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Production and characterization of novel aryl sulfotransferases and their use in bioanalytics
Petránková, Barbora ; Bojarová, Pavla (advisor) ; Křížek, Tomáš (referee)
Polyphenols are the most common natural substances found in food. The largest group is represented by flavonoids, phenolic acids and catechols. Polyphenols are metabolized in the body during the biotransformation phase II, they can be sulfated, glucuronylated or methylated. To study biotransformation, it is necessary to have defined standards. Sulfated polyphenols can be prepared by enzymatic sulfation, which needs much milder reaction conditions compared to chemical sulfation. Prokaryotic sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfate donor for sulfation, which is very expensive and difficult to regenerate. An alternative is the group of bacterial aryl sulfotransferases (EC 2.8.2.22), which catalyze the transfer of a sulfate group from a phenolic donor to a phenolic acceptor. Two bacterial aryl sulfotransferases from Desulfitobacterium hafniense and Escherichia coli have been described in the literature so far. The present thesis deals with the production, purification and biochemical characterization of two new bacterial aryl-sulfotransferases from Desulfosporosinus sp. HMP52 and Salmonella bongori. The enzymes were heterologously expressed in E. coli and purified by affinity chromatography. Their pH and temperature optimum, stability in organic solvents and...
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Analysis of sulfated flavonoids by HPLC-MS/MS
Petránková, Barbora ; Bojarová, Pavla (advisor) ; Kozlík, Petr (referee)
The aim of this work is to find the optimal separation conditions in high performance liquid chromatography for the analysis of sulfated polyphenolic substances, especially flavonoids, phenolic acids and catechols. Three chromatographic columns were used in the development of the method. ZIC-HILIC column with polar organic phase and C18 and Kinetex PFP columns with reverse phase. We focused in particular on a short time of separation, sharp tailless peaks and separation of all components of the mixture. Using a Kinetex PFP column, the optimal method for separating the test mixtures was found. The use of a mobile phase of 10 mM ammonium acetate/0,1% fromic acid and methanol was shown to be the most appropriate, with a mobile phase flow rate of 0,6 ml/min. By modifying the gradient and temperature, all components of the tested mixtures were separated, the peaks were sharp, and the separation time in most cases ranged do not exceed 10 min. The method is also directly applicable in conjunction with a mass detector. Keywords: High Performance Liquid Chromatography (HPLC), flavonoids, polyphenolic substances, sulfation, separation
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The importance of introspection for helping professions
Petránková, Barbora ; Pivoňková, Lucie (referee) ; Šnýdrová, Ivana (advisor)
intention of the work "The importance of Introspection for Helping Professions" is based on documented facts about positive action introspectional work and quality of feedback in helping professions to highlight the great importance of such action and its impact on improving the professionalism and personal life of workers in the helping professions. Based on these findings, it recommends increasing the use of introspectional techniques in the areas directly involved in the personalities of other people. It also offers an overview and description of the objectives, forms and guidelines for psychotherapeutic action and a separate chapter on supervision and other forms of feedback.
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