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Utilization of protein radical foootprinting for stuctural biology
Polák, Marek ; Novák, Petr (advisor) ; Junková, Petra (referee)
(In English) The reaction of highly reactive oxygen radicals with protein solvent-accessible residues can be utilized to map protein landscape. Fast photochemical oxidation of proteins (FPOP) is an MS- based technique, which utilizes highly reactive radical species to oxidize proteins and map protein surface or its interactions with their interaction partners. In this work, FPOP was employed to study protein-DNA interactions. First, a full-length of FOXO4-DBD was successfully expressed and purified. The ability of the protein to bind its DNA-response element was verified by electrophoretic and MS-based techniques, respectively. Optimal experimental conditions were achieved to oxidize the protein itself and in the presence of DNA, respectively. Oxidized samples were analyzed by bottom-up and top-down approach. In the bottom-up experiment, modification of individual residues was precisely located and quantified. Different extend of modification was observed for protein alone and in complex with DNA. To avoid experimental artifacts analyzing multiply oxidized protein, standard bottom up approach was replaced by a progressive top-down technology. Only a singly oxidized protein ion was isolated, and further fragmented by collision-induced dissociation (CID) and electron-capture dissociation (ECD),...
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Utilization of protein radical foootprinting for stuctural biology
Polák, Marek ; Novák, Petr (advisor) ; Junková, Petra (referee)
(In English) The reaction of highly reactive oxygen radicals with protein solvent-accessible residues can be utilized to map protein landscape. Fast photochemical oxidation of proteins (FPOP) is an MS- based technique, which utilizes highly reactive radical species to oxidize proteins and map protein surface or its interactions with their interaction partners. In this work, FPOP was employed to study protein-DNA interactions. First, a full-length of FOXO4-DBD was successfully expressed and purified. The ability of the protein to bind its DNA-response element was verified by electrophoretic and MS-based techniques, respectively. Optimal experimental conditions were achieved to oxidize the protein itself and in the presence of DNA, respectively. Oxidized samples were analyzed by bottom-up and top-down approach. In the bottom-up experiment, modification of individual residues was precisely located and quantified. Different extend of modification was observed for protein alone and in complex with DNA. To avoid experimental artifacts analyzing multiply oxidized protein, standard bottom up approach was replaced by a progressive top-down technology. Only a singly oxidized protein ion was isolated, and further fragmented by collision-induced dissociation (CID) and electron-capture dissociation (ECD),...
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Analysis of the determinants that influence the number of children which are placed in institutes for infants and homes for children up to 3 years of age
Junková, Petra ; Bolcha, Peter (advisor) ; Janíčko, Martin (referee)
This thesis analyses the possible existence of a relation between chosen determinants and number of children which are placed in institutes for infants and homes for children up to 3 years of age for social reasons in the Czech Republic during the period 1993 -- 2008. First part is focused on theory of the family by Gary S. Becker. It evaluates each part of the decision making process scheme and analyses the final choice of placing a child in the institutional care. In the empirical part of the thesis four determinants are selected based on the laid out theoretical ground and other related studies. These determinants which are the focal point of the empirical analysis are: registered unemployment, real household income, state social support benefits and number of women in prison. Regression analysis and analysis of the placement of children discharged from institutional care for children up to 3 years of age are the main methods of examination. Based on the conclusions of the examination there are possible measures and actions suggested, that can be taken in order to improve the state of institutional care.
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