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Study on protective effect of 3-hydroxybutyrate and its derivatives
Černá, Klára ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.
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Use of nanomaterials to improve optical detection of biomolecules
Špačková, Simona ; Kristýna, Pavelicová (referee) ; Bezděková, Jaroslava (advisor)
The bachelor thesis deals with modern methods of biomolecule labeling. Attention is paid especially to luminescent labeling with semiconductor quantum dots, optimization of preparation and characterization of quantum dots (QDs) and their bioconjugates with Ala4 peptide, which has antibacterial properties. The first method of preparing QDs used UV-radiation and conjugation with the peptide of interest was carried out through the thiol group of the peptide. A second type of QDs was prepared thermally and conjugation with the peptide was performed by linkers EDC/sulfo-NHS, which allowed peptide bond formation between the carboxyl group of QDs and the amino group of the peptide. The next part of the thesis is focused on the influence of conjugation the antimicrobial activity of the peptide. To verify the antimicrobial properties of the peptide, an interaction between the QDs-Ala4 conjugate and Staphylococcus aureus was observed. Conjugates thus prepared were characterized by fluorescence spectroscopy and capillary electrophoresis with absorption detection (-214 nm).
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Comparison of gel and capillary electrophoresis of proteins
Krbec, Michal ; Franěk, Tomáš (advisor) ; Kotaška, Karel (referee)
Electrophoresis of serum proteins is one of the methods used in laboratory diagnosis of pathological conditions, but also for research purposes. This is a one-step method, which involves isolation of individual proteins from a mixture (mostly blood proteins) in the electric field in the presence of a suitable buffer and a suitable carrier based on their different mobility, dependent on their surface charge and molecular weight. Originally the carrieer used to be a paper, later cellulose acetate membrane and now it is almost exclusively agarose (purified agar with low endoosmosis) or polyacrylamide (PAA). My task in this work was to analyze a serie of samples by gel electrophoresis and subsequently analyze the same serie by capillary electrophoresis. Results obtained by both methods I worked numerically, compared with each other and statistically evaluated. The output of my work is a comparison of gel and capillary electrophoresis, evaluation of the advantages, disadvantages and especially the differences between these two methods.
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Use of nanomaterials to improve optical detection of biomolecules
Špačková, Simona ; Kristýna, Pavelicová (referee) ; Bezděková, Jaroslava (advisor)
The bachelor thesis deals with modern methods of biomolecule labeling. Attention is paid especially to luminescent labeling with semiconductor quantum dots, optimization of preparation and characterization of quantum dots (QDs) and their bioconjugates with Ala4 peptide, which has antibacterial properties. The first method of preparing QDs used UV-radiation and conjugation with the peptide of interest was carried out through the thiol group of the peptide. A second type of QDs was prepared thermally and conjugation with the peptide was performed by linkers EDC/sulfo-NHS, which allowed peptide bond formation between the carboxyl group of QDs and the amino group of the peptide. The next part of the thesis is focused on the influence of conjugation the antimicrobial activity of the peptide. To verify the antimicrobial properties of the peptide, an interaction between the QDs-Ala4 conjugate and Staphylococcus aureus was observed. Conjugates thus prepared were characterized by fluorescence spectroscopy and capillary electrophoresis with absorption detection (-214 nm).
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Determination of CDT by capillary zone electrophoresis method.
BUREŠOVÁ, Kristýna
Alcohol abuse is part of very serious social problems with many social and economic impacts in developed countries. Chronical alcohol abuse may be in many cases very complicated to diagnose and good cooperation between clinician and laboratory workspace is fundamental. Routine diagnosis of chronic alcohol abuse is nowadays based on GGT and MCV determination and AST/ALT ratio. Recently, as a marker with the highest diagnostic validity is considered Carbohydrate Deficient Transferrin (CDT). The transferrin molecule consists of two polysaccharide chains where on each chain could be attached up to four sialic acids (so-calledsialization). However, sializationmay be after two weeks of increased alcohol intake significantly reduced. Detection of these less sialized transferrin molecules, known as CDTs, can be used to diagnose chronic alcohol abuse. Unfortunately, informations from literature about the diagnostic validity of CDT are highly variable. CDT validation differs among women and men and the highest diagnostic validity is achieved during determination in combination with other markers. The experimental part was done on the MINICAP instrument, which works based on the principle of capillary zone electrophoresis. Measurements of control materials and mixed serum patients were performed. From measured values, the necessary statistical values were calculated, such as arithmetic mean, standard deviation and coefficient of variation. Based on the calculations, the validation of the method on the MINICAP instrument was then performed and evaluated for CDT determination.
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Comparison of gel and capillary electrophoresis of proteins
Krbec, Michal ; Franěk, Tomáš (advisor) ; Kotaška, Karel (referee)
Electrophoresis of serum proteins is one of the methods used in laboratory diagnosis of pathological conditions, but also for research purposes. This is a one-step method, which involves isolation of individual proteins from a mixture (mostly blood proteins) in the electric field in the presence of a suitable buffer and a suitable carrier based on their different mobility, dependent on their surface charge and molecular weight. Originally the carrieer used to be a paper, later cellulose acetate membrane and now it is almost exclusively agarose (purified agar with low endoosmosis) or polyacrylamide (PAA). My task in this work was to analyze a serie of samples by gel electrophoresis and subsequently analyze the same serie by capillary electrophoresis. Results obtained by both methods I worked numerically, compared with each other and statistically evaluated. The output of my work is a comparison of gel and capillary electrophoresis, evaluation of the advantages, disadvantages and especially the differences between these two methods.
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Study on protective effect of 3-hydroxybutyrate and its derivatives
Černá, Klára ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.
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