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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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The role of small RNAs in transgenerational plant stress memory
Jaklová, Veronika ; Marková, Hana (advisor) ; Lipavská, Helena (referee)
Plants are constantly affected by various abiotic and biotic stresses, which cause a whole range of reactions.The resultcanbe increasedplantresistance tovariousstressfactorssuchasherbivoryattack or lack of water. Additionally, this resistance can also be passed on to subsequent generations through epigeneticmechanisms.Small RNAsservingas signaling moleculesof the plant's rapid response tostress can play a large part in the formationof intergenerationalandmultigenerationalstressmemory.MiRNAs are mainly regulators of gene expression,throughtheirinhibitory and degradative activitiesthey control the transcription of genes and the translation of a large number of proteins. SiRNAs could participate in the transfer of transcriptional memory through the mechanism of RNA-directed DNA methylation (RdDM). DNA methylation and histone modifications together act as chromatin marks that can be epigeneticallytransferredtosubsequentgenerations.Basedonthis,plants derivedfromstressedparent plants show large changes in gene expression compared to plants with non-stressed parents. These changesthenpersistfor varyinglengthsof time,dependingon whethergene expressionisagain induced bythe stressfactoror not.Interestinunderstandingthe mechanismsof transgenerationalstressmemory has recently grown considerably, and this...
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Histone modifications and methylation of polyomaviral genomes during the infection
Mrkáček, Michal ; Forstová, Jitka (advisor) ; Šmahelová, Jana (referee)
Similarly to other viruses, polyomaviruses require for their successful replication enzymes and other proteins encoded by their host cells. Additionally, because of their relatively small genome with only a few genes, polyomaviruses utilize for their efficient replication cellular regulation mechanisms. One of these regulations are posttranslational modifications of histones, which form nucleosomes together with viral DNA. The spectrum of these modifications is very wide, but in case of polyomaviruses, almost only ones studied are histone acetylations and methylations. Second possible regulation is a methylation of cytosine in CpG dinucleotides, which is associated with repression of gene expression. Current knowledge however suggest that polyomaviruses do not utilise this kind of modification. Moreover, because of a relatively small amount of CpG dinucleotides present in their genomes, they seem to avoid it. The goal of this work is to describe the individual types of these modifications and show their possible importance in the infectious cycle of polyomaviruses. Key words: polyomavirus, epigenetics, histone modification, DNA methylation, CpG dinucleotides
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Human endogenous retrovirus ERVWE1: transcriptional activation and modifications of promoter DNA methylation
Dobšová, Martina ; Trejbalová, Kateřina (advisor) ; Španielová, Hana (referee)
Endogenous retrovirus ERVWE1 is an integral part of the human genome. In the course of evolution, a protein encoded by the env gene of this retrovirus - Syncytin-1 - has gained unique function in human development. It mediates cell-to-cell fusion of placental cytotrophoblasts. Receptor that binds to Syncytin-1 is expressed in different cell types. Syncytin-1-mediated fusion is essential in placenta, but it can cause disruption of tissue integrity in other cell types. ERVWE1 expression is regulated by promoter DNA methylation, transcription factor GCM1 and efficient mRNA splicing. This thesis concerns the ERVWE1 expression and its regulation in non-placental tissues. It was found out that the moderate GCM1 overexpression was not sufficient to induce Syncytin-1 expression. Neither treatment with DNA demethylation agent 5-azacytidine nor with Syncytin-1 activator forskolin was able to manage Syncytin-1 expression. This thesis extends previous findings concerning high syncytin-1 expression in seminomas. In same tissues, there was found elevated TET1 expression on mRNA level in comparison with controls. The presence of the TET1 demethylation enzyme can influence ERVWE1 promoter DNA methylation. Previously unreported splicing variant of TET1 has been found during the construction of human TET1 expression...
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases.
Čepek, Pavel ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Horníková, Lenka (referee)
Abstract Background: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Its incidence in Europe is continuously rising. The highest T1D risk is associated with HLA (human leukocyte antigen) class II genes. HLA class II molecules play a key role in regulation of immune response. They contribute to the selection of T cell repertoire by presenting antigenic peptides to the CD4+ T lymphocytes. HLA class II expression is controlled by regulatory module that is situated 150 - 300 base pairs upstream of the transcription- initiation site in all HLA class II genes. Polymorphisms in this region are linked to some autoimmune diseases. There were identified several promoter alleles (named QAP) in the HLA DQA1 gene promoter region. Most of the polymorphisms appear to be conserved within haplotype. Individual QAP alleles may have a different promoter strength by which they influence expression of HLA DQA1 gene alleles. Promoter strength can be modulated by DNA methylation. Aims:Our aim was to define methylation profile of HLA DQA1 promoters and determine the mRNA expression of individual alleles of HLA DQA1 gene in T1D patients. The mRNA expression level of HLA DQA1 gene alleles was determined using quantitative PCR. Methods: 30 diabetic pacients (age range 21 to 76 years), were included in this pilot...
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Importance of epigenetic variability in evolution of clonal plants
Mareš, Štěpán ; Pinc, Jan (advisor) ; Latzel, Vít (referee)
Because of their low genetic variability and limited ability to respond to the changing environment, clonal plants are often considered an evolutionary dead end. On the other hand, numerous recent studies showed that clonal plants can react to the changing environment through epigenetic mechanisms, especially through DNA methylation. Moreover, epigenetic information in clonal plants can be transferred to future generations (so-called transgenerational memory). As a result, epigenetics can ensure better fitness of the next clonal generation. The aim of this work is to summarize the knowledge about the role of epigenetic variability in the life of clonal plants performing limited genetic variability. Key words: epigenetics, DNA methylation, epigenetics variation, clonal plants, transgeneration memory
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Histone modifications and methylation of polyomaviral genomes during the infection
Mrkáček, Michal ; Forstová, Jitka (advisor) ; Šmahelová, Jana (referee)
Similarly to other viruses, polyomaviruses require for their successful replication enzymes and other proteins encoded by their host cells. Additionally, because of their relatively small genome with only a few genes, polyomaviruses utilize for their efficient replication cellular regulation mechanisms. One of these regulations are posttranslational modifications of histones, which form nucleosomes together with viral DNA. The spectrum of these modifications is very wide, but in case of polyomaviruses, almost only ones studied are histone acetylations and methylations. Second possible regulation is a methylation of cytosine in CpG dinucleotides, which is associated with repression of gene expression. Current knowledge however suggest that polyomaviruses do not utilise this kind of modification. Moreover, because of a relatively small amount of CpG dinucleotides present in their genomes, they seem to avoid it. The goal of this work is to describe the individual types of these modifications and show their possible importance in the infectious cycle of polyomaviruses. Key words: polyomavirus, epigenetics, histone modification, DNA methylation, CpG dinucleotides
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Epigenetic mechanisms in the interferon γ signalling pathway
Fišerová, Lenka ; Reiniš, Milan (advisor) ; Javorková, Eliška (referee)
IFNγ is an important cytosine mediating imune responses, including antitumor immunity. It can affect expression of a lot of genes, which regulate different cellular processes. In tumor cells defects in signal cascade of IFNγ and mistakes in expression of genes regulated by IFNγ, for example genes for antigen adjustment and presentation (APM) or genes for major histocompatibility complex (MHC), were observed. Epigenetic mechanisms, can play a role in regulation of expression of genes for IFNγ, as well as in regulation of expression of genes regulated by IFNγ, including the components of the IFNγ signalling pathway. In lymphocytes from tumors the ability to produce IFNγ was limited by epigenetic silencing of genes for IFNγ. In tumor cells, epigenetic silencing of genes regulated by IFNγ, of genes of the IFNγ signaling cascade, for example IRF transcription factors, and other genes regulated by IFNγ, such as genes for APM, MHC or indoldioxygenase coding genes (IDO), was demonstrated. In case of their activation by IFNγ, epigenetic changes in regulation sequences of appropriate genes, were observed. IFNγ thus can be considered as an epigenetic agent. Epigenetic modulators are able to activate expression of genes regulated by IFNγ. By this way it's possible to explain some of immunomudullatory effects...
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Human endogenous retrovirus ERVWE1: transcriptional activation and modifications of promoter DNA methylation
Dobšová, Martina ; Trejbalová, Kateřina (advisor) ; Španielová, Hana (referee)
Endogenous retrovirus ERVWE1 is an integral part of the human genome. In the course of evolution, a protein encoded by the env gene of this retrovirus - Syncytin-1 - has gained unique function in human development. It mediates cell-to-cell fusion of placental cytotrophoblasts. Receptor that binds to Syncytin-1 is expressed in different cell types. Syncytin-1-mediated fusion is essential in placenta, but it can cause disruption of tissue integrity in other cell types. ERVWE1 expression is regulated by promoter DNA methylation, transcription factor GCM1 and efficient mRNA splicing. This thesis concerns the ERVWE1 expression and its regulation in non-placental tissues. It was found out that the moderate GCM1 overexpression was not sufficient to induce Syncytin-1 expression. Neither treatment with DNA demethylation agent 5-azacytidine nor with Syncytin-1 activator forskolin was able to manage Syncytin-1 expression. This thesis extends previous findings concerning high syncytin-1 expression in seminomas. In same tissues, there was found elevated TET1 expression on mRNA level in comparison with controls. The presence of the TET1 demethylation enzyme can influence ERVWE1 promoter DNA methylation. Previously unreported splicing variant of TET1 has been found during the construction of human TET1 expression...
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Molecular characterisation of novel subtype of Acute lymphoblastic leukemia with lineage switch during early phase of treatment
Dobiášová, Alena ; Fišer, Karel (advisor) ; Novotný, Marian (referee)
Leukemia is the most common malignant disease in children patients. In our laboratory (CLIP) a novel subtype of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) with lineage switch during early phase of treatment towards myeloid lineage (swALL) was recently documented. SwALL incidence is almost 4 % of all BCP-ALLs (Slámová et al., 2014). DNA methylation (presence of 5-methylcytosine) is together with post-translational histone modifications and non- coding RNAs an epigenetic mechanism which regulates gene expression without changes of genetic code. DNA methylation is easily detected by bisulphite conversion and subsequent sequencing. The aim of this work was to compare genome-wide DNA methylation patterns between patients with swALL and control BCP-ALLs. The first step in achieving that was revision and improvement of bioinformatic processing protocol for eRRBS data from massive parallel sequencing. To improve the sequence adapter trimming I tested four bioinformatic tools - FAR, cutadapt, Trimmomatic and fastx_clipper. I implemented the fastest and most effective - Trimmomatic into the processing protocol. As a next step I analysed the data with improved protocol and extended the analysis in R programming environment where the comparison of studied groups was performed. The comparison of...
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