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Infuence of natural and synthetic G4-ligands to p53 transactivatio
Perná, Kristýna ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Secondary DNA structures, such as G-quadruplexes, occur in the promoters of human genes. Dysfunctions of these quadruplex structures have been observed in several cases of cancer, and therefore these structures are the subject of research for the design of new anticancer drugs. The p53 protein is an important regulatory protein in the process of cell cycle control and DNA repair. Mutations in the gene encoding this protein occur in more than 50 % of cancer cases. In this thesis, the influence of natural ligands binding to G-quadruplexes on the binding and activity of the p53 protein was investigated. The theoretical part of the thesis describes the structure and binding properties of p53 protein, the structure and role of G-quadruplexes and describes selected G4-ligands occurring in food - curcumin, quercetin, berberine and ellagic acid. The aim of the experimental part of this thesis was to determine the effect of these substances on the transactivation activity of the p53 protein in vivo based on their interaction with G-quadruplexes using yeast isogenic systems. The interaction between selected G-quadruplex structures and ligands was first verified in vitro using the ThT assay and circular dichroism.
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Identification of probiotic bacteria with the use of PCR from plant frementation diary products
Kuljovská, Tereza ; Langová, Denisa (referee) ; Smetana, Jan (advisor)
Probiotics are encountered daily, either by targeted application of dietary supplements containing live cultures of lactic acid bacteria or by consuming traditional fermented products including dairy or non-dairy products. These cultures can significantly affect intestinal microbiota, particularly during digestive discomfort or problems associated with diarrheal diseases or infectious gastritis. This work focuses on the isolation of probiotics from fermented plant-based products that are also suitable for vegetarians and vegans, whose numbers are currently increasing. In addition to traditional lacto-fermented foodstuffs, there is another alternative in the form of Kombucha. Probiotic bacteria isolated from real samples were subjected to polymerase chain reaction (PCR) to identify them. Presence of the Bacteria domain was detected, and with more specific amplification, the presence of the genus Lactobacillus was detected too, using the specific primers. The amplification resulted in individual fragments were visualized by agarose 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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Nuclear Fuel for Pressurized Water Reactors
Smetana, Jan ; Vojáčková, Jitka (referee) ; Katovský, Karel (advisor)
This bachelor thesis deals with structural characteristics of nuclear fuels, where most of the attention is paid to pressurized water reactors. The examples of the nuclear fuels implementation for each reactor type are mentioned for reader's better understanding. A brief history of pressurized water reactors is also listed, VVER reactors particularly. The nuclear fuel development for pressurized water reactors is demonstrated on the example of the development of fuel for our nuclear power plants Dukovany and Temelin, changes of fuel structure are emphasized. In the thesis there is also carried out a computational comparison of several selected parameters of used nuclear fuel during the operation of the nuclear power plant Dukovany.
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Production and characterization of antimicrobial substances from lactic acid bacteria
Franeková, Eliška ; Smetana, Jan (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on the production of antimicrobial compounds using lactic acid bacteria, their characterization and testing of their antimicrobial activity. The theoretical part of this work deals with the characteristics of lactic acid bacteria and the bacteriocins they produce, the possibilities of bacteriocin production and the factors that influence it. In the experimental part of this work, lactic acid bacteria Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Bifidobacterium bifidum were cultivated, their growth curves and total protein content in supernatants after cultivation were measured. Cell-free culture supernatants were prepared by lyophilization and their antimicrobial activity against gram-positive and gram-negative bacteria was determined. The antimicrobial activity of commercial antimicrobial peptide nisin and a commercial preservative obtained using lactic acid bacteria was also measured. The sample obtained from Bifidobacterium bifidum was selected as the most effective of the isolates. Its antimicrobial activity was further tested on polymeric sausage packaging materials. Antimicrobial substances produced by lactic acid bacteria can be used in the food industry as preservatives or as a part of antimicrobial packaging, and in the pharmaceutical industry in materials for antimicrobial wound dressings.
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Use of polymerase chain reaction (PCR) technique for identification of probiotic microorganisms in milk diary product
Horká, Markéta ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
In this bachelor thesis, probiotic bacteria and their beneficial effects on human health were described. Several methods that are used to identify microorganisms were also stated. At the beginning of the experimental part, DNA was isolated from a selected dairy product. DNA extraction was made in two ways, both of which provided sufficiently high-quality DNA. The prepared DNA was subjected to polymerase chain reaction using specific primers. The presence of bacteria of the genus Lactobacillus, Bifidobacterium and the species Lactobacillus acidophilus, which were declared by the yogurt manufacturer, was proved by electrophoresis on agarose gel.
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Identification of probiotics in cometic product using PCR technique
Akmalova, Adelina ; Langová, Denisa (referee) ; Smetana, Jan (advisor)
This bachelor thesis deals with the identification of probiotic microorganisms in cosmetic products by polymerase chain reaction (PCR). The aim of this work was to confirm the presence of bacterial DNA in cosmetic products containing probiotic microorganisms. The theoretical part of the thesis focuses on the role of probiotic microorganisms in maintaining skin health and their effects on the human body. Different methods for the identification of probiotics are also highlighted with a focus on molecular genetic approaches. In the experimental part of the work, two cosmetic products with declared presence of probiotic microorganisms were tested. Two methods were used to extract DNA from the products: phenol extraction and commercial kit method. Furthermore, amplification of a specific region of the 16S rRNA gene was performed by PCR. The PCR products were subjected to agarose gel electrophoresis and visualized by ultraviolet light. All cosmetic products showed the presence of the Bacteria domain and the genus Lactobacillus. In addition, species-specific PCR confirmed the presence of the declared probiotic species Lactobacillus pentosus and Lactobacillus bulgaricus in the products.
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Use of advanced molecular biology techniques for detailed characterization of probiotic bacteria from a dietary supplement
Folwarczná, Tereza ; Trachtová, Štěpánka (referee) ; Smetana, Jan (advisor)
The aim of the diploma thesis was the identification of probiotic bacteria from a probiotic food supplement in the form of syrup. DNA was isolated from the complex matrix of the product in sufficient purity and quality suitable for the real-time PCR method followed by PCR-HRM. Specific primers for the genera Lactobacillus, Bifidobacterium and Streptococcus and at the species level for the species Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus thermophilus were used for amplification by real-time PCR and PCR-HRM. After optimizing the conditions for specific primers, all declared bacteria were detected in the product.
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Analysis of IFI16 protein binding to DNA
Kratochvilová, Libuše ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
This diploma thesis deals with the binding of interferon gamma-inducible protein 16 (IFI16) to DNA with the potential of G-quadruplex formation. The IFI16 protein contains two tandemly located DNA-binding HIN domains showing differential binding to DNA structures. IFI16 protein has been shown to preferentially bind G-quadruplex structures over other nucleic acid secondary structures. G-quadruplexes are secondary local structures of DNA (or RNA) that are easily formed under physiological conditions in a number of important regulatory regions of the genome, or are part of the genomes of a number of viruses and pathogens. The ability to recognize, specifically bind and stabilize G-quadruplex structures explains the involvement of the IFI16 protein in the cellular processes of replication, transcription and translation and the establishment of innate immune responses. In the first part of the thesis, the sequences of synthetic oligonucleotides with the potential for G-quadruplex formation were characterized by selected biophysical methods and the full-length IFI16 protein was isolated, which was subsequently used for in vitro binding and competitive binding experiments with characterized oligonucleotides. In the last part of the work, isogenic yeast strains differing in the sequences of the responsive element were transformed with plasmid vectors for the expression of p53 and IFI16 proteins with constitutive and GAL inducible promoters, and the one-hybrid yeast system model was optimized for the study of IFI16 protein interactions in vivo. The results show that most of the analyzed sequences are able to form G-quadruplex structures in vitro, even in the presence of only one run of three or more G-bases. While the presence of several G-runs separated by a single nucleotide spacer led to the formation of intermolecular G-quadruplex structures, mutation in the original G-quadruplex sequence induced the formation of intramolecular structures with different conformations. In vitro binding and competitive binding experiments demonstrated specific binding of the IFI16 protein to G-quadruplex structures without differences in protein binding preference to a particular G-quadruplex conformation. Stabilization of G-quadruplex structures in vivo behind the transcription factor responsive element (p53) in the gene promoter induced repression of the transcription of the given gene. In the absence of any binding site of the IFI16 protein, a protein-protein interaction between the IFI16 and p53 proteins occurred, which led to an increase in the transactivation potential of the p53 protein, while the binding of the p53 protein and initiation of reporter gene transcription was influenced not only by the presence of the G-quadruplex motif and its stabilization, but and the DNA sequence adjacent to the p53 responsive element.
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