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Use of Chiral Separations for the Determination of Enzyme Enantioselectivity
Břicháč, Jiří ; Zima, Jiří (advisor) ; Tesařová, Eva (referee) ; Flieger, Miroslav (referee)
Conclusion Enantioselective enzymes pray important rore in metaboric pathways of chirar compounds. Enantioselective metabolism of lipid peroxidation product HNE was studied using measurement of enzyme kinetics. Difference in ability to detoxiý individua| HNE enantiomers by rat brain mitochondriar ALDHs was found. Based on kinetic parameters of purified recombinant enz),Ťnes' it was concluded, that rat ALDH5A enantioselective|y oxidized (R)-HNE' whereas rat ALDH2 was not enantioserective. Moreover, direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were deveroped and validated. The indirect method involved pre-corumn derivatization with a chiral amino agent ANPAD' and subsequent separation ofdiastereomers on a Spherisorb oDS2 column. The direct separation of HNEA enantiomers was performed using the porysacharide-based CSP, Chiralpak AD-RH. Enantioserective enzymes are useful biocatalyzators a'owing cheap preparation of optically pure chemicals. screening of epoxide hydrolases produced by various microorganisms was performed and enantioselective enzymes were found using chiral GC on cyclodextrin-based CSps. In summary, this thesis describes development and optimization of the analytical methods for determination of various chiral compounds implicated in enzyme enantioselectiviý. All...
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Molecular mechanism of anti-cancer activity of selected natural compounds.
Smolová, Dagmar ; Vališ, Karel (advisor) ; Flieger, Miroslav (referee)
Molecular mechanism of anti-cancer activity of selected natural compounds Přírodovědecká fakulta Univerzity Karlovi v Praze Smolová Dagmar Nowadays, cancer is affecting more then a third of the world's population and it's the cau- se of more than 20% of all deaths. A better understanding of cancer at the epigenic, gene- tic, molecular and cellular levels is opening up lots of opportunities to intervene with the goal of preventing end-stage of the invasive disease. Recently, much attention is paid to the natural substances because of its ability to interact specifically in the tumorgenesis. This compounds are preferred before the standard treatment because of its availability and safe- ty. In this work, there were observed the effects of 6-shogaol, 6-gingerol and galangin in the leukemic cells Jurkat. The greatest efficiency was showed by 6-shogaol wich caused apoptosis in cells by activating the antiproliferative signalling of Hippo pathway and it also decreased the expression of C-MYC oncogene. There was also a decline of C-MYC onco- protein wich has a key role in the tumor cells metabolism through it's ability to provide enought of energy for the growth and active proliferation. (In Czech)
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Characterization of modern HPLC columns and their application potential
Janečková, Lucie ; Tesařová, Eva (advisor) ; Pacáková, Věra (referee) ; Flieger, Miroslav (referee)
(EN) The aim of the dissertation thesis is characterization of modern HPLC columns from the point of their interaction possibilities and demonstration of their application potential. The first part of the thesis is focused on alternative reversed-phase HPLC columns based on zirconium dioxide. These packings offer excellent chemical stability and additional interactions that can be helpful in the development of analytical methods. A detailed study of the chromatographic behaviour of biologically active nonapeptides as model analytes confirmed the substantial effect of mobile phase composition on retention mechanism. Consequently, HPLC separation systems with zirconia-based polystyrene column were characterized by distinct approaches that allowed recognition of the interactions participating in the separation process. Empirically based chromatographic tests evaluated the fundamental properties of the system - hydrophobicity and polarity. The complex model of linear free energy relationship described the prevailing interactions in different separation systems. Application of a set of basic compounds revealed the contribution of ion-exchange interactions participating in the separation systems with zirconia-based column. The second part of the thesis is devoted to new cyclofructan-based chiral...
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Regulation of C-MYC oncoprotein by natural drugs.
Filandr, František ; Novák, Petr (advisor) ; Flieger, Miroslav (referee)
Sesqiterpene lactones, a group of plant secondary metabolites which include Cnicin from Cnicus benedictus plant, have an anti-proliferative and anti-tumor effect on mammalian cells by activating specific signaling pathways while also generating oxidative stress. These factors combined drive tumor cell apoptosis. A few of these compounds have reached clinical trials and seem to be a promising chemotherapeutics. The focus of this work is to elucidate the effect of cnicin on C-MYC transcription factor and oncoprotein which is overexpressed in majority of tumor tissues, the effect of cnicin on DEAD-box RNAhelicase DDX3 and on the expression levels of several metabolic genes is also studied. Through the use of western blotting, immunodetection and qPCR it was found out, that cnicin is regulating the expression of C-MYC oncoprotein on both transcriptional and translational levels, while also lowering C-MYC protein stability probably through the effect on PIM-2 kinase. Cnicin is not affecting the total amount of DDX3 protein in cells, but it seems it is lowering its degradation rate. The possible transcriptional regulation of DDX3 by cnicin is still not clear and requires further research. With the use of LC-MS quantitative analysis and qPCR, it was found out that cnicin does not affect the metabolism of...
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Molecular mechanism of anti-cancer activity of selected natural compounds.
Smolová, Dagmar ; Vališ, Karel (advisor) ; Flieger, Miroslav (referee)
Molecular mechanism of anti-cancer activity of selected natural compounds Přírodovědecká fakulta Univerzity Karlovi v Praze Smolová Dagmar Nowadays, cancer is affecting more then a third of the world's population and it's the cau- se of more than 20% of all deaths. A better understanding of cancer at the epigenic, gene- tic, molecular and cellular levels is opening up lots of opportunities to intervene with the goal of preventing end-stage of the invasive disease. Recently, much attention is paid to the natural substances because of its ability to interact specifically in the tumorgenesis. This compounds are preferred before the standard treatment because of its availability and safe- ty. In this work, there were observed the effects of 6-shogaol, 6-gingerol and galangin in the leukemic cells Jurkat. The greatest efficiency was showed by 6-shogaol wich caused apoptosis in cells by activating the antiproliferative signalling of Hippo pathway and it also decreased the expression of C-MYC oncogene. There was also a decline of C-MYC onco- protein wich has a key role in the tumor cells metabolism through it's ability to provide enought of energy for the growth and active proliferation. (In Czech)
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