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Study of molecular organization of cytochrome P450 system
Holý, Petr ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Mixed-function oxygenase systém (MFO systém) plays a vital role in the metabolism of a variety of both endogenous substrates and xenobiotics. This membrane systém consists of cytochrome P450s, NADPH:cytochrome P450 oxidoreductase (POR), cytochrome b5 and NADH:cytochrome b5 oxidoreductase (b5R). Cytochrome P450 catalyzes a monooxygenation of a substrate, while POR and cytochrome b5 represent its redox partners. Cytochrome b5, itself having a redox partner in b5R, effects the reactions catalyzed by the MFO system in various ways, through mechanisms that are not fully understood. This paper focuses on the purification of b5R and POR from rabbit liver. The microsomal fraction obtained by differential centrifugation contained 42 mg of protein per ml. From a portion of the microsomal fraction, b5R was obtained using chromatography on DEAE-Sepharose, CM-Sepharose and 5'-ADP agarose columns. The yield was 0,3 % of ferricynide-reductase activity and the product contained several contaminants in the molecular weight range of 50-70 kDa. A second purification of b5R from the microsomal fraction was carried out using a column of DEAE-Sepharose directly connected to a 5'-ADP agarose column. The b5R product was purified with a yield of 10,9 % and it once again contained several contaminants in the molecular...
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Detection of neuronal activity associated with function of lower urinary tract with use of functional magnetic resonance imaging
Holý, Petr ; Zachoval, Roman (advisor) ; Hanuš, Tomáš (referee) ; Mašata, Jaromír (referee)
of the thesis Considerable research attention has been paid to the neural regulation of the lower urinary tract (LUT) in past three decades. The aim of this work is mapping of a brain activity by functional magnetic resonance imaging (fMRI) using refined scanning protocol with synchronously performed urodynamics. We aimed to detect neural activity associated with pelvic floor muscle (PF) contractions, filling of urinary bladder and miction. In addition we evaluated using fMRI brain activity associated with urinary bladder filling in patients with a complete spinal cord injury (SCI). We hypothesized activation of brainstem and forebrain areas in receiving information from the vagal nerves. Adjustments of urodynamic system enabled successful implementation of synchronous filling cystometry with fMRI evaluation of cortical activity. We concluded that synchronous urodynamic examination is a novel feasible method that facilitates and enhance interpretation of fMRI data acquired. The main clusters of brain activation during PF contractions were observed in the medial surface of the frontal lobe (primary motor area) and supplementary motor area (SMA). We detected neural activity associated with filling of urinary bladder and miction in middle and inferior frontal gyrus, angular gyrus, posterior and...
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Co-operativity of cytochrome P450 system and its impact on drug and carcinogen metabolism
Holý, Petr ; Hodek, Petr (advisor) ; Chmelík, Josef (referee)
The system of mixed-function oxidases (MFO system) has a significant role in metabolism of many endogenous compounds, as well as xenobiotics (for ex. karcinogens, drugs). Membrane-bound haemoproteins called cytochromes P450 are a vital part of that system. Reactions catalyzed by cytochromes P450 are influenced by another protein of the MFO system, cytochrome b5. The mechanism of this cyt b5 agency has not yet been fully described. One of methods used for study of this protein-protein interaction is covalent cross- linking. By replacing one of three methionines in the cyt b5 structure by a photo-reactive analogue (photo-methionine), an analogue of cyt b5 (photo-cyt b5) can be obtained. When activated by UV radiation, the protein covalently bonds cytochrome P450 in a membrane environment. This paper focuses on expression and isolation of a recombinant cyt b5 analogue with only one methionine position (96) in the protein structure and substitution by photo-methionine. Protein was purified in a yiedl of 6 mg from 1 liter of baterial suspension. Analysis by mass spectrometry (MALDI-TOF/TOF) showed methionine to have been substituted by the photo-reactive analogue in approx. 30 %. Photo-cyt b5 was used to fixate transient protein-protein interactions with cytochrome P450 2B4 (CYP2B4). Photo-cyt b5 was...
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Testing selected theoretical approaches concerning the building of a US radar base in the Czech Republic
Holý, Petr ; Karlas, Jan (advisor) ; Drulák, Petr (referee)
The aim of this work is to test the ability of three selected theoretical approaches to explain the response of the Czech Republic to the American offer to build the ABM radar device on the Czech soil. These theories are: neorealism, utilitarian liberalism and constructivism. Each of these approaches looks at the social reality from a different perspective which allows us to view the issue in its complexity and diversity. Each theory also uses a different set of independent variables to explain the behavior of the Czech government in this case (these variables are: autonomy and influence for neorealism; domestic interests for liberalism and international and domestic norms for constructivism). Thanks to this variety of variables we are enabled to critically evaluate which key factors played the crucial role in the Czech decision-making process in this particular issue and possibly also to point out some assumed regularities which might play an important role in forming of the Czech security policy as such. The main task of the first chapter is to provide some broader historical and political context of the case. Next three chapters are then dedicated to testing of the theories by comparing their deductively phrased hypothesis with the value of dependent variable, which for our work is represented...
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Cooperative Form of Financing of Housing Construction
Holý, Petr ; Gruber, Jan (referee) ; Breinek, Pavel (advisor)
The diploma thesis focus on evaluation of co-operative housing construction as one of the way from current credit crisis of financing of housing construction. Specifies and evaluate individual types of financig of new housing construction and apply it on model example. Evaluation is based on analysis of present circumstances of housing construction and trend of mortgage loans. In this evaluation also looks at subvene of new housing construction. At the same time looks on an alternative of non-profit co-operative housing construction.
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