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Stárnutí rybích oocytů a modifikace histonů
WAGHMARE, Swapnil Gorakh
Cellular ageing is characterized by a loss of functional abilities over time. Epigenetic changes are one of the factors that contribute to ageing. Epigenetic regulators control the cellular gene activity without any alteration of the underlying DNA sequence. Epigenetic regulators like DNA methylation, histone modifications, and non-coding RNAs have been related to the ageing process. Histone modifications are among the most crucial and common epigenetic configurations that play a critical role in the post-fertilization success and early embryo development. Oocyte carries essential information to orchestrate embryogenesis and to remodel the parental genomes. The underlying molecular pathways driving oocyte ageing remain unknown. Investigations and understanding of the molecular mechanisms behind oocyte ageing are needed. Therefore, the investigation of the oocyte ageing, focusing on histone modifications were done. Egg phenotype and functional changes during fish oocyte ageing were examined. Despite being widely used as a model organism, the impact of oocyte ageing on ploidy abnormalities was not yet studied in zebrafish (Danio rerio). Therefore, the investigation on ploidy anomalies in embryos originating from different aged oocytes in zebrafish was performed. In the molecular analysis, histone modifications with a special focus on acetylation were performed. Histone acetyltransferase activity was assessed to get more information on the dynamics of histone acetylation during fish oocyte ageing. The current study reports that maintenance and manipulation of zebrafish oocytes at 26 °C for 2 hours post-stripping (HPS) was possible without significantly reducing fertilization potential. Almost complete loss of egg viability was observed at 6 HPS in zebrafish. In addition, physical abnormalities were observed in all larvae derived from 4 and 6 HPS-old oocytes. Compared to 0 and 2 HPS, the proportion of ploidy abnormal embryos was significantly greater at 4 HPS in zebrafish. The egg eyeing and hatching rates were significantly decreased by the elapsing time of oocyte ageing in common carp (Cyprinus Carpio). A significant decrease in embryo survival rate at 1 HPS was observed in grass carp (Ctenopharyngodon idella). The specific histone acetylation on H3K9, H4K5 and H4K8 didn't exhibit significant differences in different aged oocytes of common carp and grass carp. However, the histone H3K14ac and H4K16ac didn't show a signal either in fresh or aged oocytes in common carp and grass carp. The H4K12ac increased significantly at 28 HPS in common carp while decreased significantly at 1 and 4 HPS in grass carp. The histone acetyltransferase (HAT) activity showed an increasing trend during in vivo and in vitro oocyte ageing in common carp. The HAT activity increased significantly at 30 HPS in grass carp. The observed histone acetylation marks are reported to be species-specific and oocyte stage dependant. Therefore, the obtained results are the first to report the presence of H3K9ac, H4K5ac, H4K8ac, and H4K12ac, as well as the absence of H3K14ac and H4K16ac in common carp and grass carp metaphase II oocytes. Additionally, our findings suggest the possibility of developing an epigenetic marker for egg quality associated with fish oocyte ageing.
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Elucidating the interactions of interleukin-1alpha with components of the eukaryotic transcription machinery
Zámostná, Blanka ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Mělková, Zora (referee)
4 ABSTRACT Interleukin-1α (IL-1α) is a pleiotropic cytokine and a key mediator of host immune response. It is synthesised as a 31-kDa precursor, that is cleaved by the cysteine protease calpain into the 17-kDa mature IL-1α and the 16-kDa N- terminal peptide of IL-1α (IL-1αNTP). Although IL-1α can be secreted, act on target cells through the surface receptor IL-1RI and trigger the signal transduction pathway, increasing evidence points toward the involvement of IL-1α in certain nuclear processes. IL-1αNTP is highly conserved among higher eukaryotes and contains a nuclear localisation sequence; indeed, both the precursor and IL-1αNTP are found in the cell nucleus. Previously, a genetic interaction of IL-1α with nuclear histone acetyltransferase (HAT) complexes has been reported from mammalian cells and, interestingly, also from the heterologous yeast model. This thesis extends the research of the nuclear function of IL-1α and demonstrates that IL-1α physically associates with the HAT/Core module of yeast SAGA and ADA HAT complexes. Results of the HAT subunit gene knock-out experiments followed by a set of co-immunoprecipitations also suggest a novel model of the yeast SAGA complex assembly, in which ADA appears to represent only a partly functional HAT complex. In its natural milieu of mammalian cells, IL-1α...
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