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Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
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The content of components of ATP synthasome in different rat tissues and in patients with defects in ATP synthase
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Kalous, Martin (referee)
The complexes of oxidative phosphorylation (OXPHOS) are situated in the inner mitochondrial membrane in higher structural and functional complexes, so-called supercomplexes, which facilitates substrate channeling. ATP synthase is also able to organize in higher structures. In mammalian mitochondria, ATP synthase is usually present in a dimeric form. There is evidence of its trimerization and even tetramerization. Furthermore, it seems that ATP synthase catalyzing the phosphorylation of ADP to ATP, adenine nucleotide translocator (ANT) ensuring the exchange of ADP for newly synthesized ATP across the inner mitochondrial membrane and phosphate carrier (PiC) allowing the import of inorganic phosphate (Pi) into the matrix of mitochondria are assembled in a supercomplex called ATP synthasome. This association among the components of phosphorylative apparatus seems to increase the efficiency of processes leading to the ATP synthesis. First, we studied amounts of the components of phosphorylative apparatus in connection with various ATP synthase contents among mitochondria isolated from nine rat tissues. Mitochondrial proteins were separated by denaturing electrophoresis (SDS-PAGE) and their content was analyzed using specific antibodies. In agreement with our expectations, the highest content of...
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Utilization of biosurfactants for incorporation of hydrophobic domains into modern controlled-release systems
Nešpor, Tomáš ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
This work deals with the current topic of carrier systems. Since the biggest problem is the passage of hydrophobic drugs through the bloodstream, or through universal body barriers (eg blood-brain), it is necessary to chemically modify these carriers in order to be able to administer hydrophobic substances effectively. Based on a literature search, several systems are designed and subsequently studied, in which there is a presumption of possible use for carrier systems and at the same time they have biosurfactants incorporated in them due to their ability to solubilize hydrophobic molecules. The theoretical part of this work will describe the individual biosurfactants, the process of their production, their physicochemical properties, and the possibility of their use in carrier systems. At the same time, the individual carrier systems, the procedure of their preparation, the possibilities of their use are described, and their advantages and disadvantages are also compared. In the practical part, the screening of both individual substances and their mutual interactions, as well as methods used to study the emerging structures is then performed. The study of molecular interactions is primarily performed using the technique of dynamic light scattering. The next part describes the optimization of hydrogel formation with incorporated biosurfactants in their structure and then the formed gels are subjected to rheological and solubilization tests. The study of the internal structure of these gels is performed using a scanning electron microscope.
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Solubilization in catanionic surfactant systems
Konečná, Anna ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the solubilization of hydrophobic dyes in systems of positively charged catanionic vesicles. The aim of the work was to determine their solubilization capacity. Vesicular systems were prepared from oppositely charged surfactants CTAB and SDS. To increase the stability of the vesicles, cholesterol and the positively charged double-chain surfactant DODAC were added to their structure. DODAC also gave the system a positive charge. To determine the solubilization capacity, the dyes 1-Naphthol, Sudan Red G and Oil Red O were used. For sample preparation two methods were used, spontaneous and direct solubilization. The measurement was performed after 1, 2, 3 and 7 days from the preparation of the samples by UV-VIS spectrophotometry. The solubilization capacity was determined from the measured absorbance by calculation from the calibration curves. The results indicate that direct solubilization is slightly more effective than spontaneous solubilization. Of the selected dyes, 1-Naphthol is the substance with the smallest molecule, and therefore it seems to be 100% solubilized in the vesicle system.
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Heterologous expression and purification of human NADPH: cytochrome P450 oxidoreductase
Kostelanská, Marie ; Černá, Věra (advisor) ; Kavan, Daniel (referee)
NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...
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Hydrosolubilization of BODIPY for optical labelling of biomolecules
Bartoň, Jan ; Kotek, Jan (advisor) ; Vrábel, Milan (referee)
1 Abstract This work aims at showing synthesis and potential use of water-soluble fluorescent probes based on BODIPY. The preparation of probes containing bioorthogonal mono- and heterobifunctional functional groups was demonstrated. Ground work was done at the optimisation of reliable, scalable and fast sulfonation of BODIPY in 2,6-positions. A protocol for handling sulfonated BODIPY has been established; especially for the exchange of counterions. In counterion se- lection, their relation to synthetic pathway and biocompatibility were taken into consideration. The second part of the work shows series of water-soluble fluorescent probes, into which can be easily introduced bioactive or bioorthogonal functional groups. This can be used for click chemistry in connection with turn off/on probes or fluorescent sensing of molecules or ions. All this can be done in aqueous solution without organic solvents, which is relevant for biochemical, analytical and imaging applications. Keywords BODIPY, bifunctional, water-soluble, fluorescent probe, solubilization, biocompa- tible probes, bioorthogonal reaction, BODIPY sulfonation
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Bifunctional BODIPY for optical labelling of biomolecules
Bartoň, Jan ; Kotek, Jan (advisor) ; Henke, Petr (referee)
The water-soluble derivate of BODIPY was prepared, which will be further modified in order to prepare bioorthogonal bifunctional BODIPY. Target application of this derivate is fluorescent probe for labelling of biomolecules. Main goals of this thesis were optimalization of synthesis of BODIPY core and sulfonation to positions 2,6. It was found out that sodium salt of sulfonated BODIPY shows good solublility in water and methanol, but it is poorly soluble in less polar solvents. Based on these findings DIPEA salt of sulfonated BODIPY was prepared, which shows outstanding range of solubility from water to dichlormethane. These findings will be used for synthesis of bifunctional water- soluble BODIPY. Further, the potential of Pd-BODIPY complex for detection of CO was reasserted which will be further investigated. Keywords BODIPY, bifunctional, water-soluble, fluorescent probe, fluorescence, protein labelling, solubilization
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Releasing of the solubilized substances from the phase-separated hydrogels
Havlíková, Martina ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on releasing of the solubilized substances from the phase separeted hydrogels. The aim of this work is to determine the solubilization capacity of these hydrogels. Preparation of them were based on interaction between hyaluronan and cationic surfactant carbethopendecinium bromide. Hydrogels were prepared by two possible ways, „wet“ and „dry way“. For solubilization experiments were used fluorescein and acridine orange as fluorescent probes. Primarily UV-VIS spectrophotometry was used to determine if these probes are released form hydrogel or not. Hydrogels were monitored after 24, 48 and 72 hours from their preparation. But this method proved to be inappropriate and inaccurate, because of cloudy supernatants over the hydrogels and also because of very low concentrations of fluorescent probe in this solution. For these reasons instead of this metod was used method, which is based on fluorescence. The instrument is called MicroTime 200. It´s very accurate method and results of this are considered like relevant. It was proved that fluorescein as a negatively charged molecule is released less than acridine orange.
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Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
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