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Studying of Gene Expression Involved in Hyaluronic Acid Synthesis in Streptococcus Equi Subsp. Zooepidemicus Using DNA Microarrays and Real-Time PCR
Hrudíková, Radka ; Šeda,, Ondřej (referee) ; Bobek,, Jan (referee) ; Velebný, Vladimír (advisor)
Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.
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Reimplementation of the Connectivity Map with Accessibility Enhancements
ARICANLI, Evren
The Connectivity Map (CMap) is a biomedical research tool which leverages modern biotechnology and bioinformatic methods to provide a platform for comparing the a subject gene expression profile against a library of drug-treatment gene expression profiles. This comparison allows for retrieval of drugs from the CMap's library which induce biological activity similar (or dissimilar) to a given subject expression profile -- thereby making it a tool which allows for the finding of repositionable drugs. Herein, a number of adaptations are proposed -- most focally being changes which would enhance the tool's accessibility (e.g. a simplified UI/workflow, expanded queryable gene identifiers). Using the R statistical computing environment and numerous libraries available through it, an adapted CMap tool was created from the publically-accessible CMap data, which housed the proposed adaptations. To evaluate success of the accessibility adaptations, the adapted CMap tool was focus tested on several participants -- which ultimately yielded great favor for the changes made, allowing them to be conclusively suggested for future iterations of the CMap.
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Studying of Gene Expression Involved in Hyaluronic Acid Synthesis in Streptococcus Equi Subsp. Zooepidemicus Using DNA Microarrays and Real-Time PCR
Hrudíková, Radka ; Šeda,, Ondřej (referee) ; Bobek,, Jan (referee) ; Velebný, Vladimír (advisor)
Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.
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The study of mutual interaction between pathogenic yeasts of the genus Candida and bacterium Pseudomonas aeruginosa during cocultivation
Mynářová, Lenka ; Hodek, Petr (advisor) ; Papoušková, Klára (referee)
The genus Candida includes several opportunistically pathogenic species which are common causative agents of the yeast infections in humans. Although current medical research is focused mostly on cancer, AIDS or Alzheimer disease, the problem of systemic candidiases cannot be neglected. These infections represent a real threat to the immunocompromissed patients, they are connected with a high mortality rate and expensive medication with poor prognosis. Pseudomonas aeruginosa could be an inspiration in a way of how to eliminate the pathogenic yeasts. The bacterium can inhibit growth of the most common yeast species of the genus Candida, C. albicans. This effect is based on production of toxic substances by the bacterium and on interaction of the bacterium with the C. albicans cell wall, which leads to the lysis of the yeast cells and which is not fully understood. Nevertheless, coexistence of these microorganisms is also possible and their relationship is affected by various factors. Knowledge of these inter- microbial interactions was obtained from studies of diseases and pathologies, during which C. albicans + P. aeruginosa coinfections occur. In this thesis I studied mechanisms of interaction between pathogenic yeast C. albicans and bacterium P. aeruginosa by a) C. albicans gene expression...
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DNA čipy a jejich využití pro molekulární diagnostiku a predikci u onkologických onemocnění
VORÁČKOVÁ, Lucie
Many visible factors produced during carcinogenesis refer to oncological disease, e.g. modified gene's expression or increased number of miRNA. These factors can be diagnosed by analyzing of gene's expression. For this diagnostics, there are used many methods, of whom DNA microarray analysis is the most significant progress in genetic research. A large number of DNA sequences can be detected DNA with microarrays on a single microarray in a relatively short time. This analysis means a positive turnover for the diagnosis and prediction of disease.
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