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Human F1Fo-ATPsynthase deficiency
Suldovská, Sabina ; Tesařová, Markéta (advisor) ; Černá, Leona (referee)
F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...
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Influence of cultivation conditions on the production of recombinant proteins
Gardošová, Zuzana ; Nováčková, Ivana (referee) ; Brázda, Václav (advisor)
Recombinant proteins are produced by using genetic modifications. In this process is insert contained gene encoding a certain protein in a cloning vector cloned into the host organism. Recombinant proteins are expressed after the transformation of the cloning vector into a host, the host organism. The expression of recombinant proteins in bacterial cells is one of the most efficient ways to manufacture these proteins. The p53 protein is a tumor suppressor protein, the main role in cells is to react on DNA damage. Due to the reaction to various intracellular and extracellular stimuli, including DNA damage, the p53 protein shows different biological functions, including regulation of senescence, cell cycle, or apoptosis. The theoretical part of the thesis part presents the basic properties of proteins, methods of recombinant protein expression, methods of protein isolation, and characterization of p53 protein. The aim of the experimental part was to determine the effect of incubation temperature on recombinant p53 protein production. The work involves the isolation of plasmid DNA and its transformation into E. coli production cells. The produced proteins were successfully isolated and subsequently characterized by SDS-PAGE and Western Blotting.
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The effect of temperature and drought on the glutenin proteins content in two genotypes of wheat
Vybíral, Lukáš ; Vespalcová, Milena (referee) ; Hrstka, Miroslav (advisor)
This bachelor thesis deals with the influence of water and temperature deficiency on protein content of gliadin fraction and on glutenin/gliadin ratio in two wheat varieties. The varieties selected for the experiment are called Pannonia and RGT Reform. Samples were cultivated at three different temperatures, 26 °C, 32 °C and 38 ° C, and irrigation was checked at the time of flowering. For wet-grown samples, the soil had a moisture content greater than 70 %, and drought-grown specimens grew on soil with a moisture content not exceeding 30 %. After harvest, the grains were ground into flour and glutenins and gliadins were extracted from the individual samples. SDS-PAGE was used for separation and protein quantification was performed by computer densitometry. A significant effect of the variety on individual gluten proteins was found. In the Pannonia variety, it has been found that the interaction of higher temperature and drought has no major effect on glutenin content and glutenin/gliadin ratio. The RGT variety was significantly affected by these conditions, resulting in a decrease in glutenin content and a significant change in ratio. In both varieties, the glutenin content was higher when cultivated at higher humidity but only at two higher cultivation temperatures.
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Nutritional benefit of cricket flour
Šťastná, Martina ; Zemanová, Jana (referee) ; Němcová, Andrea (advisor)
Edible insects have been part of the diet of many countries across the world for several decades. It is becoming increasingly popular in the europian developed countries, mainly because of the content of nutritionally important substances. The theoretical part of the thesis deals with the characterization of edible insects in terms of nutritional benefits, explains the reasons and method of breeding edible insects. For the analysis of nutritionally active substances, flour of house cricket (Acheta domesticus) was used. A. domesticus belongs to the representatives of insects legalized from 1.1. 2018 on the EU market. Of the significantly nutritional substances, proteins, lipids, fatty acids, fiber and selected minerals were analyzed. In this study, optimalization of vertical electrophoresis SDS-PAGE was performed to determine protein fragment sizes. In the second part the influence of acricket flour addition protein bars was investigated by sensory analysis. In the experimental part that was found that cricket flour contains protein, lipids and fatty acids in significant amounts. It also contains important minerals for human health such as magnesium, potassium, iron or zinc. However, the sensory properties of protein bars for consumers were not very attractive.
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Human F1Fo-ATPsynthase deficiency
Suldovská, Sabina ; Tesařová, Markéta (advisor) ; Černá, Leona (referee)
F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...
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Impact of temperature and drought on gliadins and glutenins contents in four varieties of wheat
Tomasz, Teresa ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
This diploma thesis deals with an influence of high temperature and water shortage on the protein content of gliadin and glutenin fractions in four varieties of winter wheat: Bohemia, Tobak, Pannonia and var. Syria with designation S46 (IG142780). The crop was grown at 26, 29, 32, 35, 38 and 41 °C during anthesis under control irrigation treatment (with soil moisture higher than 70 %) or under drought stress (with soil moisture lower than 30 %). To separate gliadins, the A-PAGE method was used, and glutenins were separated by SDS-PAGE method. Proteins were quantified by computer densitometry. Significant influence of genotype on the gluten proteins was found. Variety Pannonia has high content of -, 5-gliadins, LMW and HMW glutenins, but low content of other gliadin fractions. It was the opposite in the other varieties. Due to temperature, as well as drought, there was an increase in the content of all gluten fractions, especially of HMW glutenins, 1,2-gliadins and total gliadins. The largest increase in the gluten fractions due to drought was observed in Syria variety. In other varieties simultaneous exposure to drought and heat caused decrease in gliadin content, but increase in glutenin content. Drought at high temperatures reduced gliadin-to-glutenin ratio, mostly in Bohemia variety. This ratio has increased due to the temperature, especially in Tobak variety. For Syria variety, no effect of stress conditions was found on gliadin-to-glutenin ratio.
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Analysis of low-molecular proteins in barley by the SDS-PAGE method during malting
Myslivcová, Pavla ; Svoboda, Zdeněk (referee) ; Mikulíková, Renata (advisor)
This diploma thesis deals with the analysis of low molecular weight proteins in barley during malting by SDS-PAGE method. Attention was paid to PR proteins, specifically LTP proteins and thionins, considered to be connected with gushing effect. Barley samples, malt intermediates and malt samples taken in 10 consecutive days to cover the whole malting process were used for the experiment. In total, 5 samplings were used for the analysis. Proteins extracted from the samples were separated by SDS-PAGE using a Tris-tricine buffer system. The protein lines of LTP proteins and thionins were identified in the resulting gels. The relative optical density values of the selected protein bands were obtained to assess the effect of malting on these proteins. A similar pattern of change in the content of mentioned low molecular weight proteins during the malting process was observed. This was confirmed by finding a statistically significant positive correlation between the relative optical density values of LTP proteins and thionins. Furthermore, the relationship between the low molecular weight protein content and the gushing potential and the microbiological contamination of the samples was investigated, but was not confirmed.
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Analysis of glutenin fraction in winter wheat cultivated in ambient and elevated carbon dioxide concentrations
Čileková, Marta ; Vránová, Dana (referee) ; Hrstka, Miroslav (advisor)
Increasing carbon dioxide concentrations in the atmosphere has influence on plant’s photosynthesis and its metabolits. This bachelor thesis is focused on analysis of glutenin fraction in winter wheat (Triticum aestivum) var. Bohemia grown in ambient (AC) and elevated (EC; 700 mol.mol-1) carbon dioxide concentrations. Plants were cultivated in conditions with nitrogen fertilization (N+; 200 kg.ha-1) and without fertilization (N), in presence of UV-B radiation (UV+) and absence of UV-B radiation (UV), at drought (DRY) and with ambient irrigation (WET). Grain was harvested at 2014. The glutenin proteins were extracted from flour by isopropanol and DTT then separated by SDSPAGE and quantified by computer densitometry. Elevated carbon dioxide concentration decreased glutenin concentration in variant UV+ otherwise differences between AC and EC variants were nonsignificant. The nitrogen fertilization markedly increased glutenin content. Higher concentrations of glutenins were found in variants UV– WET and UV+ DRY. The highest concentrations of glutenins were found in conditions with presence of UV radiation, drought and nitrogen fertilization.
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Protein calculator
Sedlo, Petr ; Maděránková, Denisa (referee) ; Škutková, Helena (advisor)
This work is focussed on studying and analysis of protein qualities while using experimental methods which are next confronted with numerical analysis, which have been introduced by the expansion in computer engineering. Within that task I built up a program for calculation of isoelectric point and molecular mass of an entered chain of amino acids. The program will draw a graph of pH dependance of molecular charge in the wide range of pH values in accordance with the given criteria. We can gain results very quickly and easily in this way, however, these results are just a rough estimate. We´re assumed to be aware of this fact because incorrect use of that program where exact analysis is required would cause errors.
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