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Effect of chromatin on the repair of double-strand DNA breaks after cleavage by CRISPR/Cas and other programmable nucleases in plants
Trojan, Jakub ; Přibylová, Adéla (advisor) ; Procházková, Klára (referee)
Plants are highly resistant to ionizing radiation, also thanks to a high-quality repair system for repairing double-stranded breaks. Double-strand breaks in plants are repaired by four repair pathways. Most often, double-strand breaks are repaired by non-homologous end joining (NHEJ), which joins the broken ends without further processing. More accurate but slower and more complex is repair through homologous recombination (HR), which repairs the break using a homologous sequence. HR repair takes place preferentially in a region with active transcription and during the S and G2 phases of the cell cycle. Alternatively, repair further proceeds through single-strand annealing (SSA) or Theta mediated end joining (TMEJ). Both pathways are based on short homology between the overlapping ends of the double-strand break. An often neglected part of repairs is the overcoming of repressive chromatin, which protects the genome from DNA damage and prevents access of nucleases but also acts as a barrier for repair proteins. This work summarizes the current knowledge about DNA repair in plants. Furthermore, describe the influence of chromatin not only on the repair but also on the activity of programmable nucleases used in genetic engineering, such as zinc finger nucleases (ZFNs), transcription activator-like...
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Epigenetic regulation of DQB1 gene in patients with type 1 diabetes mellitus
Gécová, Dominika ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Holá, Dana (referee)
Background: Type 1 diabetes mellitus is a multifactorial disease caused by beta cell destruction of Langerhans pancreatic islets. From the genetic aspect the main predisposition lays on HLA class II genes (40 - 50%), molecules of which present exogenous peptides to CD4+ T lymphocytes. Enviromental factors play a crucial role in the etiopathogenesis of T1DM. Through epigenetic regulation (e.g. DNA methylation) the genetic and enviromental factors communicate. The level of methylation in the regulatory regions can significantly affect expression of these genes. Aims: The aim of the diploma thesis was to define methylation profile of HLA DQB1 alleles in type 1 diabetes mellitus patients and determine their expression. Methods: The genotyping of HLA class II genes (HLA-DRB1, HLA-DQA1, HLA-DQB1) was performed using sequence specific primers. DNA was treated with sodium bisulfite, regulatory region of HLA DQB1 was amplified and cloned into E.coli, strain DH5α/XL1-Blue. Positive clones were sent for sequencing and results analyzed. RNA was transcribed to cDNA by reverse transcription and the level of expression was analyzed by quantitative PCR. Results: Statistically significant differences were found in total methylation of DQB1*0201 and *0302 alleles in the B section of DQB1 gene. Difference in...
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Epignetic Modifications of the Sperm and the Application in Clinical Practice of Human Assisted Reproduction Therapy
Štiavnická, Miriama ; Nevoral, Jan (advisor) ; Kloudová, Soňa (referee) ; Vašíček, Jaromír (referee)
Basement of healthy embryo development comes from quality of oocytes and spermatozoa. Today, when percentage of couples suffering infertility together with assisted reproductive therapy (ART) is increasing, understanding to gamete biology and heritable epigenetic code is crucial. The study is focused on promising epigenome based markers that could serve as indicators of gamete quality for either their screening or selection for ART. Accordingly selected markers were used for the investigation of environmental pollutant bisphenol S (BPS) effect on gametes quality. To obtain these aims, we have used human semen samples, boar semen samples and ICR mice gametes. Samples were analyzed by flow cytometry, immunocytochemistry and western blot analysis. All experimental work was in accordance with Ethics committee University Hospital in Pilsen and approved experimental designs for appropriate experimental animal project. In the study, we detected the dimethylation of histone H3 on lysine K4 (H3K4me2) as potential epigenetic marker of sperm quality and chromatin immaturity. Secondly, we observed the role of the gasotransmitter hydrogen sulphide (H2S) as anti-capacitating agents, slowing down capacitation possibly through post-translational modification of proteins. Thirdly, SIRT1 histone deacetylase was...
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Epigenetic regulation of DQB1 gene in patients with type 1 diabetes mellitus
Gécová, Dominika ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Holá, Dana (referee)
Background: Type 1 diabetes mellitus is a multifactorial disease caused by beta cell destruction of Langerhans pancreatic islets. From the genetic aspect the main predisposition lays on HLA class II genes (40 - 50%), molecules of which present exogenous peptides to CD4+ T lymphocytes. Enviromental factors play a crucial role in the etiopathogenesis of T1DM. Through epigenetic regulation (e.g. DNA methylation) the genetic and enviromental factors communicate. The level of methylation in the regulatory regions can significantly affect expression of these genes. Aims: The aim of the diploma thesis was to define methylation profile of HLA DQB1 alleles in type 1 diabetes mellitus patients and determine their expression. Methods: The genotyping of HLA class II genes (HLA-DRB1, HLA-DQA1, HLA-DQB1) was performed using sequence specific primers. DNA was treated with sodium bisulfite, regulatory region of HLA DQB1 was amplified and cloned into E.coli, strain DH5α/XL1-Blue. Positive clones were sent for sequencing and results analyzed. RNA was transcribed to cDNA by reverse transcription and the level of expression was analyzed by quantitative PCR. Results: Statistically significant differences were found in total methylation of DQB1*0201 and *0302 alleles in the B section of DQB1 gene. Difference in...
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