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Interaction of selected natural substances used in food industry with DNA and its structural motifs
Gardošová, Zuzana ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
G-quadruplexes represent secondary DNA structures formed in guanine-rich nucleic acid regions. These structures are involved in many biological processes, including DNA replication, transcription, and telomere maintenance. Several natural substances interacting with G-quadruplex structures have been described. Many of them can be used in the treatment of cancer or other areas of therapeutic practice. G-quadruplexes are dynamic structures whose stability can be affected by a variety of different factors, including chemical modifications to DNA. One of these modifications is DNA methylation, which is an important epigenetic mechanism regulating gene expression. DNA methylation can affect the function and stability of G-quadruplex structures. The theoretical part of the present work focuses on DNA secondary structures, characterization of G-quadruplexes and their ligands and describes the relationship between DNA methylation and G-quadruplex structures. In the experimental part, the binding ability of the natural substances quercetin, berberine, piperine, and caffeine to G-quadruplex structures formed in telomeric oligonucleotide sequences and sequences derived from the proto- oncogene c-Myc was confirmed. Furthermore, the ability of berberine and quercetin was proven to stabilize G-quadruplexes in the aforementioned sequences. Bioinformatics analysis showed that the frequency of G4 is higher in CpG regions than in their surroundings, and the highest frequency of G4 within CpG regions was observed on chromosome 19. Global methylation assays demonstrated that the breast cancer cell line exhibited hypomethylation compared to the non-tumor human dermal fibroblast cell line. After treatment with berberine, the analyzed DNA of both cell lines showed hypermethylation, whereas DNA after interaction with quercetin showed hypomethylation.
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The effect of indole alkaloids on selected cell lines and DNA structures
Dobrovolná, Michaela ; Hoová, Julie (referee) ; Brázda, Václav (advisor)
G-quadruplexes (G4) are secondary DNA structures formed by a cluster of guanines that have been shown to play a role in many biological functions, including regulation of replication timing and repression of oncogene expression. In this work, four plant alkaloids (harmine, harmane, harmaline, and brucine) were tested as potential G4 ligands that could regulate, induce, or convert different G4 topologies. Furthermore, their effect on fibroblast cell line HDF164 and breast cancer cell line MCF-7 was studied, in which case a dose-dependent growth inhibition mechanism was demonstrated. Interaction with the parallel G4 promoter of the proto-oncogene c-Myc was confirmed for harmine, harmaline, and brucine, and with the hybrid G4 of telomeric DNA for harmine, harman, and harmaline. In addition, the ability of the tested compounds to interact with calf thymus double-stranded DNA (ctDNA) was verified by UV-Vis absorption spectroscopy. The methylation assay showed that the DNA of MCF-7 cells treated with harmine and brucine was hypermethylated.
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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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Development of methods for genetic analysis of plant foods
Fialová, Lenka ; Brázda, Václav (referee) ; Doškař, Jiří (referee) ; Márová, Ivana (advisor)
Multiplex real-time PCR-HRM is an approach which has gained some attention in recent years. It has already found applications in clinical diagnostics and food authenticity and safety control. Compared to its corresponding singleplex PCR assays, an optimized multiplex PCR assay provides the same information in a fraction of time. First part of this work dealt with isolation of DNA from both fresh fruits and processed commercial products. Six different DNA isolation protocols were tested with fresh fruits – three silica column-based kits, two magnetic carrier-based kits and one conventional protocol. One method was chosen as the most suitable and was applied to DNA isolation from commercial products. These experiments also involved optimisation of the chosen method. The second part of this work was focused on the development of a triplex real-time PCR assay for simultaneous detection of blueberry, strawberry and raspberry, and its application on DNA isolated from commercial products. During DNA isolation, calcium chloride was shown to be a promising agent for removal of pectin from samples. In several samples, presence of raspberry DNA was confirmed by singleplex PCR. We found out that for accurate results of food analysis by this assay, further optimization of its parameters would be needed.
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Eucaryotic cells systems and their biotechnologycal applications
Porubiaková, Otília ; Obruča, Stanislav (referee) ; Vorlíčková, Michaela (referee) ; Brázda, Václav (advisor)
The submitted dissertation is divided into several parts. The first part deals with the interactions of the p53 protein and its isoforms with different potential DNA substrates under different experimental conditions. These are predominantly DNA and its non-canonical structural motifs, such as G-quadruplexes or cruciform structures, whose interactions have been studied in yeast isogenic systems or by in vitro methods. The seconds part deals with the bioinformatic analysis of the mentioned secondary DNA structures in different organismal groups, and the result is a set of publications that show their non-random distribution in the genome and their relationship with regulation. The last part of the work contains unpublished results, including the results of testing the effect of natural and synthetic substances on aging in model human cells.
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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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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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Polyphenols in nutritions and their effect on DNA
Osorio, Juan ; Černayová, Diana (referee) ; Brázda, Václav (advisor)
Epidemiologické studie prokázaly vliv konzumace rostlinných potravin v prevenci široké škály nemocí. Přírodní antioxidanty přítomné v těchto potravinách, mezi nimiž jsou velmi důležité polyfenoly, mohou být zodpovědné za tuto činnost podporující zdraví. Cílem bakalářské práce je ukázat interakci určitých polyfenolů s genetickým materiálem prostřednictvím různých signálních mechanismů, zejména pokud jde o stabilizaci nekanonické struktury DNA G-kvadruplex a poukázat tak na nejselektivnější látku pro inhibici biochemických procesy. Dále práce obsahuje podrobné informace, které mohou pomoci pochopit, jak mohou polyfenolové sloučeniny interagovat s DNA prostřednictvím epigenetických mechanismů a G4 struktur, a které faktory mohou ovlivnit jejich účinnost. Různé experimenty, biologickým a experimentálním opakováním, byly použity k potvrzení interakce mezi sloučeninami a DNA.
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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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Influence of storage on the microbial composition of French Saint-nectaire cheese
Šislerová, Lucie ; Mikulíková, Renata (referee) ; Brázda, Václav (advisor)
The aim of my work is the comparison of microbial composition between farmtype and dairytype of Saint-nectaire cheese and the influence of storage time and temperature on the development of microbial composition, content of fatty acids and aromatic substances. Selected microorganisms were identified by RT-PCR. In addition, Penicillium roqueforti and fuscoglaucum have been identified in the Saint-nectaire farm type compared to the dairy type. In both types of cheese, the highest amount of selected microorganisms was detected in fresh cheese. When stored at 20 °C, an increase over fresh cheese occurred in the following microorganisms: Streptococcus thermophilus, Lactobacillus bulgaricus, Cladosporium herbarum and Penicillium commune and camemberti, and the presence of contaminants and pathogens was noted. After one week of storage at 20 °C, they were Micrococcus luteus, Salmonella enterica and Staphylococcus aureus, and after another two weeks of storage, Listeria monocytogenes was identified. The fatty acid and volatile compounds were compared for five samples: fresh cheese, cheese stored in the refrigerator for one week and three weeks and cheese stored at 20 °C for one week and three weeks. The content of bound and free fatty acids was measured, both by GC-FID. The content of bound fatty acids was comparable in all measured samples. The highest content of free fatty acids was in the cheese after three weeks of storage at 20 °C. The most common fatty acid is palmitic acid. Volatiles were determined by HS-SPME-GC-MS. The most volatiles were identified in the cheese after three weeks at 20 °C and in the cheese after one week in the refrigerator. The most represented groups were alcohols, ketones and acids.
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