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Use of molecular techniques to characterize yeasts of the genus Metschnikowia
Schneiderwindová, Nicole ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
This diploma thesis deals with the possibilities of implementation and use of molecular methods for the characterization of yeasts of the genus Metschnikowia and the application of methods in biotechnology or the food industry. The theoretical part focuses on a brief description of yeast, specially selected species that were used during the practical part of the work, the possibilities of their use, and especially on a detailed description of all molecular techniques used. The practical part focuses on the optimization of the molecular methods, namely the method of pulsed gel electrophoresis and the method of denaturing gradient gel electrophoresis. Initially, yeast was cultured under optimal conditions that are specific to this genus. Furthermore, their DNA was isolated using isolation techniques, which were subsequently processed using PFGE and PCR–DGGE methods. The DNA isolation procedure needed to be optimized the most. Several optimizations of the concentration of lysis enzymes, especially the lyticase enzyme, were performed. It was also necessary to determine the correct ratio of low-melting agarose and isolated DNA, which was essential for the correct consistency of the isolated DNA blocks and their further application in PFGE analysis. Finally, the PFGE method was optimized, which brought the correct distribution of chromosomes, and it was possible to describe the individual chromosomes according to their size according to the standard used CHEF of the yeast Hansenula wingei. To properly optimize the DGGE analysis process itself, it was first necessary to isolate the yeast DNA using a kit, then it was used as a template for the PCR reaction. The annealing temperature was also optimized for the individual groups of primers. The amplicons obtained by this reaction were separated by the DGGE method. This technique mainly required the optimization of basic parameters such as the range of the denaturation gradient or the total separation time. According to the measurement results, it can be determined that the process of yeast DNA isolation and their subsequent analysis using molecular methods of pulsed gel electrophoresis and denaturing gradient gel electrophoresis was successful. We were able to describe the genome and determine the number of chromosomes in all used yeast species of the genus Metschnikowia at least partially.
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Mutant p53 protein and its binding and transactivation properties
Vojsovič, Matúš ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The "genome guardian" protein p53 plays an important role in cancer growth. P53 mutations occur in more than 50 % of human cancers. Mutated proteins significantly affect the proper functioning of cells. Due to the mutation, proteins can gain, but also lose, some of their functions, which also help them in modulating cell metabolism. Mutant forms of p53 may be involved in indirect binding or direct binding to DNA. They appeared to have a lower binding activity to the DNA than non-mutated p53. The experimental part of the thesis focuses on measuring the binding properties of selected p53 mutants using gel retardation analysis and using an atomic force microscope and monitoring the transactivation potential. The results were compared with the wild-type form of p53. It has been found that binding to the most common types of local DNA structures reduces the binding activity of p53 mutants over the wild-type. P53 mutants has been shown to have a lower intensity of transactivation than the wild-type p53 by studying their transactivation abilities and also they are able to reduce the intensity of transactivation when co-expressed with p53.
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Analyses of inverted repeats localization in bacterial genomes
Šedý, Michal ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
Inverted repeats (IR) are common part of DNA of all living prokaryotic and eukaryotic organisms. Inverted repeats plays an important role in the regulation of basics cells processes. They are responsible for formation of cruciform structures. Inverted repeats also cause genomic instability and can be a source of numerous mutations. Cruciform structures can be recognized by DNA-binding proteins and can also act as a transcriptional regulators. Using the Palindrome Analyser tool, the frequency of IR and localization of inverted repeats in bacterial genomes was analyzed. The frequency of IR across the bacterial genome is variable. The frequency of short inverted repeats shows an approximately quadratic dependence on the %GC content in the genome with a minimum of about 50% of GC content. The localization of inverted repeats with respect to “annotated features” show a non-random distribution. The frequency of IR for most features is higher “outside” than “inside”.
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Influence of various cosmetic polysaccharides as prebiotics on skin microbiome
Pelánová, Lenka ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
The presented master thesis deals with the monitoring of the influence of polysaccharides which are used as an additive in the cosmetic products, on the monitored types of bacteria which are part of the skin microbiome. And it also deals with the study the effect of polysaccharides as prebiotics on selected species of bacteria that are part of the skin microbiome. Two polysaccharides were selected to determine the effects on the skin microbiome: Nanomoist and PoLevan S. The first part of the thesis focuses on the literature search which deals with skin anatomy, skin diseases and skin microbiome and its function. The experimental part is focused on monitoring changes in the quantity of selected microorganisms of the skin microbiome, before and after application of polysaccharides to the skin using qPCR. Staphylococcus epidermidis, Cutibacterium acnes, Escherichia coli and Micrococcus luteus were monitored. The PCR products were detected by agarose gel electrophoresis. The bacterium Staphylococcus epidermidis was detected on the skin to the greatest extent, especially after the application of the polysaccharides Nanomoist and PoLevan S. Thus, a positive effect of both polysaccharides on the growth of this bacterium was found.
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Bioinformatic analysis of PHA synthases of thermophilic bacteria
Brondová, Zuzana ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.
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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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Influence of cultivation conditions on the production of recombinant proteins
Gardošová, Zuzana ; Nováčková, Ivana (referee) ; Brázda, Václav (advisor)
Recombinant proteins are produced by using genetic modifications. In this process is insert contained gene encoding a certain protein in a cloning vector cloned into the host organism. Recombinant proteins are expressed after the transformation of the cloning vector into a host, the host organism. The expression of recombinant proteins in bacterial cells is one of the most efficient ways to manufacture these proteins. The p53 protein is a tumor suppressor protein, the main role in cells is to react on DNA damage. Due to the reaction to various intracellular and extracellular stimuli, including DNA damage, the p53 protein shows different biological functions, including regulation of senescence, cell cycle, or apoptosis. The theoretical part of the thesis part presents the basic properties of proteins, methods of recombinant protein expression, methods of protein isolation, and characterization of p53 protein. The aim of the experimental part was to determine the effect of incubation temperature on recombinant p53 protein production. The work involves the isolation of plasmid DNA and its transformation into E. coli production cells. The produced proteins were successfully isolated and subsequently characterized by SDS-PAGE and Western Blotting.
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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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Detection of probiotic bacteria in diary food products using PCR technique
Klaška, Dominik ; Brázda, Václav (referee) ; Smetana, Jan (advisor)
In the bachelor thesis, DNA was isolated from commercially available white yogurt. The isolated DNA, which was gained by two different methods, was performer analyse by a spectrophotometer. Both methods provided sufficiently concentrated and high-quality DNA for further analysis by PCR. Precisely defined sections of isolated DNA were amplified using specific primers. The presence of the bacterium domain was detected, and in the case of genus specific amplification, the presence of bacteria of the genus Lactobacillus was detected too, by gel electrophoresis.
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Analysis of local structures in DNA molecules
Nyczová, Adéla ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Local DNA structures are alternative DNA conformations which can be formed aside from typical B-DNA conformation. These structures often play pivotal roles in regulation of basic biological processes, such as DNA replication, transcription or binding of specific ligands. This biological significance makes alternative DNA secondary structures a potential drug target. In this diploma thesis, local structures in genomes of viruses from Flaviviridae and Retroviridae families are analysed using bioinformatics tools. Furthermore, these structures are visualised using atomic force microscopy.
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