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Optimization of methods for the isolation and analysis of beta-glucans produced by carotenogenic yeasts
Šimková, Eva ; Márová, Ivana (referee) ; Szotkowski, Martin (advisor)
The aim of this thesis was to optimize the methods of isolation and analysis of -glucan polysaccharides produced by carotenogenic yeasts. As part of the experiment, the yeast Rhodotorula toruloides, which was cultivated in laboratory bioreactors, was selected as the production strain. Theoretical part describes the subject of carotenogenic yeasts and the metabolites they produce. The main metabolite that this work deals with is the polysaccharide -glucan. -glucans are polysaccharides that form the cell wall in the yeast cell. They are known for their immunostimulating effects, antioxidant effects, promote wound healing and help lower cholesterol and blood glucose levels. The experimental part of the work deals with the cultivation of yeast biomass and its characterization. Furthermore, optimization of the enzymatic determination of -glucans using a commercial enzymatic kit and isolation of -glucans from yeast biomass. As part of the experiment, yeast biomass extraction methods were also further developed, enabling the separation and analysis of -glucans, lipids, lipidic substances and proteins from a small sample of yeast biomass in one extraction procedure.
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Introduction of spectrophotometric determination of urinary citrates and oxalates into routine clinical laboratory testing.
FEJT, Martin
Determination of the level of citrates and oxalates in urine is of great importance in the diagnosis and subsequent monitoring of patients with urolithiasis. Data about urine concentrations of these substances, together with other metabolic tests, help to determine the likelihood of recurrence of urolithiasis. At the same time, they help physicians find appropriate regime measures for patients with the disease. This bachelor thesis deals with the introduction of the determination of citrates and oxalates in the routine operation of a clinical laboratory. The theoretical part first briefly describes the issue of urolithiasis, its distribution, etiopathogenesis, treatment and prophylaxis. Emphasis is placed on the role of citrates and oxalates. Furthermore, individual types of urinary stones and possibilities of their analysis are characterized. The following is an approach to the process of verification of the measuring procedure and associated performance parameters. The related analytical properties of the methods are also listed and described here. Within the description of the implementation process the setting of parameters in the laboratory and hospital information system is briefly explained. In the laboratory of ÚKBH FN Plzeň we chose enzymatic methods and an automatic analyzer Beckman Coulter AU480 to determine the concentration of citrates and oxalates in urine. The theoretical part of the bachelor thesis continues with a description of the spectrophotometry and operation of the instrument used. We tested reagents from foreign manufacturers INstruchemie (Netherlands) and LTA (Italy). The following is an explanation of the principles of the methods from the above manufacturers and briefly lists other possible methods for the determination of these analytes. Reagents from INstruchemie were selected for routine use. As this manufacturer has already validated its methods on Beckman Coulter spectrophotometers, only their verification has taken place at ÚKBH. The practical part of this bachelor thesis deals first with the preparation of reagents for analysis and continues with programming the parameters of individual methods into the analyzer. The main content of this work is a detailed description of the implementation of individual phases of analysis, which are required by the verification process. After successful completion of all measurements and evaluation of their results, appropriate verification protocols were prepared and the methods were put into routine operation of the laboratory.
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