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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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Myosin I and actin binding proteins in cell
Krásna, Hana ; Hozák, Pavel (advisor) ; Motlík, Jan (referee) ; Pěknicová, Jana (referee)
Many studies have established the presence and essenciality of actin in the nucleus. Recently, actin has been associated with processes in the nucleus ranging from chromatin remodeling to transcription, splicing or nuclear transport. To ensure the dynamics of the nuclear processes, actin is coupled with one of the main motor protein such as myosin. This study demonstrates a structural role of actin and the nuclear myosin I (NMI) take in the transcription of ribonuclear genes by RNA polymerase I (Pol l). We suppressed the transcription Pol I in vitro by microinjections of antibodies anti actin and anti MNI. The series of in vitro experiments confirm transcript Pol I inhibition after applying antibodies anti actin and MNI on pure DNA as well as on pre-assembled chromatine template. The co-immunoprecipitation experiments reveal direct bound between actin, NMI and rRNA genes and transcription complex Pol I. As actin binds to the primer and elonged Pol I molecule, NMI interacts with subunit of Pol I and is capable of assembling into productive initiation complex by binding up to TIF-IA, transcriptional factor responsible for regulation rRNA synthesis. There are known number of hypothesis on the form of nuclear actin. Recent research suggests actin exists in equilibrium between its monomeric and...
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Role of modified autophagosomal function in patophysiology of Huntington's disease.
Kotrčová, Eva ; Motlík, Jan (advisor) ; Sládková, Jana (referee)
Huntington's disease, an autosomal dominant neurodegenerative disease, affects the cell in several toxical ways. One of them is accumulation of protein aggregates in cytoplasma, which could become a serious problem especially for long-lived cells such as neurons. Autophagy (macroautophagy) is an important catabolic pathway, crucial for cell survival. If fully functional, it should eliminate protein aggregates and reduce the toxic effect on the cell. However, recent works show that this pathway might be defective, most probably in the cytoplasmic cargo recognition. In my work I used a transgenic miniature pig model of Huntington's disease to verify the hypothesis of autophagical dysfunction in individuals suffering from Huntington's disease. I studied levels of autophagosomal markers - LC3 and p62 in mesenchymal stem cells after different autophagy stimulation treatments, and ammonium chloride was found the most effective. In addition I evaluated the effect of age of the animals on autophagic function, but no significant changes were identified, even if animal genotype was considered. Moreover I had an opportunity to study proteins levels in three porcine brain tissues - cortex, cerebellum and striatum. Even though there is no significant diference, we can observe a trend of LC3 II and p62 increase in...
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Generation of porcine induced pluripotent stem cells - a model of Huntington disease.
Svobodová, Eliška ; Motlík, Jan (advisor) ; Fulka, Josef (referee)
Stable porcine ES cell lines have not been succesfully established yet. Ability to selfrenew or to differentiate has been limited in different porcine ES-like cell lines so far. PiPSCs represent an alternative to pESCs. PiPSCs can be generated by reprogramming of somatic cells by introduction of several transcription factors on viral vectors and were established by several groups. However, the majority of piPS cell lines depend on transgene expression because of incomplete reprogramming and weak activation of endogenous pluripotency genes. Transgene expression can infuence differentiation potential of piPSCs. Therefore, we have used integrative and reexcisable PiggyBac transposons to generate viral free piPSCs. At the same time, small molecules (low-molecular inhibitors) with potential to increase reprogramming efficiency and to activate endogenous pluripotency genes were used in the reprogramming media. This strategy has a potential for generation of naive piPSCs. Successful excision of transgenes would generate transgene-free piPSCs with uncompromised differentiation potential. Pig (Sus Scrofa) is at the same time an important animal model in preclinical stage research of the diseases. Somatic cells used for generation of piPSCs were isolated from pigs carrying mutated huntingtin. Integration of the...
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Changes in beta-catenin expression during ontogenesis in the transgenic minipigs for human mutant huntingtin
Žižková, Martina ; Motlík, Jan (advisor) ; Jarkovská, Karla (referee)
Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG repeat sequence within the huntingtin gene. Huntingtin associates with ubiquitin-proteasome system that ensures degradation of particular proteins including β-catenin which is an important molecule whose equilibrated degradation is necessary for the proper functioning of the Wnt signaling pathway. The binding of β-catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β-catenin. The main goal of my thesis was to determine whether the accumulation of β-catenin due to the presence of mutant huntingtin is also characteristic of Liběchov minipigs, a large animal model of Huntington's disease stably expressing N-truncated human mutant huntingtin. Using immunoblot and specific antibodies, we have revealed age-dependent accumulation of mutant huntingtin in transgenic minipigs. Unlike endogenous huntingtin, no decrease of the level of mutant huntingtin was observed in the striatum of transgenic animals. Surprisingly, this was followed by a decrease of phosphorylated β-catenin. Nevertheless, our results demostrate the accumulation of β-catenin in mesenchymal stem cells isolated from the oldest boars during ontogenesis. Furthermore, we have revealed a...
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Huntington's disease modeling and stem cell therapy in spinal cord disorders and injury
Hruška-Plocháň, Marián ; Motlík, Jan (advisor) ; Bjarkam, Carsten (referee) ; Roth, Jan (referee)
Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...
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Analysis of pluripotent gene expression program in early embryos and embryonic stem cells
Moravec, Martin ; Svoboda, Petr (advisor) ; Motlík, Jan (referee)
Pluripotence je schopnost buňky diferencovat do jakéhokoliv buněčného typu. Formuje se během časného embryonálního vývoje u savců a její vznik je spojen s reprogramací genové exprese na globální úrovni. Proces přirozeného vzniku pluripotence není stále zcela pochopen. Pro získání nového pohledu na události, které vedou ke vzniku pluripotence u savců, studovali jsme změny v genové expresi během oocyt-zygotického přechodu u myši. V tomto modelovém systému, oplodněné vajíčko podstoupí reprogramaci, která vede k vytvoření pluripotentních blastomer. Tyto blastomery zakládají samotné embryo. Cílem mé diplomové práce bylo analyzovat aktivaci transkripce během časného vývoje a vyvinout metodu pro monitorování exprese genů v oocytech, časných embryích a embryonálních kmenových buňkách. Metoda využívá kvantitativní PCR a umožnuje změřit expresi až 48 vybraných genů, které slouží jako markery pro maternální degradaci, aktivaci pluripotentního programu a diferenciaci do zárodečných linií. Dále ukazujeme, že náš systém monitoruje dynamiku transkriptomu během oocyt-zygotického přechodu, a získané výsledky jsou srovnatelné s daty naměřenými pomocí jiných metod. Díky našemu bioinformatickému přístupu jsme navíc identifikovali nové oocyt-specifické a zygotické nekódující RNA. Klíčová slova: pluripotence,...
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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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Biomedical models of Huntington disease
Žižková, Martina ; Motlík, Jan (advisor) ; Moravec, Jan (referee)
Huntington's diease is a dominant inherited neurodegenerative disorder that is caused by an expansion of a CAG repeats within a huntingtin gene. Mutant protein causes a neuron degeneration in a brain of HD pacients which leads to a motor abnormalities and personality decay. This disease is very malign because of its late onset. An equal therapy does not exist yet, but a lot of research teams focus on designig a suitable medical treatment. It is necessary to create animal models of Huntington disease which can be used for testing the therapies. In my work I aim to summarize the animal models of HD which are used in research. A rodent model is the most common due to its low price and easy breeding. However, more important are human related large animals like sheep, pigs or non-human primates. The principal criterion of animal model is its method of creation. We can divide the models into two categories, genetic and non-genetic. The memebers of the first one are able to reproduce better expression of human Huntington disease. Generation of animal models of HD leads to better comprehension the principles of HD, and developing an equal therapy for HD pacients.
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Cell therapy in animal models - preclinical studies
Juhásová, Jana ; Motlík, Jan (advisor) ; Grim, Miloš (referee) ; Jendelová, Pavla (referee)
The progress of cell therapy can be greatly facilitated by using suitable experimental models. It is essential to verify the clinical usefulness of new healing procedures obtained in studies on laboratory animals by using a large animal model. One of suitable models well acceptable in medical community is undoubtedly the miniature pig, which resembles humans in terms of physiology and body proportions. This PhD thesis presents the summary of our experimental studies relating to possible exploitation of mesenchymal and neural stem cells in the healing of locomotive apparatus and neural tissue disorders in humans or animals. The first part of the thesis briefly describes the current issue of cell therapy and animal models, mesenchymal cells and/or their combination with new types of scaffolds, neurogenesis, neural stem cells and their potential application in therapy of spinal cord injury. The second part is focused on the goals and methodology, the individual publications being listed in the third part. Our experiments with iatrogenic physeal defect in rabbits, which served as a model of the occurrence of valgous deformation in the clinical practice, showed the positive preventive and therapeutical effects of a new type of scaffolds seeded with allogeneic mesenchymal stem cells in animals without...
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