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Analysis of rRNA genes in variets Brassica napus
Dofková, Květoslava ; RNDr.Roman Matyášek, CSc. (referee) ; Kovařík,, Aleš (advisor)
Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.
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Nucleotide density for finding CpG islands
Sikorová, Eva ; Provazník, Ivo (referee) ; Maděránková, Denisa (advisor)
This bachelor thesis deals with searching for CpG islands in the DNA sequences using the nucleotide density vectors. The first part includes view of the DNA structure, the genetic information expression, more detailed analysis of CpG islands and primarily their detection methods. In the practical part the algorithm for graphical representation of nucleotides on the basis of their densities and detections of CpG islands of artificially created and real DNA sequences was realized in the MATLAB program. The thesis includes the analysis of twenty sequences with the expected content of CpGIs and the comparison of results between the created program and two search engines.
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DNA Methylation Patterns in patients with Neurodevelopmental Disorders
Vítková, Magdalena ; Steiner Mrázová, Lenka (advisor) ; Krejčík, Zdeněk (referee)
Neurodevelopmental disorders (NDDs) are diseases characterized by abnormal development of the central nervous system and brain. Simultaneously, the NDDs may also be associated with specific changes in DNA methylation. The rapid development of sequencing techniques, particularly whole genome sequencing (WGS) and whole exome sequencing (WES), has helped to uncover a number of genes associated with NDDs. Determining the pathogenicity of the variants found remains a challenge in the diagnosis of patients with NDDs. Finding the DNA methylation patterns by microarray or methylation sequencing in patients with NDDs carrying a variant of uncertain significance (VUS) may provide valuable information to address this problem. This thesis demonstrates the feasibility of using these approaches to functionally classify found variants in the KDM5C, ARID1B, ATRX, and BRWD3 genes. The aim was to determine whether there are changes in the DNA methylation patterns of patients with NDDs compared to probands and healthy controls, which could be used to confirm the diagnosis. Key words: DNA methylation patterns, DNA methylation, histone modifications, NDDs, KDM5C, ARID1B, ATRX, BRWD3
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Regulation of histone post-translational modifications and its use in the diagnosis and treatment of lung cancer
Marada, Kryštof ; Janoštiak, Radoslav (advisor) ; Červená, Klára (referee)
The aim of this bachelor's thesis is to summarize the epigenetic mechanisms, namely DNA methylation, non-coding RNA and post-translational modification of histones, within the scope of which the five currently most researched types are described, i.e. acetylation, methylation, phosphorylation, ubiquitinylation and sumoylation. Furthermore, a summary of the modifying enzymes whose function is to add and remove these epigenetic components. And also the description of the proteins that recognize these components. The following is a description of how epigenetic processes are involved in the course of cancer. The remaining part of the work is devoted to lung cancer, its most common causes, what methods are currently used to treat it, and how post-translational modifications of histones could be used in its treatment. Most of this chapter is devoted to histone deacetylase inhibitors, as these inhibitors represent potential as a new component of lung cancer treatment, which also makes them the target of much research.
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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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Transgenerational effects of plant biotic interactions
PUY GUTIÉRREZ, Javier
This thesis focuses on the transgenerational effects triggered by plant biotic interactions and explores their relevance on ecological and evolutionary processes. The following sections document novel results that show their important consequences on different aspects. Primarily, we established the necessary methodology to be able to explore these questions and to disentangle the mechanisms originating the transgenerational plasticity by validating a demethylation method. Then, we checked whether the biotic interactions alter the phenotype via within-generation and transgenerational plasticity, examining the magnitude and direction of the response on each specific "response traits". Lastly, the potential role of transgenerational plasticity for adaptation, species coexistence, creating biodiversity and population and ecosystem functioning is tested.
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Epigenetic regulation of HLA class II genes in relation to senescence of organism
Říhová, Adéla ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Slavčev, Antonij (referee)
Introduction: Glycoproteins of the major histocompatibility complex (MHC) are an irreplaceable part of immune response regulation and immune homeostasis maintenance. The regulation of the expression plays an important role in adaptive immune response. Recently, DNA methylation in regulatory areas, crucial for DNA availability to transcription factors, is one of the most researched mechanisms of this type of regulation. The DNA methylation is, among others, related to the aging processes. Increased predisposition age-related immunosenescence in higher age could result from the changes in methylation status of regulatory areas of MHC class II genes. Aims: The aim of this thesis is to analyze the methylation status of regulatory areas of DQB1 gene and to compare the differences between generations and specific alleles. The differences in the levels of DQB1 gene mRNA transcription between generations and specific alleles is also compared. Methods: Both DNA and RNA were isolated from blood samples obtained from donors of three different age groups. DNA was genotypized and modified by bisulfite conversion. The regulatory areas of DQB1 genes were then amplified and subcloned into bacteria. The positive clones were selected and subjected to DNA methylation analysis. RNA was reverse transcribed into cDNA...
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Role paternálního H4K12ac při utváření pronukleí a v časné embryogenezi u myší.
Dudková, Barbora ; Hortová, Kateřina (advisor) ; Stopka, Tomáš (referee)
During the process of spermatogenesis, histones are replaced by protamines, basic proteins enabling transmission of DNA to the oocyte during fertilization. In mouse sperm, there is only 1% of remaining histones whose N-terminal tails contain post-translationally modified residues. In this study, I was interested in contribution of paternal histone H4 acetylated on lysine K12 residues (H4K12ac) that is present in mature sperm head in remaining nucleosomes. Physiologically, H4K12ac has an important role in transcription factor accumulation and in regulation of gene expression. The presence and abundance of H4K12ac modification in various pronuclei stages of 1-cell embryo and parthenotes were assessed by imunnoflourescent detection with utilization of anti-H4K12ac antibody. Altogether, the paternal pronucleus exhibits a strong acetylation signal on H4K12 since its formation, while in the maternal one, there is a slow continual increase of H4K12ac getting on the same level as paternal pronucleus till the pronuclei fusion. Simultaneously DNA methylation status in both pronuclei was detected. In paternal pronucleus there is a continual decrease in the DNA methylation detectable as a decrease of 5mC and an increase of 5hmC signal. Meanwhile, the maternal pronucleus stays widely methylated. DNA...
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