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ADP-ribosylation in ARH3-deficient cells and its impact on cellular functions
Kuttichová, Barbora ; Hanzlíková, Hana (advisor) ; Valihrach, Lukáš (referee)
ADP-ribosylation is a crucial post-translational modification that regulates various cellular processes, including DNA repair. It is catalysed by poly-ADP-ribose polymerases (PARPs) and involves the transfer of ADP-ribose moieties from the redox cofactor NAD+ to proteins, including histones. To maintain cellular homeostasis, ADP-ribose chains need to be rapidly degraded by ADP-ribosyl glycohydrolases. While poly-ADP-ribose glycohydrolase (PARG) is highly efficient, it cannot cleave the terminal ADP-ribose moiety. For the removal of the terminal mono-ADP-ribose, two glycohydrolases, TARG1 and ARH3, are involved. This removal process is necessary because it enables DNA repair factors to access the site of DNA damage. The primary goal of this thesis is to characterise cells derived from patients with homozygous ARH3 mutations and to develop appropriate tools to improve our understanding of the molecular mechanism by which ARH3 mutations affect ADP-ribosylation and how it contributes to the onset of the associated neurological disease. To achieve this, I measured the levels of ARH3 protein and detected increased mono-ADP-ribosylation in ARH3-mutated patient-derived fibroblasts. Furthermore, I assessed the sensitivity of these cells to different PARP inhibitors, which hold potential for the therapeutic...
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DNA damage response in Huntington disease
Vachová, Veronika ; Šolc, Petr (advisor) ; Roth, Jan (referee)
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, which leads to loss of striatal neurons in basal ganglias. It is characterized by involuntary movements and progressive cognitive impairment. HD is a relatively rare disease and the prevalence is approximately 0,01 % of the population of Western European. HD is caused by a CAG repeat expansion in the huntingtin gene (HTT). This mutation results in an elongated stretch of glutamin. Mutant huntingtin (mHTT) expression leads to accumulation of DNA double-strand breaks (DSB) due to reduced ability of effective reparation, which contributes to the pathogenesis of HD, however this mechanism is not fully understood. There are several angles of view how mHTT impaires DNA damage response (DDR). Some studies say that the expression of the mHTT initiates excessive activation of the DDR including p53 signaling pathway leading to apoptosis. Other studies represent results for dysfunction of non-homologous end joining after recognition of DSB or that the cell is not able to recognize DSB. All theories would explain cell death as a consequence of high level of unrepaired DNA damage. The understanding of these mechanisms is important for the development of therapeutical strategies. Key words: Huntington's disease, huntingtin, DNA...
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The effect of maternal age on the level of DNA damage in oocytes
Koubovská, Markéta ; Šolc, Petr (advisor) ; Tlapáková, Tereza (referee)
Female germ cells are oocytes, whose number defines the length of the reproductive period. Besides quantity the quality of oocytes is also an important factor. The quality is not the same throughout whole life, but it decreases with increasing age. The loss of quality is mainly due to the accumulation of DNA damage. The most serious damage is considered to be double-strand breaks (DSBs) because of both DNA strands break and subsequent translocation or deletion. There are also products caused by reactive oxygen species, UV or γ radiation, modified DNA section or mismatch sequence. All these lesions are repaired by oocyte repair mechanisms. However, the older a woman is, the more gene expression of DNA repair genes is decreased. That is why the repair is not efficient and DNA damage is not removed. Higher age is associated with unsuccessful attempts to conceive, a higher risk of miscarriage or an ectopic pregnancy. To avoid the risks that pregnancy in old age represents, the reproductive ability has been lost. In humans, it ends around the age of 50 with so- called menopause.
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DNA damage response in mammalian oocytes
Vachová, Veronika ; Šolc, Petr (advisor) ; Nevoral, Jan (referee)
During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...
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Role of Polo-like kinases in the cell cycle and DNA damage response
Kudláčková, Radmila ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Within the process of cell division, genetic material must be equally distributed between the two daughter cells. In the next phase, the missing portion of the genome must be synthesized. The entire cycle is regulated by cyclin-dependent kinases (Cdks) in cooperation with cyclins. If the DNA is damaged during the cell cycle, signaling pathways of checkpoints supress cycle progression and enforce the cell cycle arrest until the damage is repaired. Malfunction of the checkpoints results in tumorigenesis. Polo-like kinases (Plks) are, much like Cdks, important regulators of the cell cycle. Plks play significant role mainly in the mitosis and also in a response to the DNA damage. This thesis is focused on human homologues, nevertheless conservation of homologues among organisms is considerable, thus presented findings are of general relevance. Human cells express five proteins from the family of Polo-like kinases, from which Plk1 corresponds to Polo-like kinases of lower eukaryotes. Knowledge on the remaining four kinases is still on the rise.
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DNA damage response in Huntington disease
Vachová, Veronika ; Šolc, Petr (advisor) ; Roth, Jan (referee)
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, which leads to loss of striatal neurons in basal ganglias. It is characterized by involuntary movements and progressive cognitive impairment. HD is a relatively rare disease and the prevalence is approximately 0,01 % of the population of Western European. HD is caused by a CAG repeat expansion in the huntingtin gene (HTT). This mutation results in an elongated stretch of glutamin. Mutant huntingtin (mHTT) expression leads to accumulation of DNA double-strand breaks (DSB) due to reduced ability of effective reparation, which contributes to the pathogenesis of HD, however this mechanism is not fully understood. There are several angles of view how mHTT impaires DNA damage response (DDR). Some studies say that the expression of the mHTT initiates excessive activation of the DDR including p53 signaling pathway leading to apoptosis. Other studies represent results for dysfunction of non-homologous end joining after recognition of DSB or that the cell is not able to recognize DSB. All theories would explain cell death as a consequence of high level of unrepaired DNA damage. The understanding of these mechanisms is important for the development of therapeutical strategies. Key words: Huntington's disease, huntingtin, DNA...
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