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LINC complex: The link between chromatin integrity and sperm motility
Šanovec, Ondřej ; Komrsková, Kateřina (advisor) ; Lánská, Eva (referee)
The LINC complex (Linker of the Nucleoskeleton and Cytoskeleton) is a protein structure located in the nuclear membrane that connects the cytoskeleton with the nucleoskeleton. This complex can be found in every mammalian cell including the gametes. However, here the LINC complex is more diverse and less studied than in the somatic cells. In this thesis, the LINC complex and its role in spermiogenesis have been studied in wild-type and Protamine 2 knockout (Prm2-/- ) mice. Protamines are small proteins that replace histones during spermiogenesis. The mouse model generated by the group of prof. Hubert Schorle has a deletion in Prm2 in exon 1 and its sperm possess a surprising phenotype including complete loss of motility. Therefore, it was hypothesized that the LINC complex might be responsible for miscommunication between the sperm head and tail which leads to the loss of sperm motility. Results from this study suggest that the LINC complex is not influenced by Prm2 deletion, however, actin dynamics, cytoskeletal motor proteins and tubulin acetylase/ histone deacetylase activity might be impaired. Prm2-/- sperm have a significantly higher abundance of β-actin compared to the wild type. Next, Prm2-/- sperm also show a different pattern of acetylation of α-tubulin but no change in the abundance of...
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Structural characterization of interactions between subunits of the GBAF chromatin remodeling complex
Naušová, Karolína ; Veverka, Václav (advisor) ; Rozbeský, Daniel (referee)
Epigenetics investigates heritable phenotypic changes that are not caused by alterations in DNA sequence. One of the major epigenetic tools is chromatin remodeling mediated by ATP-dependent chromatin remodeling complexes, which fundamentally affect gene expression and thus cellular fate. A mammalian variant of the ATP-dependent chromatin remodeling SWI/SNF complex is the BAF complex, which is involved in both activation and repression of gene expression. There have been identified three major variants of BAF complex one of which is non-canonical BAF, also known as GLTSCR1 containing BAF, GBAF. Each complex consists of up to 15 subunits, some of which are specific only to one of the complex variants. Mutations in genes coding subunits of BAF lead to several genetic disorders or to the cancer development. The GBAF complex contains specific subunits, BRD9 (Bromodomain containing protein 9), GLTSCR1 (Glioma tumor suppressor candidate region 1) and GLTSCR1L (GLTSCR1-like), which are essential for its formation. Although the exact function of the GBAF complex has not been elucidated yet, it is often associated with synovial sarcoma and rhabdoid tumors. Two of the three complexes are impaired in those tumors and gene expression is maintained only due to the GBAF complex. If GBAF would additionally loose...
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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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I.Synthesis of acyclic nucleoside phosphonates with a thiadiazole base mimicking 5-azacytosine as compounds with potential biological activitiesII.Development of small molecules targeting c-MYC oncogene.
Pomeislová, Alice ; Krečmerová, Marcela (advisor) ; Jindřich, Jindřich (referee) ; Voltrová, Svatava (referee)
The first part of the thesis regards synthesis of N-[2-(phosphonomethoxy)ethyl] (PME) and (S)-3-hydroxy-2-(phosphonomethoxy)propyl ((S)-HPMP) derived acyclic nucleoside phosphonate (ANP) analogues carrying 5-amino-1,2,4-thiadiazol-3(2H)-one as a nucleobase that is supposed to mimic cytosine or 5-azacytosine. A series of 1,2,4-thiadiazole derivatives bearing at the N2 position PME- or (S)-HPMP-moiety and NH2-protecting group (benzoyl, ethoxycarbonyl, or Fmoc) were obtained as chemically stable ANP congeners. Their synthesis was performed via stepwise construction of the thiadiazole ring and required the use of two newly prepared synthons, PME-amine and (S)-HPMP-amine. However, all attempts to prepare the intended PME- and (S)-HPMP-thiadiazole phosphonic acids with free amino moiety failed due to instability of the N2 -substituted thiadiazole ring. Biological evaluation of twenty-one selected thiadiazole compounds towards two human cysteine- dependent enzymes, cathepsin K and glycogen synthase kinase 3ß, revealed that several compounds inhibited both enzymes in the low micromolar range. Some of these efficient inhibitors had also favourable toxicity profile at 100 µM, which makes them appropriate for further development of potential drug candidates. In the second part of the dissertation, design and...
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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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Epigenetické regulace u autoimunitních onemocnění se zaměřením na revmatoidní artritidu
Horková, Veronika ; Daňková, Pavlína (advisor) ; Hušáková, Markéta (referee)
Exact cause of rheumatoid arthritis, as well as other autoimmune diseases has not been identified yet. In last twenty years, epigenetics showed a new face of immune system. DNA methylation, modification of histones - proteins around which DNA is wrapped, or interference of small RNA sequences - microRNAs, these all are heritable changes outside the DNA sequence that provide another component involved in autoimmunity. Presented epigenetic mechanisms alter gene expression and thus facilitate production of pro- inflammatory factors leading to autoimmune reactions. Moreover, genes regulating apoptosis are also frequently targeted by epigenetic modifications. Not only these mechanisms provide another level of immune defense, they also explain higher female susceptibility to autoimmune diseases and the influence of environment on pathogenesis of these diseases.
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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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The study of epigenetic regulation of gene HLA II. Clas within family relationships
Chmel, Martin ; Černá, Marie (advisor) ; Urbanová, Jana (referee)
Introduction: At our post-genomic era the studies of epigenetic regulation constitutes one of the tools for understanding the function of genes. Epigenetic regulation can directly control the temporal and spatial gene activity or silencing. The molecular basis of these regulations are DNA bases modifications, chromatin remodeling and RNA interference. At the same time, these mechanisms have a special way of transferring genetic information to subsequent generations called epigenetic inheritance. It has been proven epigenetic deregulation of certain genes as cause for many disease. For this reason, the study of epigenome HLA genes seems particularly important because these genes play a fundamental role in regulating the immune system. Aims: The aim of this work is to create a description of epigenetic modifications within families. It is an analysis of histone modifications and DNA methylation in the promoter region of the gene HLA DQA1. The aim was also to compare the differences in epigenetic modifications between alleles and compared the differences in these modifications between generations. The results will be compared with the analysis of the level of expression of the gene HLA DQA1. Methods: From collected peripheral blood of donors were isolated DNA, RNA, and leukocytes. DNA was used for...
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Function of Zinc finger protein 644 (Zfp644) in mouse organism.
Szczerkowska, Katarzyna Izabela ; Sedláček, Radislav (advisor) ; Komrsková, Kateřina (referee) ; Blahoš, Jaroslav (referee)
ZNF644 (Zinc Finger Protein 644) is a C2H2 zinc finger gene encoding a putative transcription regulator, of which a point mutation (S672G) is associated with inherited high myopia in humans. It is also described to be a partner of the G9a/GLP (G9a- euchromatic histone- lysine N-methyltransferase 2, EHMT2; GLP - euchromatic histone-lysine N-methyltransferase 1, EHMT1) complex, known for its essential role in histone methylation, specifically H3K9me1and H3K9me2. It was reported that another transcription factor, WIZ (Widely-Interspaced Zinc Finger-Containing Protein), can bind to this complex and cooperate in gene silencing simultaneously. In order to study Zfp644 impact on myopia, we generated a mouse model, Zfp644S673G that mimics human mutation. In addition, a mouse with a persuasive truncated form of the protein, Zfp644Δ8 was created. Both mouse models went through an examination of retinal function and morphology. Moreover, with use of ultrasonography, different ocular parameters were examined. We conclude, that Zfp644 gene is causative for myopia in mice. Further examinations of Zfp644Δ8 animals show severe symptoms in metabolism and female fertility. To describe the impact of Zfp644 in mouse fertility we performed various experiments including analysis of expression of Zfp644 in reproductive...
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Epigenetic mechanisms in psychostimulant addiction
Kubátová, Eliška ; Novotný, Jiří (advisor) ; Rydzyková, Tereza (referee)
Psychostimulants are highly addictive substances that act in a brain reward center by increasing extracellular levels of monoamines. In western civilization, they have gained popularity during the 20th century. Psychostimulant addiction is a persistent social problem even today since therapy is often unsuccessful and former users relapse frequently. Alterations in gene expression occur after psychostimulant is administered. These changes are long-lasting as they can often persist for weeks and months. This work focuses on the epigenetic changes in a brain associated with the development of addiction after psychostimulant administration. Key words: Epigenetics, psychostimulants, amphetamine, methamphetamine, cocaine, addiction
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