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Epigenetic mechanisms in the regulation of the B7-H1 and IRF-1 expression in tumour cells.
Hrušková, Veronika ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
Interferon γ is an important T-cell helper type 1 (Th1) cytokine involves in antimicrobial immunity. It is a part of the inflammatory immune response in the site of infection. However, for its proper function, the regulation of immunity is necessary to avoid injury of the tissue caused by long-term inflammation. While interferon γ triggers expression of proinflammatory genes, it also regulates genes which inactivate immune response. The B7-H1 molecule belongs among these inhibitory regulators. Furthermore, antitumour effect of interferon γ is well-known as well. After extensive experiments, interferon γ was tested as an immunotherapeutic drug against melanomas in clinical trials. However, the trials had to be terminated prematurely because of unsuccessful results. It started to be clear that interferon γ could have also a protumour effect. Interferon γ upregulates the expression of B7-H1 molecule which aids tumour in escape from immunity. The B7-H1 molecule possesses a binding site for interferon regulatory factor 1 (IRF-1) in its promoter region. This IRF-1 is induced by interferon γ - JAK/STAT signalling pathway. In our previous research, we observed interferon γ induced DNA demethylation of promoters in genes that are involved in antigen presenting machinery. Additionally, DNA methylation of...
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The prognostic significance of molecular alterations in pediatric gliomas of central nervous system
Mišove, Adéla ; Zápotocký, Michal (advisor) ; Netuka, David (referee) ; Kolenová, Alexandra (referee)
The prognostic significance of molecular alterations in pediatric gliomas of central nervous system Abstract The purpose of this dissertation is to delve into the molecular and genetic underpinnings of low-grade gliomas in children, focusing on a cohort treated over the past two decades. It aims to correlate these findings with the clinical course and outcomes of the disease. The study was conducted through a collaborative effort between the Clinic of Pediatric Hematology and Oncology and the Laboratory of Molecular Pathology at the Charles University, highlighting the significance of a multidisciplinary approach in diagnosing. This work highlights the importance of diagnosis as a synthesis of insights from various disciplines, underscoring the significance of molecular-genetic factors in the prognosis and treatment of low-grade gliomas in children. Through a detailed study involving methods such as PCR, Sanger sequencing, RNA panel sequencing, MLPA, and DNA methylation profiling, key genetic alterations were identified, including frequent BRAF fusions and mutations, changes in FGFR genes, and alterations in MYB/MYBL genes, among others. The study revealed the diverse molecular landscape of pediatric low-grade gliomas, emphasizing the need for integrated diagnostics in determining the appropriate management...
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New possibilities to distinguish monozygotic twins
Letková, Kristína ; Kulichová, Iva (advisor) ; Priehodová, Edita (referee)
The necessity of distinguishing monozygotic twins from a genetic point of view is important not only for determining paternity, when it is decided which of two identical twins is the father, but also, for example, for criminal cases, where it is necessary to identify the perpetrator who is one of the monozygotic twins. In addition to analyzing and evaluating molecular biological methods, the work also deals with monozygotic twins themselves, their emergence and the causes of their emergence. The topic affects both forensic genetics and medical fields.
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Chromatin modifications and small regulatory RNA in plant intergenerational and transgenerational stress memory: experiments in plants subjected to drought
Macek, Jan ; Holá, Dana (advisor) ; Čermák, Vojtěch (referee)
Water is important for many vital processes, and a lack of water in plants causes drought stress. In case plants survive drought, they can be better adapted for subsequent drought periods. This is defined as stress memory, which will last for a certain period after the stress ended, and can even be transmitted to offspring to help it deal with stressful environment. In such case it is called an inter- or transgenerational stress memory and is probably mediated by chromatin modifications or non-coding RNAs. In this thesis, I collected and discussed original papers dealing with the molecular basis of this phenomenon in drought-stressed plants, focusing mostly on various methodical aspects. It is evident that the maintenance of chromatin modifications across generations is quite a complex process. Currently available information is still scarce, concerns mostly DNA methylation, is incomplete and conflicting. To better understand this topic in the future, the methods of analysis of these modifications need to be adjusted (and focus also on histone modifications), the general design of the experiments has to be improved (analysed generations subjected to stress/control conditions, drought verification, number of replicates) and some factors that can affect plant stress memory should be purposefully...
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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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