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Purification and characterization of membrane-bound reductase / / human liver tissue
Pelcová, Vendula ; Wsól, Vladimír (advisor) ; Kvasničková, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Vendula Pelcová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of rigorous thesis: Purification and characterization of membrane-bound reductase from human liver. This study was focused on implementation of experimental techniques for purification of human liver microsomal fraction. There were chosen three basic steps of purification process. As a first step, ion-exchange chromatography was chosen followed by two purification steps on the base of gel filtration. Before purification the microsomal fraction had to be solubilized to release proteins from membrane bounds and conditions had to be optimized. Single fractions were subsequently incubated with specific substrate of reductases oracin and the quantity of metabolite 11-dihydroracin was assessed by achiral HPLC analysis. For the most active fractions the SDS-PAGE analysis was done to set size of unknown enzyme. By use of antibody against 11beta-HSD1 was axcleded the presence of this reductase in all analyses fractions. By use of described purification techniques we were successful to concentrate fraction with unknown human liver reductase.
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Antimicrobial activity of cobalt bis(dicarbollide) aminoderivatives
Kvasničková, E. ; Masák, J. ; Šícha, Václav
We carried out experiments focused on the determination of the antimicrobial acitivity of selected representatives of cobalt bis{dicarbollide) aminoderivates. lt was proved, that studied yeast strain C. parapsilosis DBM 2165 is not sensitive to the cobalt bis{dicarbollide) or its aminoderivatives. Gram-negative bacteria P. aeruginosa B-59188 is weakly susceptive to cobalt bis(dicarbollide), but no significant inhibition of growth by cobalt bis(dicarbollide) aminoderivatives was observed. The highest effect on inhibition of microbial growth was confirmed in the case of gram-positive bacteria 5. aureus. This observation demonstrates the real possibility of using these substances in the treatment of infection caused by gram-positive bacterial strains.
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Molecular characterization of selected clostridial strains isolated from cheeses
Chroboková, Maria ; Kvasničková, Eva (referee) ; Rittich, Bohuslav (advisor)
The study was focused on molecular characterization of 42 clostridial strains. DNA was isolated by fenol-chloroform extraction procedure and precipitated with ethanol. After DNA isolation, PCR amplifications with specific primer sets were used for genus and species identification. Finally 19 strains were clasified as Clostridium tyrobutyricum and 3 strains were clasified as Clostridium butyricum. Presence of hydrogenase gene hydA was tested by PCR amplification using specific primer set HGf and HGr. Presence of hydrogenase gene was detected within 21 strains. (GTG)5 primer (rep-PCR) and Pr1 and Pr6 primers (RAPD) were used for differentiation of clostridial strains. Next, the cultivation of Clostridium tyrobutyricum S5 was studied under different conditions. The cultivation was carried out in liquid Reinforced Clostridial Medium (RCM) with lactose and cheese whey instead of glucose under anaerobe conditions. Growth was observed at laboratory temperature (20 to 23 °C) and at 37 °C, pH values ranging from 4.0 to 8.0 with 0.5 unit.
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Probiotics in food products for children
Vozárová, Petra ; Kvasničková, Eva (referee) ; Španová, Alena (advisor)
Despite of the high level of science and medicine, people are trying to return to the nature and use it for their lives. For example, they are using probiotics, which are live cultures able to reside in the human intestine and there can start their role. The teoretical part of this bachelor thesis deals with probiotic bacteria, their properties and those properties which are important in the food industry and the main part focuses on the probiotics used in baby food. The experimental part of the thesis targets on an analysis of two probiotic supplements for children (BioLac Baby and Probacílium+). There was a DNA isolated from these two products by method of phenol extraction and using magnetic media. The whole DNA isolated from these products was amplificated by genus-specific PCR.
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Hydrogen production by the bacteria of the genus Clostridium
Filová, Dagmar ; Kvasničková, Eva (referee) ; Rittich, Bohuslav (advisor)
Bachelor thesis deals with hydrogen production using bacteria of Clostridium genus - specifically C. butyricum and C. tyrobutyricum. Theoretical part analyzes means of hydrogen production divided into biological and non - biological methods. Thesis is further aimed at practical use of these microorganisms for hydrogen production purposes and introduces cheese whey as one of utilizable growth medias. In experimental part, there was used polymerase chain reaction for identification of bacteria genus Clostridium and species Clostridium butyricum and Clostridium tyrobutyricum. PCR products were reamplified using the same primers for specifity confirmation purposes. Presence of gene producing hydrogenase A enzyme was proved by PCR, too.
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