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The role of SIRT1 during in vitro maturation of oocytes
Landsmann, Lukáš ; Nevoral, Jan (advisor) ; Šolc, Petr (referee)
SIRT1 histone deacetylase acts towards many epigenetic and non-epigenetic targets. The involvement of SIRT1 in oocyte maturation is assumed and the importance of ooplasmic SIRT1 pool for further destiny of matured oocyte is strongly suggested. We hypothesized that SIRT1 play role of the signal molecule in mature oocyte through selected epigenetic and non- epigenetic regulation. We observed SIRT1 re-localization in mature oocyte and the association with spindle microtubules. In matured oocyte, SIRT1 shows a spindle-like pattern and spindle- specific SIRT1 action is supported decreasing α-tubulin acetylation. Based on the observation of histone code in immature and matured oocytes, we suggest that SIRT1 is mostly predestined for epigenetic mode of action in germinal vesicle (GV) of immature oocyte. Accordingly, SIRT1- driven trimethylation of histone H3 on lysine K9 in matured oocyte is considered to be an inheritance of GV epigenetic transformation. Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocyte capable to be switched from epigenetic to the non- epigenetic mode of action readily depending on meiosis progress. Keywords: oocyte, SIRT1, histone, developmental competence, tubuline, epigenetics
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Function of nuclear phosphoinositides and their binding partners in gene expression
Uličná, Lívia ; Hozák, Pavel (advisor) ; Šolc, Petr (referee) ; Macůrková, Marie (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...
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Double strand DNA breaks response in Huntington´s disease transgenic minipigs
Vaškovičová, Michaela ; Šmatlíková, Petra ; Herbert, A. ; Motlík, Jan ; Šolc, Petr
Huntington’s disease (HD) is progressive neurodegenerative disorder caused by presence of CAG expansion in the huntingtin gene, which gives rise to mutated form of huntingtin protein (mHtt). There is a strong evidence that DNA damage response is compromised by presence of mHtt in cells and increase of double strand DNA breaks (DSBs) is an early event in HD pathology. It was shown, that level of γH2AX is significantly higher in R6/2 mice compared to wild-type animals. Moreover, level of γH2AX is higher also in striatal neurons and fibroblasts of human HD patients. Furthermore, protein p53, key player in DNA damage response, is hyperactivated in cells expressing mHtt and inhibition of p53 or ATM ameliorates phenotypes of HD animal models. However, exact mechanism of mHtt action is not clear and therefore further investigation of mHtt effects on DSBs response is very important for the understanding of HD pathology.
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Double strand DNA breaks response in Huntington´s disease
Šolc, Petr
There are strong evidences that DNA damage response (DDR) signalling significantly underline the molecular pathology of polyglutamine (polyQ) diseases, including Huntington´s disease (HD) [1-4]. Double strand DNA breaks (DSBs) are the most deleterious DNA lesions.\nIn this talk I will review how DDR on DSBs is affected in HD.\n
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Cell signaling pathways controlling meiotic maturation of mammalian oocytes
Šolc, Petr ; Motlík, Jan (advisor) ; Petr, Jaroslav (referee) ; Dráber, Pavel (referee)
4 2 Summary in English The female germ cells called oocytes arise from the primordial germ cells during embryogenesis. They are essential for the reproduction. Already during embryogenesis oocytes enter meiosis, however, they arrest at the dictyate stage of prophase I. After onset of sexual maturity luteinizing hormone induces the resumption of meiosis of follicle enclosed oocytes (GV stage) in animals (in vivo) but removing of oocytes from follicles and culture in a suitable medium allows the spontaneous resumption of meiosis in vitro. Nuclear envelope break down (NEBD or GVBD) is the first visible mark of the meiosis resumption. Later after GVBD, the metaphase I (MI) spindle forms and after all chromosome bivalents are correctly attached to microtubules (MTs) anaphase I occurs. Following meiosis I completion, oocytes enter directly meiosis II and arrest at metaphase II (MII). These oocytes are fertilizable and sperm trigger meiosis II completion. The development from GV to MII oocytes is governed mainly by meiosis promoting factor (MPF) that consists of cyclin dependent kinase 1 (CDK1) and cyclin B (CCNB). On the mouse oocytes, we have shown using functional studies (RNA interference, mRNA microinjection) that phosphatases CDC25A and B cooperate in the induction of CDK1 activity and resumption of meiosis....
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Gamma-Tubulin forms and functions in microtubule organization and beyond
Kourová, Hana ; Binarová, Pavla (advisor) ; Šolc, Petr (referee) ; Bíšová, Kateřina (referee)
Microtubules are highly dynamic structures forming complex arrays changing accordingly to cellular requirements. In most eukaryotes, microtubules are nucleated from defined organizing centres like centrosomes or spindle pole bodies. Despite conservation of the major cytoskeletal components in plants and animals, plant cells lack centrosome-like structures and have evolved a unique mechanism to control microtubule assembly and organization. γ-Tubulin is an essential component of microtubule organizing centres, highly conserved in all eukaryotes with a prominent role in microtubule nucleation. However, growing body of evidence suggests γ-tubulin as a multifunctional protein. Next to microtubule nucleation, γ-tubulin controls microtubule plus-end dynamics, regulates gene expression, associates with DNA repair proteins and is implemented in mitotic and cell cycle regulation. We focused on γ-tubulin complexes and aimed to characterize interactors of γ-tubulin and their function in Arabidopsis. Next to characterizing microtubule dependent functions, we analyzed cell cycle and division. We also studied DNA damage response and involvement of E2F/RBR (retinoblastoma related) pathway and possible link to γ-tubulin. We characterized NodGS protein as a novel interactor of γ-tubulin in Arabidopsis and proposed...
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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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Regulation of cell cycle and DNA damage response in mouse oocytes.
Mayer, Alexandra ; Šolc, Petr (advisor) ; Macůrek, Libor (referee) ; Macek, Milan (referee)
A specific feature of mammalian oocytes is a long prophase I arrest, which can be maintained for many years in humans. The oocytes must ensure robust mechanisms, which can keep them in prophase I, but effectively trigger meiotic resumption when required. Consequently, throughout the maturation of an oocyte, non-erroneous chromosome segregation is a prerequisite for the generation of healthy offspring. In this study we aimed to investigate the new roles of Aurora A (AURKA) and polo-like kinase 1 (PLK1) in the regulation of the cell cycle progression. For this purpose, we used transgenic mice that specifically overexpress wild type (WT-) or kinase-dead (KD-) AURKA in oocytes only, and to study PLK1 we treated oocytes with BI2536, a small molecule inhibitor known to specifically inhibit PLK1 in somatic cells. Our data show, that both AURKA and PLK1 are not essential for meiotic resumption, however they participate in this process. Active AURKA regualtes the increase in microtubule organizing centers (MTOC) in prophase I, which is the first visible marker of resumption of meiosis in oocytes. AURKA activation is biphasic, and the initial increase in MTOC is transient, while full AURKA activation needed for the stability of MTOC requires the activity of Cyclin-dependent kinase 1 (CDK1). We show that PLK1...
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Localization and function of phosphoinositides in the cell nucleus
Kalasová, Ilona ; Hozák, Pavel (advisor) ; Stopka, Tomáš (referee) ; Šolc, Petr (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids. Their inositol head can be phosphorylated at three positions generating seven differently phosphorylated species. Cytoplasmic phosphoinositides regulate membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generate second messengers. In the nucleus, PIs are implicated in pre-mRNA processing, DNA transcription and chromatin remodelling. However, their nuclear functions are still poorly understood. Here we focus on nuclear phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We describe their localization and interaction with proteins involved in regulation of DNA transcription. PI(4)P localizes to nuclear membrane, nuclear speckles and nucleoplasm. The majority of nuclear PI(4)P is associated with chromatin and colocalizes with H3K4me2. PI(4,5)P2 localizes to nucleoli and nuclear speckles. Besides, 30 % of nuclear PI(4,5)P2 forms small nucleoplasmic PI(4,5)P2 islets. They have carbon rich core, which is probably formed by lipids, and are surrounded by proteins and nucleic acids. The active form of RNA polymerase II associates with PI(4,5)P2 islets and DNA is actively transcribed in the vicinity of PI(4,5)P2 islets. Moreover,...
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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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