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Charakterizace oocytů afrických rypošů
KOPECKÁ, Eva
This study is focused on characterisation of oocytes of long-lived rodents, naked mole-rat and giant mole-rat, that are emerging models of long-term oocyte and ovarian quality. In the second part, the effect of repeated ovulations on aging-associated quality decline of oocyte is studied, comparing mouse and giant mole-rat.
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Effect of cancer therapy on fertilization parameters in testicular germ cell tumour patients.
Kruf, Annabelle Elisabeth ; Vondráková, Jana (advisor) ; Nevoral, Jan (referee)
Testicular germ cell tumours (TGCTs) are a rare type of cancer. It is the most common type of cancer in the age group of 15-35 years. The consequences that TGCT and the treatment of this disease impose to these young men in terms of sperm parameters and fertility is yet unclear. This study involves 84 semen samples from 46 patients sorted in three time groups - before treatment, 3 months after the treatment and 6 months after the treatment. Several sperm parameters were studied and compared within the three time points. Adding on to this, the study takes two patients with complete set of semen samples and studies them individually. The results mostly show great differences in patients and this study underlines the individual characteristics that need to be taken into consideration when it comes to patients' fertility journeys after battling TGCT. Sperm viability, concentration, apoptosis, DNA damage, acrosomal integrity and leukocyte concentration nor histone modification evaluation showed any significant changes between said groups. DNA damage was correlated to the level of acrosomal permeability. IgA antibody levels rose significantly in samples after orchiectomy. IgG antibody levels did not show the same trend. A novel approach was optimized and applied using JUNO and FcRL3 double transfected...
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Epigenetic Dysregulation through Histone Modifications in Lymphoma
Hostaš, Ondřej ; Havránek, Ondřej (advisor) ; Malík, Radek (referee)
Lymphomas are a diverse group of malignant tumors that arise from lymphocytes, commonly affecting lymph nodes or the spleen. They are one of the most common types of tumors worldwide. Unfortunately, many subtypes have a poor prognosis, or are not currently fully curable with standard therapeutic approaches. Mutations in enzymes responsible for posttranslational modifications of histones are very common in certain subtypes of lymphoma, as well as in many other cancer types. These enzymes directly affect gene expression by changing the condensation state, and thus the accessibility, of chromatin. Some of these enzymes have been found to play an important role in the formation of germinal centers in lymphoid follicles. Therefore, their mutations can lead to uncontrolled proliferation and cancer development. Since many conventional therapeutic strategies are incapable of curing a large portion of lymphomas, novel and more targeted approaches are needed. Inhibition and/or modulation of the function of the aforementioned enzymes may be a basis for such approaches. Key words: lymphomas, epigenetic regulation, histone modifications, KMT2D, EZH2, CREBBP, EP300
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Effect of estrogens on in vitro models of testicular tissue and spermatogenesis
Jursová, Pavlína ; Děd, Lukáš (advisor) ; Tlapáková, Tereza (referee)
Although estrogens are primarily known for their functions in female reproductive system, their effect on male reproductive functions has also been well established. Physiological estrogen concentration is essential for a proper spermatogenesis. Estrogens regulate many functions in testicular tissue, including proliferation and apoptosis of all testicular cell types, dynamic restructuring of cell-cell junctions in the testis, and post-translation modifications of histones. Hence, the aim of this thesis was to study effect of estrogens on in vitro models of testicular tissue and spermatogenesis and thus to address their functions in testicular tissue more deeply. This project includes testicular organoid cultivation for further usage as in vitro model of spermatogenesis. To addresss the effect of various avaliable estrogen forms, experiments on MCF-7 cell line were done. Finally experiments with in vitro model of testicullar tissue - TM4 Sertoli cell line were done. In order to fulfill the aims and verify the hypotheses, some advanced methods such as CLARITY volume confocal imaging and holographic microscopy were used. It was found that estrogens can affect Sertoli cell morphology and the expression of some genes involved in cell-cell junction dynamics. Furthermore the process of spermatogenesis was...
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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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Role of Smarca5 (Snf2h) during transcription of transfected DNA template.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Smetana, Karel (referee)
Cellular and tissue characteristics are results of dynamic regulation of gene expression. DNA wrapped into proteins, referred to as chromatin, requires involvement of mechanisms guiding accessibility of specific sequences. In higher organisms, chromatin remodeling proteins are indispensable in regulating chromatin structure including ISWI ATPase SMARCA5. SMARCA5 is involved in almost any transaction on DNA including transcription, however precise in vivo role of SMARCA5 in these processes remains unknown. To advance understanding of specific role of SMARCA5 in the development of chromatin structure during transcription we devised cellular model in which SMARAC5 level is manipulated while chromatin structure development and transcriptional response are monitored. Our data indicate that the transfected DNA template that is transcribed is enriched with histone H3 and its specific methylation of Histone H3 lysine (K) 4, a mark of active chromatin structure. Overexpression of SMARCA5 results within the reporter gene coding sequence in ~2,5-3 fold increase of both H3 occupancy an its modification H3K4Me3. Increased DNA template commitment into chromatinization is associated with repression of reporter gene expression. These results are supported by studies indicating dynamic development of nucleosomal...
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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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Recombination hot-spots in various organisms
Stočesová, Lucie ; Holá, Dana (advisor) ; Forman, Martin (referee)
Meiotic recombination is an important process which occurs in sexually reproducing organisms and creates new allelic combinations. Frequency and distribution of crossing-overs (COs) are affected by many internal and external factors. Regions with higher frequency of COs are called recombination hot-spots and in this thesis, they are divided into ancestral and derived hot-spots. Ancestral hot-spots are the more original ones and they are determined by histone modifications, nucleosome-depleted regions, promoters and sequence motifs. This type of hot-spots occurs, e.g., in yeasts, birds and plants. Derived hot-spots are determined by the PRDM9 protein, which searches for specific sequence motifs and creates histone modifications. These hot-spots are typical for most mammals except the canines which lost functional PRDM9 due to the mutation of the Prdm9 gene. Activity of PRDM9 destroys primary locations of hot- spots via gene conversion. This process is called "hot-spot paradox" and is solved by the rapid evolution of alleles of the Prdm9 gene. This thesis summarizes basic information on distribution and determination of hot-spots among various eukaryotes. It particularly focuses on the PRDM9 protein, its structure, function and evolution. A determination of location of recombination in species...
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Polyomavirus minichromosome structure
Satratzemis, Christos ; Forstová, Jitka (advisor) ; Mělková, Zora (referee)
The polyomavirus genome is present in the host cell as circular double-stranded DNA associated with nucleosomes. Consequently, the expression of polyomavirus genes is affected by the location of nucleosomes on DNA and histone modifications. This thesis reviews the current state of knowledge regarding the polyomavirus minichromosome structure and the effects of nucleosome phasing and histone modifications on polyomaviral replication cycle. In addition, factors conditioning these phenomena are discussed. Drawing on available literature, neither nucleosome phasing nor histone modifications appear to be random. However, not all viral DNA molecules are identical in these respects. Processes such as early and late transcription, replication and encapsidation thus occur only within certain fractions of the set of DNA molecules
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Effect of epigenetic modifications on replication and transcription of hepatitis B virus DNA
Hofman, Tomáš ; Hirsch, Ivan (advisor) ; Roučová, Kristina (referee)
The hepatitis B virus forms in a nuclei of infected hepatocytes a stable cccDNA resistant to degradation and capable to persist for many years. The main role in the persistence of cccDNA play the epigenetic mechanisms that regulates viral transcription and replication in various stages of the infection. Major epigenetic mechanisms involved in the regulation of HBV include CpG islands methylation, cccDNA-bound histone acetylation and methylation and RNA interference. CpG methylation is associated with the activity of capsid protein, which binds to the cccDNA and promotes transcription and structurally distinguishes viral and the host genome. HBx protein besides CpG methylation mainly affects histone modifications via its interaction with host proteins and thus eliminates the inhibitory effects and promotes the active modifications favoring virus transcription. This work summarizes current knowledges concerning the cccDNA epigenetic regulation. Keywords: HBV, cccDNA, replication and transcription of HBV DNA, histone modifications, methylation of CpG, CpG islands, HBx, HBc, IFNα, IL-6
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