanks to their properties, proteases are used for therapeutic purposes but found their used also in industry and research. This work deals with the production of recombinantly prepared neprosin - a newly discovered prolyl endopeptidase isolated from the carnivorous plant Nepenthes ventrata. It is an acidic protease with potential for use in structural biology, proteomics and medicine. Due to its ability to cleave primarily C-terminal proline residues, neprosin become a promising option for celiac enzyme therapy. (In Czech)
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Stereotypes about bilingualism
ŠMÍDOVÁ, Aneta
The topic of this bachelor thesis is the phenomenon of bilingualism, specifically stereotypes about bilingualism and bilingual upbringing. The aim of the thesis is to describe these stereotypes and either disprove the existing prejudices or, on the other hand, confirm them as relevant. The thesis comprises a general introduction in bilingualism, predominantly in children, typology of bilingualism, theoretical knowledge about bilingual upbringing and, finally, strategies of upbringing in bilingual families. Also discussed will be the stereotypes and myths which are generally part of our notions about bilingual children. The empirical part of the thesis will analyze the speech of bilingual children up to the age of eight. Their speech will be used to test the verity of selected stereotypes and prejudices.
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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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Stability of the catalytic domain of neutral trehalase studied by differential scanning fluorimetry
Šmídová, Aneta ; Obšilová, Veronika (advisor) ; Kacířová, Miroslava (referee)
This bachelor thesis is part of a project aiming for the crystallization of trehalase domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is to optimize the purification protocol of two different constructs of yeast Nth1 and determine the optimal buffer for crystallography of these constructs using the differential scanning fluorimetry DSF. Trehalases are important, highly conserved enzymes found in many organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and their common feature is hydrolytic cleavage of trehalose molecule into two glucose subunits. Trehalose is a naturally occurring non-reducing disaccharide which serve in yeast cells as storage carbohydrate and energy and stress metabolite. Trehalose and its hydrolysis is very important in the life cycle of the insect, as it is present as the main sugar component of insect hemolymph, so some trehalase inhibitors could be used as insecticides. By this time, however, only one structure of trehalase from a prokaryotic organism Escherichia coli was solved. For this reason it is very important to solve the structure of trehalase from a eukaryotic organism. Keywords: Nth1, expresion, purification, DSF Key subjects: Protein biochemistry, biophysical chemistry
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