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Influence of makroelements from food on DNA and epigenetic profile
Veselý, Zdeněk ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
The macroelements contained in food have an important function for the human body. They are involved in several of biochemical reactions in the body and their abundance can prevent serious diseases. The theoretical part of the bachelor thesis describes the function of minerals in the human body, the function of DNA and epigenetic mechanisms such as DNA methylation or histone modification. The influence of nutrition and function of selected macroelements – sodium, magnesium, calcium, potassium on epigenetic modifications and on the stability of G-quadruplexes was described. The aim of the experimental part of this work was to study the effect of these substances on DNA structures in vitro and to prepare them experimentally for in vivo studies.
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Extraction of arabinoxylans from wheat bran
Koblasová, Dana ; Kouřilová, Xenie (referee) ; Diviš, Pavel (advisor)
This diploma thesis is focused on the use of wheat bran as a raw material for extraction of arabinoxylans. Wheat bran is a waste product of mill processing of wheat into flour and is mainly used as a livestock feed. However, the amount of bran produced significantly exceeds its consumption as feed, which opens up space for research into their further use. Polysaccharides such as cellulose, hemicelluloses and lignin are mainly present in the bran. The most common type of hemicelluloses are arabinoxylans, which form the main structural component of wheat bran. Alkaline extraction disrupts hydrogen and covalent bonds and releases polysaccharides of various molar weights from the crosslinked cell wall structure. Hydrolysis is required to obtain arabinose and xylose. Trifluoroacetic acid was chosen for the hydrolysis. The acid breaks down glycosidic bonds of the poly- and oligosaccharides to give the corresponding monosaccharides. The experiments made during this thesis have shown that the yield of multi stage extraction is twice as high as the yield of single stage extraction. Hydrolysis of the solid fractions after alkaline extraction revealed that a relatively large amount of arabinoxylans and other carbohydrates still remain bound in the complex matrix of the bran cell wall. Thus, alkaline extraction alone may not be the best option for obtaining arabinoxylans on a larger scale, or process optimization must be considered. Extraction with the addition of hydrogen peroxide appears to be effective for large-scale extraction processes.
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Screening of biotechnological potential of selected members of the genus Geobacillus and other related genuses
Kouřilová, Xenie ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.
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Analyses of inverted repeats in human patogen genomes
Dobrovolná, Michaela ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
Helminth parasites are highly prevalent in humans in developing countries. According to WHO, approximately 2 billion people are infected worldwide. The etiological agents of parasitic infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms), causing inflammatory responses, malnutrition, and anemia that are the primary cause of mortality. Drug resistance is accelerated by the overuse of human anthelmintics, as well as poor infection prevention and control. The therapeutic potential of small molecule ligands binding G-quadruplexes (G4s) has been demonstrated. For instance, that it can be used to stabilize the quadruplex structures and eliminate drug-resistant pathogens. G4s are secondary structures formed in guanine-rich nucleic acid sequences, which can regulate the process of gene expression, DNA damage repair, transcription, and translation of oncogenes. Here we used the G4Hunter Web Tool to identify and compare G-quadruplex sequences (PQS) in the nuclear and mitochondrial genomes of six Platyhelminth and four Nematode species to identify targets for G4 ligands to predict new drug targets and more effective drugs. We found that PQS are nonrandomly distributed in these genomes. Most of the G-quadruplexes are in the proximity of genes, suggesting their role in genetic regulation. Interestingly, less infective Platyhelminthes were found enriched with PQS, compared to highly infective species with a lower PQS frequency. In contrast, a Nematoda, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This highly infective species can tolerate high-stability G4 structures, which are not counter-selected at all in contrast to Caenorhabditis elegans.
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Isolation of PHA from bacterial biomass and their subsequent purification
Zlochová, Barbora ; Kouřilová, Xenie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the isolation and characterization of the bacterial polymer PHA isolated from the bacterial strain Halomonas halophila (CCM 3662) by osmotic stress in distilled water, ie hypo shock, when the cells crack and release PHA. The addition of biosurfactants simultaneously solubilizes the proteins and lipids of the residual biomass, theoretically achieving a purer PHB product without the use of toxic substances such as chloroform. The theoretical part of the work focuses on PHA in terms of their general properties and use, the mechanism of synthesis of PHA microorganisms and their subsequent degradation and the existing strategy for isolating PHA from bacterial biomass. The experimental part describes the production of PHB bacteria Halomonas halophila and the subsequent process of isolation of PHB by hypo shock of halophilic bacteria in distilled water with the addition of selected biosurfactants. Biosurfactants used in the work are coconut soap, lecithin, rhamnolipids, sophorolipids, coco glucoside, lauryl glucoside and cocoamidopropyl betaine. The properties of the isolated PHB material were analyzed by gas chromatography with FID detection and SEC-MALS and FTIR methods. Based on the results, we chose the pair of coco glucoside and lauryl glucoside as the most suitable biosurfactants that can be used to isolate PHB from bacterial biomass. In combination with washing the material in ethanol, at concentration of biosurfactant 5 g/l and isolation in 70 ° C, it was possible to achieve a purity of 90 % of material using cocot glucoside and 89 % purity of material using lauryl glucoside.
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Study on PHA production in extremophiles from genus Bacillus and related genuses
Reinohová, Nikola ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This Bachelor thesis is focused on study of production od polyhydroxyalcanoteas by extremophilic bacteria of genus Bacillus and related genera. In this thesis were studied microorganisms from german and czech collections Ureibacillus composti DSM 171951, Alkalibacillus haloalkaliphilus DSM 5271, Alicyclobacillus acidocaldarius DSM 446, Halobacillus halophilus CCM 3527, Thermobacillus composti DSM 18247, Bacillus licheniformis CCM 2206 and isolated microorganisms from natural sources Aneurinibacillus thermoaerophilus LK7, Aneurinibacillus thermoaerophilus F109, Aneurinibacillus thermoaerophilus AFn2, Geobacillus thermodenitrificans F101, Geobacillus thermodenitrificans F102, Geobacillus stearothermophilus A12, Geobacillus sp. AH11. The theoretical part describes extremophilic microorganisms, polyhydroxyalkanoates and their applications. In the experimental part, the detection of PHA production at the genotype level was performed using the PCR method, where the presence of first and fourth class PHA synthases was detected. Detection of presence of the 16SrRNA gene was performed by PCR. PHA production was also tested at the phenotype level, where the ability of utilization of different carbon sources and the ability of microorganisms to form 4HB and 3HV copolymers with different precursors at different temperatures was monitored. Copolymers are very interesting because of their properties, which make them suitable for a wide range of applications. The microorganism Aneurinibacillus thermoaerophilus AFn2 proved to be the best representative for PHA production in this work, producing PHA up to 1,99 g/l and 3HV copolymer up to 0,49 g/l.
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Study on PHa biosynthesis by cyanobacteria
Drinka, Jakub ; Kouřilová, Xenie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis concerns with the effect of selected environmental stresses towards the viability of cyanobacterial species Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Both strains were studied via flow cytometry using the appropriate fluorochromes while also observing the applicability of these stains and precision of this method in the analysis of cyanobacterial cultures. In spite of the optimization being carried out, fluorescein diacetate was not suitable for the analysis of these cells because of their autofluorescence interfere with the fluorochrome emissions. Therefore, SYTOXTM Blue Dead Cell stain was used for the further experiments. A cryoprotective effect of polyhydroxybutyrate was observed during the freezing-thawing cycles. On the other hand, the culture rich in PHB seemed to be less resistant towards the oxidative stress induced by the hydrogen peroxide, probably justifiable by the state of chlorosis. Furthermore, this thesis deals with the increase/decrease in biomass concentration and intracellular PHB pool as a response to the external carbon source addition, analysed by the gas chromatography paired with FID detection. The most intesive cell growth in Synechocystis sp. PCC 6803 was detected in the presence of sodium acetate combined with gamma-butyrolactone while the highest PHB content in the medium supplied with acetate and 1,4-butanediol. Nevertheless, Synechocystis salina CCALA 192 showed markedly higher polyhydroxybutyrate accumulation under both photoautotrophic and mixotrophic conditions, the only non-suitable substrate was gamma-butyrolactone.
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Comparison of growth and productivity of autotrophic algae and red yeasts
Kouřilová, Xenie ; Rapta, Marek (referee) ; Márová, Ivana (advisor)
The submitted bachelor thesis focuses on the comparison of growth and productivity of red yeasts Rhodotorula aurantiaca and Xanthophyllomyces dendrorhous and autotrophic algae Chlamydomonas reinhardtii and Chlorella vulgaris under different physical conditions. The theoretical part contains the characteristics of selected micro-organisms, stress factors caused by temperature, radiation, combination of aeration and radiation and osmotic stress. Furthermore, knowledge on metabolites and the possibilities of their analysis are mentioned. Physical stresses, namely 2%, 3%, 4% and 5% solution of sodium chloride, intense radiation, absence of lighting, 8°C and 30°C, were applied to selected strains in cultivation. Under these conditions, changes in production of carotenoids, fatty acids, ergosterol, ubiquinone and proteins were being observed. A significant inhibitive effect on all the above mentioned micro-organisms was brought about by the use of 5% solution NaCl. The highest production of metabolites with X. dendrorhous was discovered when 4% solution of NaCl was used. R. aurantiaca demonstrated higher substance utilization at 30°C. For the selected algae strains were the used stresses lethal. Within the cultivation process, the possibilities of co-cultivation of yeasts and algae were also tested, which will be the subject of further studies.
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Study on metabolic properties of the bacterium Schlegelella thermodepolymerans with respect to biosynthesis of polyhydroxyalkanoates
Chvátalová, Viktorie ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This bachelor's thesis deals with the metabolic properties of the thermophilic bacterium Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates (PHA). The aim of the study was to understand the metabolism and utilization of different carbohydrates (xylose, cellobiose, glucose and their combinations) depending on the cultivation time. In the experimental part, two bacterial strains (M15344 and DSM15344) were used. The increase in optical density was monitored during the measurements for the bacteria. Furthermore, the content of P(3HB) from the biomass was determined using GC-FID and the molecular weight of the polymer was determined using SEC- MALS analysis. By measuring residual sugar using HPLC-RI, the course of carbohydrate utilization was determined as a function of time. The results of both bacteria were compared and evaluated. DSM15344 showed better results in P(3HB) production than M15344, and cellobiose and xylose appeared to be the most suitable substrates for P(3HB) production.
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Influence of makroelements from food on DNA and epigenetic profile
Veselý, Zdeněk ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
The macroelements contained in food have an important function for the human body. They are involved in several of biochemical reactions in the body and their abundance can prevent serious diseases. The theoretical part of the bachelor thesis describes the function of minerals in the human body, the function of DNA and epigenetic mechanisms such as DNA methylation or histone modification. The influence of nutrition and function of selected macroelements – sodium, magnesium, calcium, potassium on epigenetic modifications and on the stability of G-quadruplexes was described. The aim of the experimental part of this work was to study the effect of these substances on DNA structures in vitro and to prepare them experimentally for in vivo studies.
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