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Biotechnological production of PHA by selected bacterial isolates
Matějka, Filip ; Šedrlová, Zuzana (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the production of polyhydroxyalkanoates (PHA) using thermophilic bacterial isolates with designation 34, 35 and BŽ. Bacterial straines were isolated from activated sludge and compost The theoretical part contains a description of PHA, extremophilic bacteria and detection techniques for the determination of polyhydroxyalkanoates. In the experimental part, the presence of the phaC gene, which is crucial for the ability to produce PHA, was first determined by PCR and bacterial strains were also subjected to DNA sequencing of 16S rRNA gene which enabled preliminary taxonomical classification of the isolates. In the next part of the experimental work, the conditions for biomass growth and PHA production were optimized. Suitable carbon source, the ideal temperature for cultivation and the influence of precursors on the production of copolymers were studied and identified. The composition and proportion of PHA were determined spectrophotometrically and by GC-FID. Finally, visual screening of PHA accumulation inside bacterial cells was performed using fluorescence microscopy.
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Utilization of physicochemical and spectroscopic techniques in study of stress-response of cyanobacteria
Skoryk, Maksym ; Šedrlová, Zuzana (referee) ; Sedláček, Petr (advisor)
Tato bakalářská práce je soustředěná na zkoumaní cyanobakterií, vystavených hyper a hyposmotickým stresům. Na základě literární rešerše byly navžené vhodné analytické metody určené ke studiu dvou modelových organismu - Synechocystis sp. PCC 6803 a Synechocystis sp. salina Wislouch CCALA 192. Celkem čtyři metody byly použité k charakterizaci osmoticky zatížených bakterií. Průtoková cytometrie byla použita k vyhodnocení viability. Fluorescenční sonda SYTOX Blue poskytla důvěryhodnou informaci ohledně viability bakterií. Propidium jodid naopak poskytl nezřetelnou informaci. Optické vlastnosti cyanobakterií byly prozkoumané pomoci UV-VIS absorpčních a turbidimetrických měření. Termogravimetrická analýza byla použita pro mapování změn obsahu vody v osmoticky stresovaných buňkách. Tato metoda ukazala. že PHB-positivní Synechocystis sp. PCC 6803 jsou pravděpodobně vice odolné vůči hyperosmotickým stresům než PHB-negativní. Plynová chromatografie byla použita ke kvantifikaci vnitrobuněčného PHB, který činil přibližně 1-2 % suché hmoty PHB-positivních buněk.
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Analysis of C. necator genome changes after evolutionary adaptation
Vojsovič, Matúš ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The p53 protein is a transcription factor that belongs to the group of the tumour suppressor proteins. p53 functions include cell cycle regulation, interaction with DNA, and responses to damaged DNA that may lead to apoptosis or even senescence may occur. The theoretical part summarizes the latest information about the chemical structure and functions of the protein p53 and allosteric modifications that the p53 protein may possess in cells. It also describes selected methods of isolation and characterizes proteins. The aim of the experimental part was to isolate and characterize -isoforms of the p53 protein, which were isolated from the microorganism E.coli. Protein production was preceded by preparation and transformation of plasmids into production cells. From the production cells, 4 -isoforms of p53 were isolated by affinity chromatography, due to the polyhistidine anchor that the proteins contain. The isolated proteins were subsequently characterized by SDS-PAGE and western blotting. Moreover, the transactivation potential in the yeast system was monitored by luciferase assay under conditions involving the use of various culture media, as well as expressed or coexpressed under the inducible GAL1 promoter and the constitutive GPD promoter.
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Study on gene expression in Cupriavidus necator and other selected polyhydroxyalkanoates producers
Centnerová, Radmila ; Šedrlová, Zuzana (referee) ; Pernicová, Iva (advisor)
The aim of this bachelor thesis was study on gene expression in bacterium Cupriavidus necator H16 that is known as a model bacterium for the metabolism of polyhydroxyalkanoates. In the first part of this thesis, the optimalization of RT-qPCR method was performed. The optimized method was implemented on the study on gene expression. Furthermore, there were tested several commercial isolation kits for the genomic DNA isolation, the RNA isolation and the reverse transcription of the RNA and synthesis of the complementary DNA. These kits were compared in order to choose the one that would have provided the most relevant results and also the kit handling would have been simple and safe. There were different results accomplished for all kits. This means the kit used for the isolation had unneglectable impact on the quality of the isolated nucleic acid and therefore also on the success of the whole measurement. Isolated genomic DNA was used for optimalization and calibration. Isolated RNA and complementary DNA were used in the second part of the thesis. In this part, the studied bacterium was cultivated under various conditions and carbon sources. Subsequently, the optimized RT-qPCR method was performed and used for study on gene expression of chosen genes involved in the biosynthesis of polyhydroxyalkanoates. There were more significant differences in gene expression observed for fructose as a carbon source, compared to -butyrolacton as a carbon source. The greatest increase of the gene expression for fructose as a carbon source was measured for gene encoding 4-hydroxyphenylacetate-3-hydroxylase. There were more considerable differences in gene expression observed for -butyrolacton as a carbon source only for gene encoding 4-hydroxybutyrate dehydrogenase. Therefore, the choice of the carbon source impacts fundamentally the gene expression.
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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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Sequences forming G-quadruplexes in the amyloid beta precursor human gene and its homologues
Stránská, Anna ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The APP gene encodes the transmembrane protein amyloid beta precursor, which is expressed in many cell types, including neurons. Its functions have not yet been fully described, but it is clear that it is being cleaved before being exported to the extracellular space. It is degraded by various degradation pathways also undergoes homodimerization, which can produce particles with protective neuronal function as well as fragments that are toxic and cause nerve cell death. The formation of harmful amyloid beta plaques and their accumulation among neurons in the brain is closely linked to the onset and progression of Alzheimer's disease, a neurodegenerative brain disease manifested by death and loss of neurons, which leads to dementia, i.e. loss of cognitive functions. There is currently a lot of research that deals with the links between neurodegenerative diseases and the occurrence of G-quadruplexes in genes that are involved in disease manifestations. G-quadruplexes are non-canonical DNA and RNA nucleic acid secondary structures that arise in guanine-rich regions. They are important mainly in terms of their connection with biological processes such as the regulation of gene expression in genes and mainly in oncogenes because they occur in important regions of the gene such as the promoter. It is possible to stabilize them with small molecules, and it is this ability that is used in research into the therapeutic treatment of various diseases. A bioinformatics analysis of both the human gene and 346 other gene homologs was performed to determine the importance of G-quadruplexes localization and conservation in the human APP gene. For this purpose, the G4Hunter program was used, which provided information about the found sequences with the potential to form a G-quadruplex, such as their location or G4Hunter score. In vitro analysis was performed using thioflavin T reagent, which tested the ability of the found sequences to form G-quadruplexes under physiological conditions. The results confirmed the presence and evolutionary importance of G-quadruplexes found in the APP gene of Homo sapiens and their ability to assemble into quadruplex structures in the presence of salts such as sodium and potassium.
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The presence and localization of local DNA structures in the genome of Schizosaccharomyces pombe
Kubínová, Michaela ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The thesis focuses on the study of local DNA structures (cruciforms and G quadruplexforming sequence) in the genome of Schizosaccharomyces pombe, a yeast used in the food industry. The analysed local structures are non-randomly distributed within the genome. Based on previous studies, it has been found that they often colocalize with regulatory regions of genes and that the role of these secondary structures in the regulation of basic cellular processes (e.g. replication or transcription) is significant. This analysis was performed using specialized bioinformatics tools (G4Hunter and Palindrome Analyser) that allowed me to identify and analyze these structures in terms of their presence and localization. Many times less IR was found in mtDNA compared to the occurrence of IR in chromosomes. The number and frequency of PQS in mtDNA was also found to be very low. It is very different from the yeast Saccharomyces cerevisiae in this respect. It was also found that the number of IRs found decreases with increasing IR length and about 17% of IRs do not have a loop. A large enrichment of IRs was observed in the repeat_region and rRNA, and in the case of PQS in the rRNA and mRNA regions, i.e. sequences important for cellular processes.
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Sequences forming G-quadruplexes in the amyloid beta precursor human gene and its homologues
Stránská, Anna ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The APP gene encodes the transmembrane protein amyloid beta precursor, which is expressed in many cell types, including neurons. Its functions have not yet been fully described, but it is clear that it is being cleaved before being exported to the extracellular space. It is degraded by various degradation pathways also undergoes homodimerization, which can produce particles with protective neuronal function as well as fragments that are toxic and cause nerve cell death. The formation of harmful amyloid beta plaques and their accumulation among neurons in the brain is closely linked to the onset and progression of Alzheimer's disease, a neurodegenerative brain disease manifested by death and loss of neurons, which leads to dementia, i.e. loss of cognitive functions. There is currently a lot of research that deals with the links between neurodegenerative diseases and the occurrence of G-quadruplexes in genes that are involved in disease manifestations. G-quadruplexes are non-canonical DNA and RNA nucleic acid secondary structures that arise in guanine-rich regions. They are important mainly in terms of their connection with biological processes such as the regulation of gene expression in genes and mainly in oncogenes because they occur in important regions of the gene such as the promoter. It is possible to stabilize them with small molecules, and it is this ability that is used in research into the therapeutic treatment of various diseases. A bioinformatics analysis of both the human gene and 346 other gene homologs was performed to determine the importance of G-quadruplexes localization and conservation in the human APP gene. For this purpose, the G4Hunter program was used, which provided information about the found sequences with the potential to form a G-quadruplex, such as their location or G4Hunter score. In vitro analysis was performed using thioflavin T reagent, which tested the ability of the found sequences to form G-quadruplexes under physiological conditions. The results confirmed the presence and evolutionary importance of G-quadruplexes found in the APP gene of Homo sapiens and their ability to assemble into quadruplex structures in the presence of salts such as sodium and potassium.
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Study on gene expression in Cupriavidus necator and other selected polyhydroxyalkanoates producers
Centnerová, Radmila ; Šedrlová, Zuzana (referee) ; Pernicová, Iva (advisor)
The aim of this bachelor thesis was study on gene expression in bacterium Cupriavidus necator H16 that is known as a model bacterium for the metabolism of polyhydroxyalkanoates. In the first part of this thesis, the optimalization of RT-qPCR method was performed. The optimized method was implemented on the study on gene expression. Furthermore, there were tested several commercial isolation kits for the genomic DNA isolation, the RNA isolation and the reverse transcription of the RNA and synthesis of the complementary DNA. These kits were compared in order to choose the one that would have provided the most relevant results and also the kit handling would have been simple and safe. There were different results accomplished for all kits. This means the kit used for the isolation had unneglectable impact on the quality of the isolated nucleic acid and therefore also on the success of the whole measurement. Isolated genomic DNA was used for optimalization and calibration. Isolated RNA and complementary DNA were used in the second part of the thesis. In this part, the studied bacterium was cultivated under various conditions and carbon sources. Subsequently, the optimized RT-qPCR method was performed and used for study on gene expression of chosen genes involved in the biosynthesis of polyhydroxyalkanoates. There were more significant differences in gene expression observed for fructose as a carbon source, compared to -butyrolacton as a carbon source. The greatest increase of the gene expression for fructose as a carbon source was measured for gene encoding 4-hydroxyphenylacetate-3-hydroxylase. There were more considerable differences in gene expression observed for -butyrolacton as a carbon source only for gene encoding 4-hydroxybutyrate dehydrogenase. Therefore, the choice of the carbon source impacts fundamentally the gene expression.
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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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