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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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Characterization of immune cells and monitoring changes of inflammatory proteins in minipig model of Huntington's disease
Butalová, Nikola ; Motlík, Jan (advisor) ; Janda, Jozef (referee)
The Huntington disease (HD) is a hereditary neuro-degenerative disorder caused by a mutation of the huntigtin gene that codes a protein of the same name. The mutated form of the huntigtin gene plays its part in many pathological interactions and influences a number of cellular mechanisms, including the immune system that could serve as a modifier of the neuropathology of the disease. The cells of the monocyte-macrophage system express cytokines whose production changes in relation to the activation of the cell. The presence of the mutated huntingtin protein in these cells renders them hyper-responsive to immunity incentives leading to changes in the production of cytokines. These differences are discernible a few years prior to the appearance of the symptoms. Therefore, the changes in the levels of certain cytokines could serve as appropriate biomarkers for monitoring of the onset of the disease and its progression. The HD pathogenesis includes an inflammation of the central neutral system. Inflammatory changes in peripheral tissues could reflect inflammatory processes in the central neural system. A miniature TgHD pig could represent an appropriate model organism for studying of the impact of the mHtt on the immune system. This model enables to observe a slow progression of the disease. Changes in...
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Healing wound as a model for the study of cell interactions
Gál, Peter ; Smetana, Karel (advisor) ; Motlík, Jan (referee) ; Brábek, Jan (referee)
Healing wound as a model for the study of cell interactions Abstract Galectins play an important role in the processes of cell proliferation, differentiation, migration and extracellular matrix formation. Furthermore, galectins are able to transfer cellular signals and to participate in cell interaction. It has been proven that galectins play an important role in the microenvironment formation of a tumor and/or healing wound. This study demonstrated significant role of galectins, in particular Galectin-1, in wound healing and cell interactions (endothelial cells, fibroblasts and keratinocytes) forming a part of the granulation tissue and tumor stroma. We have demonstrated that the extracellular matrix rich on Galectin-1 creates a suitable environment for the cultivation of keratinocytes. Galectin-1 also induces differentiation of fibroblasts into myofibroblasts. The knowledge of above mentioned processes is important to better understand the complexity of cancer biology and its parallel to wound healing. Key words: tissue repair, regeneration, galectin, tumor
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Effect of selected pollutants on mammalian organisms in vivo and cells in vitro and preparation of specific monoclonal antibodies for their detection
Dorosh, Andriy ; Pěknicová, Jana (advisor) ; Linhart, Otomar (referee) ; Motlík, Jan (referee)
Environmental pollution and its effect on the living organisms has attracted lots of attention recently. There is a growing body of evidence that we are exposed to environmental pollutants at low concentrations in everyday life. The cells and organisms have tools to identify, neutralize and excrete the majority of the toxic compounds. The most dangerous are those that can escape this process or act at low trace concentrations. Endocrine disruptors (EDs) belong to the latter group. Endocrine disruptors can be of natural and anthropogenic origin. EDs target corresponding hormonal receptors and can act at low concentrations. A wide family of nuclear receptors recognize steroid hormones. The majority of EDs can pass through the cytoplasmic membrane, use the hydrophobic nature of the receptor-ligand binding, trigger hormone response and change the expression of the sensitive genes. By interfering with estrogen and androgen signaling, EDs can have effect on the whole organism, but the reproductive system is influenced most. In the present work, our aim was to develop the methods for ED detection and monitoring, analyze the estrogenic potency of EDs, and evaluate the effects of natural estrogens and EDs on male reproductive functions, including sperm and testicular physiology and endocrine functions. First, we...
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Analysis of inflammatory biomarkers in the transgenic minipig model of Huntington's disease
Valeková, Ivona ; Motlík, Jan (advisor) ; Janda, Jozef (referee) ; Ondráčková, Petra (referee)
Huntington's disease (HD) is an inherited monogenic neuropsychiatric degenerative, progressive, and fatal condition. The disease onset is in middle age of the patient. The most prominent clinical features are motor impairment, progressive decline in cognitive functions, and personality changes. Any preventive or disease-modifying therapies are not available so far. Therapeutic interventions can only target symptoms. It is believed that the primary pathology of HD results from massive degeneration of neurons in the basal ganglia. However, the expression of mutant huntingtin was detected in all tissues. Thus the mutation in non- neuronal cells of the brain and in peripheral tissues contributes to the pathology of HD. Neuroinflammation, especially microglia activation, is involved in the pathogenesis of HD. Given evidence that mutated huntingtin is expressed in peripheral immune cells, it is possible that inflammatory changes detected in peripheral tissues may reflect the inflammatory process in central nervous system (CNS). Several recent studies indicated that the immune system could act as a modifier of HD neuropathology. In order to monitor the success of any disease- modifying drugs in the pre-manifest stage of HD it is important to identify robust biomarkers of the onset and disease progression....
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Pathogenesis of Huntington's disease in peripheral tissues
Vachútová, Dominika ; Motlík, Jan (advisor) ; Fiala, Ondřej (referee)
Huntington's disease (HD) is an autosomal dominant inherited disorder with manifest of symptoms around the age of 40. This disorder is caused by an expansion of CAG repeats in huntingtin gene, Huntingtin (Htt) is a protein expressed in almost all tissues. HD is mainly characterized by neurodegeneration in the basal ganglia and cerebral cortex, but mutation in huntingtin have also serious influence on peripheral tissues. Many studies show serious heart dysfunction, weight loss, altered glucose homeostasis, impairment of energetic metabolism and muscular atrophy in HD patients and animal models. Till now, mechanism of these changes has not been sufficiently described and there is nor an adequate treatment yet. Key words: Huntington's disease, mutated huntingtin, CAG repeat, peripheral tissue
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