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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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Kinázová signalizace v meióze I savčích oocytů
Brzáková, Adéla ; Šolc, Petr (advisor) ; Dráber, Pavel (referee)
PLK1 belongs to the extended family of serine/threonine kinases controlling the cell cycle. It is well known for its role in the control of mitosis and contributes also to the regulation of meiotic division. On a basis of Live Cell Imaging (LCI) experiments we can describe the phenotype of the oocytes with PLK1 inhibited by small molecular inhibitor BI2536. PLK1 inhibition leads to delayed nuclear envelope breakdown (NEBD) and chromatin condensation (CC) and also causes desynchronization of NEBD and CC; in contrast to control oocytes, PLK1 inhibited oocytes break down their nuclear envelope with chromatin almost fully condensed. Also duration of these two early nuclear events is prolonged in oocytes with inhibited PLK1. In contrast to somatic cells, PLK1 inhibition in mouse oocytes does not prevent assembly of spindle with two distinct poles but affects the final spindle volume. Similar to somatic cells, mouse oocytes with PLK1 inhibited from the beginning of the meiotic maturation stay arrested in metaphase I but in the case of mouse oocytes, this block is not dependent on Spindle Assembly Checkpoint (SAC) persisting activity. When mouse oocytes are synchronized on metaphase I/anaphase I transition by proteasome inhibition and then PLK1 kinase activity is inhibited, about 2/3 of the oocytes stay arrested...
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The involvement of mitotic kinases AURKA and PLK1 in the oocyte meiotic maturation
Brzáková, Adéla ; Šolc, Petr (advisor) ; Šebková, Nataša (referee)
Aurora A and Plk1 belongs to the extended family of serine/threonine kinases controlling the cell cycle. Both are involved in the control of mitosis and contribute also to the regulation of meiotic division. Although the role of Aurora A in the resumption of meiosis after the first meiotic block seems to be, according to present studies, a bit questionable matter, it is virtually certain that AURKA plays an irreplaceable role in the construction of the spindle. In the oocytes with inhibited (by RNAi or small molecular inhibitors) or overexpressed Aurora A, abnormal spindles with aberrant number of poles or otherwise unusually shaped spindles are created. Disruption of Plk1 function in oocytes also leads to the appearance of damaged spindles. In addition, Plk1, almost certainly, plays a role in the timing of the nuclear membrane breakdown - at GVBD.
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Cell signaling pathways controlling meiotic maturation of mammalian oocytes
Šolc, Petr
5 2 Summary 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. After...
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Regulation of PKB/AKT phosphorylation during resumtion of meiosis
Böhmová, Tereza ; Krylov, Vladimír (referee) ; Šolc, Petr (advisor)
PI3K-PKB signal pathway participates in the CDK1 activation, which is necessary for meiosis resumption of mouse oocytes. That's why we wanted to examine the role of PKB in this process more in the details. The activity of PKB is associated with its phosphorylation at Thr308 and Ser473. These phosphorylations are probably independent and influence PKB function. Thr308 phosphorylated PKB is implicated in resumption of meiosis (GVBD), whereas Ser473 phosporylation is not - as oocytes with reduced phosphorylation on Thr308 have delayed GVBD kinetics and oocytes with inhibited phosphorylation of Ser473 reinitiate meiosis comparably to control oocytes. Conversely, oocyte treatment with synthetic biologically active PtdIns(3,4,5)P3 leads to stronger phosphorylation at Thr308 and accelerated GVBD kinetics. It was also found, that the kinase responsible for Ser473 phosphorylation in mouse oocytes is ATM. Powered by TCPDF (www.tcpdf.org)
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Using simulation to predict defects in and cast Al-alloy castings
Šolc, Petr ; Bařinová, Dagmar (referee) ; Čech, Jaroslav (advisor)
The aim of this work is comparing three casting process simulation programs for porosity and microstructure prediction capabilities for die-casting. After confronting these results with experimentally measured data taken from real castings it could be said that simulation is pretty accurate for DAS microstructure prediction and hot-spot areas. Amount of measured porosity could not be compared with predicted values because specimens were not taken from the exact hot-spot areas.
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CDC25A je schopna indukovat znovuzahájení meiosy, ale inhibuje metafase I – metafase II přechod
Šolc, Petr ; Šašková, Adéla ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein is present at metaphase I (MI) and metaphase II (MII) stages. CDC25A mRNA is stable during entire meiotic maturation. Exogenous CDC25A was sufficient to overcome cAMP-mediated GV-stage block. Using microinjection of GFP-CDC25A and GFP-CDC25B mRNAs constructs we have revealed that CDC25A is exclusively nuclear protein until nuclear envelope break down (NEBD). In contrast CDC25B localizes to cytoplasm at GV-stage oocytes and translocates to nucleus shortly before NEBD. Overexpression of GFP-CDC25A, to interfere with CDC25A degradation during meiotic maturation, resulted in MI block characterized with problems in chromosome congression and spindle formation. This MI block was accompanied with the transient reduction of both CDK1 and MAPK activities. RNAi mediated CDC25A knock-down resulted in a reduced ability to resume meiosis and to reach MII. These data demonstrate that behavior of CDC25A during female meiosis differs significantly from mitosis and CDC25A is involved in both, resumption of meiosis and also in metaphase I spindle formation as a prerequisite for correct MI-MII transition. It is evident that CDC25B is not only important CDC25 phosphatase for meiotic maturation but also CDC25A has its meiotic specific role.
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Aurora-A je zhrnuta v znovuzahájení meiosy a formaci metafáze I spindlu
Šašková, Adéla ; Šolc, Petr ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized entirely to the centrosome (MTOC) shorly before germinal vesicle breakdown (GVBD). Compared to somatic cells, where active Aurora-A is at the centrosomes and the spindle poles, active Aurora-A is strictly localized on MTOCs at metaphase I in oocytes. We show that activation of centrosomal Aurora-A is independent on PI3K-PKB and CDK1 signaling pathways. This was proved by cultivation of oocytes in presence of roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor). Treated oocytes show high phosphorylation of Aurora-A on T288 and centrosome amplification despite the presence of intact nuclear envelope. Silencing of Aurora-A by RNA interference induces incorrect spindle assembly. Oocytes are arrested in prometaphase I and unable to reach metaphase II. After microinjection of eGFP-Aurora-A mRNA into GV-stage oocytes, overexpression of Aurora-A leads to distortion of MI spindle organization as well. Our results indicate that Aurora-A is the key centrosomal player in meiotic maturation, essential for proper spindle formation and metaphase I - metaphase II transition.
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Aurora-A je zhrnuta v znovuzahájení meiosy a formaci metafáze I spindlu
Šašková, Adéla ; Šolc, Petr ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized entirely to the centrosome (MTOC) shorly before germinal vesicle breakdown (GVBD). Compared to somatic cells, where active Aurora-A is at the centrosomes and the spindle poles, active Aurora-A is strictly localized on MTOCs at metaphase I in oocytes. We show that activation of centrosomal Aurora-A is independent on PI3K-PKB and CDK1 signaling pathways. This was proved by cultivation of oocytes in presence of roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor). Treated oocytes show high phosphorylation of Aurora-A on T288 and centrosome amplification despite the presence of intact nuclear envelope. Silencing of Aurora-A by RNA interference induces incorrect spindle assembly. Oocytes are arrested in prometaphase I and unable to reach metaphase II. After microinjection of eGFP-Aurora-A mRNA into GV-stage oocytes, overexpression of Aurora-A leads to distortion of MI spindle organization as well. Our results indicate that Aurora-A is the key centrosomal player in meiotic maturation, essential for proper spindle formation and metaphase I - metaphase II transition.
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CDC25A je schopna indukovat znovuzahájení meiosy, ale inhibuje metafase I – metafase II přechod
Šolc, Petr ; Šašková, Adéla ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein is present at metaphase I (MI) and metaphase II (MII) stages. CDC25A mRNA is stable during entire meiotic maturation. Exogenous CDC25A was sufficient to overcome cAMP-mediated GV-stage block. Using microinjection of GFP-CDC25A and GFP-CDC25B mRNAs constructs we have revealed that CDC25A is exclusively nuclear protein until nuclear envelope break down (NEBD). In contrast CDC25B localizes to cytoplasm at GV-stage oocytes and translocates to nucleus shortly before NEBD. Overexpression of GFP-CDC25A, to interfere with CDC25A degradation during meiotic maturation, resulted in MI block characterized with problems in chromosome congression and spindle formation. This MI block was accompanied with the transient reduction of both CDK1 and MAPK activities. RNAi mediated CDC25A knock-down resulted in a reduced ability to resume meiosis and to reach MII. These data demonstrate that behavior of CDC25A during female meiosis differs significantly from mitosis and CDC25A is involved in both, resumption of meiosis and also in metaphase I spindle formation as a prerequisite for correct MI-MII transition. It is evident that CDC25B is not only important CDC25 phosphatase for meiotic maturation but also CDC25A has its meiotic specific role.
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