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Protein synthesis and protein degradation in mammalian oocyte development
Šušor, Andrej ; Motlík, Jan (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Concr,usroNs We anďysed eucaryoticinitiation t,anslation factorsthat are responsiblefor protein qmthesisin mammalianoocýes. As well we examinedthe affinity.of this factorsto.'mCap, Sepharoseduring in útro conďtion...Inour'laboratorywas optimisedthe protocol for generation of poreine parttrenotesthat serve us as a model for anďysis of. cellular processos in .rygotes with biochemical approaches.. : , , - Antibod}r micÍoanay analysis was used to investigate the: regulation of signalling pathways primarily during meiotic maturationof pig oocytesand subsequentlyin comparisonto other species(bovine,frog andseastar). on the base of proteomeanďysis of maturationof oocýes was chosenUCH-LI molebuleandis studiedin detail in mammalian oocyte. Abnost thorougily was analysed function of UCH-LI in porcine and bovine oocyte using specific inhibitors and overexpression. The mechanism that is involved UCH-LI in antipolyspermydefencein mammalian oocyte was unveiled in our laboratory.
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Following the phenotype development of TgHD minipigs by invasive and noninvasive approach
Ellederová, Zdeňka ; Baxa, Monika ; Vidinská, Daniela ; Bohuslavová, Božena ; Vochozková, Petra ; Šmatlíková, Petra ; Klíma, Jiří ; Valeková, Ivona ; Ardan, Taras ; Juhás, Štefan ; Juhásová, Jana ; Konvalinková, R. ; Klempíř, J. ; Pokorný, M. ; Krupička, R. ; Kauler, J. ; Hansíková, H. ; Motlík, Jan
Recent promising treatments for Huntington’s disease (HD) may require pre-clinical testing in large animals. In 2009, we generated HD transgenic (TgHD) minipigs with one copy encoding the N-terminal part (548 aa) of human huntingtin (HTT) with 124 CAG/CAA repeats integrated into chromosome 1 q24-q25. The successful germ line transmission occurred through four successive generations.
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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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Oxidative stress in primary porcine fibroblasts expressing mutated huntingtin
Šmatlíková, Petra ; Askeland, G. ; Vaškovičová, Michaela ; Klíma, Jiří ; Motlík, Jan ; Eide, L. ; Ellederová, Zdeňka
Molecular events, such as protein aggregation, mitochondrial dysfunction, and transcriptional dysregulation have been linked to Huntington’s disease (HD) pathogenesis. Oxidative stress has been considered as one of the key players in disease progression. Though, it is still not clear whether oxidative stress causes HD, or if it is a consequence of other primary events.
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Mitochondrial phenotype in minipig model transgenic for N-terminal part of human mutated huntingtin
Hansíková, H. ; Rodinová, M. ; Křížová, J. ; Dosoudilová, Z. ; Štufková, H. ; Bohuslavová, Božena ; Klíma, Jiří ; Juhás, Štefan ; Ellederová, Zdeňka ; Motlík, Jan ; Zeman, J.
Huntington’s disease (HD) is neurodegenerative disorder caused by an abnormal expansion of CAG repeat encoding a polyglutamine tract of huntingtin (htt). It has been postulated that mitochondria dysfunction may play significant role in the pathophysiology of the HD. But it is still not known yet in detail how mitochondria are able to cover energy needs of the cells during the progression of the HD.
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Establishing preclinical proof-of-concept of gene therapy for Huntington disease
Miniariková, J. ; Juhás, Štefan ; Caron, N. ; Spronck, L. ; Vallés, A. ; De Haan, M. ; Blits, B. ; Ellederová, Zdeňka ; van Deventer, S. ; Petry, H. ; Southwell, A. ; Déglon, N. ; Motlík, Jan ; Konstantinová, P. ; Evers, M.
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene. The translated expanded polyglutamine repeat in the huntingtin protein is known to cause toxic gain-of-function, affecting numerous cellular processes. Our approach involves a new therapeutic modality by developing a single (one-time) treatment for HD based on a gene therapy lowering the expression of the toxic huntingtin using the RNA interference (RNAi) mechanism. Huntingtin lowering is achieved using gene transfer of a cassette encoding an engineered microRNA targeting human HTT, delivered via adeno-associated viral vector serotype 5 (AAV5-miHTT).
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AAV-mediated delivery in large animals
Blits, B. ; De Haan, M. ; Evers, M. ; Spronck, E. A. ; Motlík, Jan ; Bohuslavová, Božena ; Ellederová, Zdeňka ; Lewis, O. T. ; Johnson, D. ; Woolley, M. ; Gill, S. ; van Deventer, S. ; Konstantinová, P. ; Petry, H.
Gene therapy is an attractive option for treatment of neurological diseases. Delivery of the therapeutic gene at the proper location is key for an effective treatment and remains challenging, especially in larger animals. For translation from smaller (rodents) to larger animals, dimensions are different, but also the immune system plays a more prominent role in larger animals. Direct intracranial parenchymal infusions usually result in local transduction of tissue, whereas intrathecal infusions result in a more widespread transduction in the brain. Depending on the indication, the desired expression pattern of the therapeutic gene is to be elucidated and is dictating the route of infusion.
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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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