La neurodégénérescence fait partie intégrante de la maladie de Huntington (MH) dont les premiers symptômes moteurs et cognitifs apparaissent vers l’âge de 30 à 40 ans. Cette maladie incurable est causée par une mutation dans le gène codant pour la protéine huntingtin (htt). L’activation de la caspase-6 (casp6) est observée au stade présymptomatique chez l’humain et les modèles murins MH faisant de la casp6 un joueur majeur dans la neurodégénérescence précoce associé à la MH. De plus, le clivage de htt mutant par la casp6 produit un fragment N-terminal neurotoxique essentielle au développement de la MH. Des résultats préliminaires ont permis de révéler l’interaction et le clivage des protéines proapoptotiques Serine/Threonine Kinase 3 (STK3), Death-Domain Associated Protein (DAXX) par la casp6. Des effets proapoptotiques sont associés à leurs fragments et leur production par les caspases pourrait influencer la neurodégénérescence observée dans diverses maladies neurodégénératives et dans le vieillissement normal. Nos résultats dans les souris C57Bl/6 démontrent que l’expression de DAXX varie fortement avec l’âge selon l’organe analysé. Ses divers fragments ne suivent pas la même tendance d’un organe à l’autre suggérant des fonctions différentielles à travers l’organisme et une importante régulation de ses fonctions par des modifications post-traductionnelles. Chez les modèles murins de la MH, les souris YAC128, nous avons constaté une augmentation des fragments à 65 et 70 kDa dans le cortex et une diminution de DAXX entier et du fragment à 70 kDa dans le cervelet soulignant la possibilité de fonctions spécifiques selon les régions cérébrales. Nous avons aussi démontré pour la première fois le clivage de STK3 par la caspase-7 et la production différentielle de fragments par les caspase-3, 6 et 7. L’expression protéique de STK3 augmente globalement à travers l’organisme avec l’âge et dans le cervelet des souris YAC128. Par contre, une diminution de l’expression de STK3 est observée dans le cortex des individus atteints de la MH et des souris YAC128. Finalement, par l’induction de différents stress cellulaires, nous avons constaté la présence d’un mécanisme adaptatif des neurones modèles de la MH impliquant STK3. En conclusion, l’expression de DAXX et STK3 varie avec l’âge à travers l’organisme et est altérée dans la maladie de Huntington. Plus particulièrement, STK3semble être impliqué dans un mécanisme protégeant les neurones de la mort cellulaire dans la MH.

This Master's Thesis deals with the famous work of a Clash of Civilizations by Samuel Huntington. The topic is described to full extent. First of all, there are defined basic terms connected with culture. Afterwards, the conception of a Clash of Civilizations is described in its basic concepts, whereas there are not omitted consequences connected with the author's other works as well as his life. A special chapter is dedicated to the impact that this work caused. This chapter is devided into two parts. In the first part, there are presented general references of several authors. In the other part, there are discussed comprehensive conceptions of certain authors. Concretely, they are represented by Francis Fukuyama, Immanuel Wallerstein, Alvin Toffler and Michael Novak. After description of basic ideas of these big thinkers, there is made a comparison of their conceptions, which is based on the following terms -- universalism, democracy a capitalism, world order and modernization. In the last part of this Master's Thesis, there are deduced conclusions from the comparison of these conceptions that deal with the international system, which makes this issue more clear for both public and academic people.

Les astrocytes deviennent réactifs dans les maladies neurodégénératives (MND) comme la maladie d’Alzheimer (MA) et de Huntington (MH) mais les conséquences fonctionnelles de cette réactivité sont peu connues. Dans cette étude, nous avons évalué 1) les voies de signalisation impliquées dans la réactivité astrocytaire, 2) la contribution des astrocyte réactifs (AR) à la dysfonction neuronale dans des modèles de MND et 3) les caractéristiques fonctionnelles des AR.Nous avons montré que la voie JAK2/STAT3 est responsable de la réactivité astrocytaire dans des modèles murins de la MA et la MH. Nous avons développé de nouveaux vecteurs viraux ciblant cette voie dans les astrocytes, in vivo. Grâce à ces outils, nous avons étudié la contribution des AR à la dysfonction neuronale dans deux modèles murins de la MH. Nos résultats suggèrent que les AR ne jouent pas un rôle central dans ces modèles de pathologie. En ciblant la voie JAK2/STAT3, nous avons induit la réactivité astrocytaire chez la souris sauvage et avons montré que cette voie régule la transcription de gènes impliqués dans des fonctions cellulaires importantes. De plus, nous avons observé que l’activation des astrocytes conduit à une diminution de la plasticité synaptique dans le cerveau de souris.En conclusion, nous avons montré que la voie JAK2/STAT3 est une voie centrale dans les AR. Nous avons développé des vecteurs viraux innovants pour évaluer 1) la contribution des AR à la dysfonction neuronale dans des modèles de MND et 2) les propriétés fonctionnelles des AR in vivo. L’étude des AR permettra d’identifier de nouvelles cibles moléculaires pour manipuler ces cellules pléiotropes à des fins thérapeutiques. / Astrocyte reactivity is a hallmark of pathological conditions in the CNS including neurodegenerative diseases (ND) such as Alzheimer’s (AD) and Huntington’s (HD) diseases. Reactive astrocytes (RA) are identified by morphological changes but their functional features and influence on neurons are poorly understood, especially in ND. Therefore, we aimed at 1) identifying the signaling cascades involved in astrocyte reactivity in ND, 2) evaluating RA contribution to disease phenotype in ND models and 3) deciphering RA functional features. The JAK2/STAT3 pathway is a known trigger of astrocyte reactivity in CNS injuries. Here, we show that this pathway is a common inducer of astrocyte reactivity in AD and HD models. We developed new viral vectors to target this cascade in astrocytes and manipulate astrocyte reactivity in vivo. We used these vectors to determine the contribution of RA to neuronal dysfunction in HD mouse models. We found that RA do not primarily influence disease phenotype in HD. Last, we targeted the JAK2/STAT3 pathway in WT mice to characterize RA functional features in vivo. We show RA undergo transcriptional changes of numerous genes involved in metabolism, protein degradation pathways and immune response. Moreover, we show that astrocyte reactivity alters synaptic plasticity in the mouse hippocampus. Our results identify the JAK2/STAT3 pathway as a central cascade for astrocyte reactivity. The viral vectors developed in this project represent powerful tools to decipher the roles of RA in various ND models and to characterize RA functional features in vivo. Better understanding RA functions may lead to the identification of new therapeutic targets for ND.

The aim of this diploma degree thesis called The Hungarian uprising 1956 in light of transitological theories is an application of these theories on a process which took a place in a period 1953 - 1956 and ended in uprising in 1956. These events happened before the creation of the transitological theories. The application of these theories provides politological analysis of the whole process, examines the transitological theories, above all their universality. In the first part, transitology as politological analyses is outlined, including its creation and development as well as the authors and their studies which supported the development of these theories. I emphasized the best known theories from Samuel Huntington and Adam Przeworski. The second part is focused on analyses of position of opposition outside regime, reformers and hardliners in regime using these theories. I paid my attention to Soviet Union. The soviets tanks repressed the Hungarian uprising in the end. Despite this fact I studied if the year 1956 could have constituted the democracy or some hybrid form of democracy. I analyzed this idea with application the transitology theories on Hungarian example. On the basis of these theories I reached the conclusion that the situation in 1956 could constitute some form of hybrid democracy....

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Kocurová Supervisor: Prof. MUDr. Jaroslav Dršata, CSc. Title of diploma thesis: Investigation of proteolytic enzymes expression in different tissues at the transgenic animal model of Huntington's disease by means of biochemical and immunohistochemical methods Background: Huntington's disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the huntingtin (Htt) protein. Because it is known that mutant Htt and especially its small proteolytic fragments are toxic to neurons (particularly those in the striatum and cortex), it has been suggested that proteolysis of mutant huntingtin (mHtt) might play an important role in HD pathogenesis. Therefore, the aim of the present study was to examine the expression of endogenous and mtHtt and possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMPs), kallikreins (KLKs) and calpains in HD pathology of brain tissue. Methods: In this study we used WT and TgHD minipigs for N-terminal part of the human mtHtt (548aaHTT-145Q, both F2 generation, age 36 months; F3 generation, age 48 months in additional experiment), R6/2 mice were used as...

Under flera år har offentlig verksamhet upplevt en tilltagande byråkratisering inom statsförvaltningen. Byråkratiseringen upplevs flytta fokus från kärnverksamhet till administration och därigenom minska efterfrågan på expertis inom offentlig verksamhet. Det kallas ibland för en pågående avprofessionalisering. Detta har även börjat märkas inom militären där frågor kring vad som är professionellt börjar förekomma. Det finns även farhågor att denna process inom militären kan leda till att den civila kontrollen förloras över militären. Syftet med studien är att undersöka om förvaltningsstyrning, och speciellt tillitsbaserad styrning enligt Bringselius ger den kontroll av militärmakt som eftersträvas enligt Huntingtons teori om objektiv kontroll.  Kombinationen av Huntingtons objektiva kontroll och Bringselius ramverk för tillitsbaserad styrning, ger ett teoretiskt ramverk för dennas studie. Med en deduktiv ansats undersöker studien hur styrdokumenten för Försvarsmakten leder till civil kontroll.  Analysen av dokumenten genom fyra teman skapade ur det teoretiska ramverket visar att förvaltningsstyrningen med sina många detaljstyrningar, och avsaknad av delegering av ansvar gör att styrningen inte svarar mot Huntingtons objektiva kontroll.  En starkare officersprofession skulle i samspel med politiken kunna tydliggöra den delegering av ansvar som behöver ske för att tillitsbaserad styrning ska kunna införas som styrmodell mellan regeringen och Försvarsmakten. Införs tillitsbaserad styrning som förvaltningsstyrning finns goda möjligheter att stärka officersprofessionen och samtidigt bibehålla objektiv kontroll.

Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disease that specifically affects the striatum of the human brain. HD is characterized by a chorea-like movement disorder, cognitive decline, and psychiatric symptoms. In Europe, it has a relatively high prevalence of about 2.17-7.33 per 100,000 people compared with other continents. By far, there is no cure for HD. The mean survival time of patients after the diagnosis of HD is 15 to 20 years. Although the mutant form of the Huntingtin (HTT) as the cause of HD has been confirmed for decades, the exact pathogenesis of HD is still elusive. More recently, large global genome-wide association studies (GWAS) and several other studies provided new insights for HD mechanism, by highlighting several genes involved in DNA damage repair mechanisms as modifiers of age at onset and disease severity in HD. Thus, this project focused on the investigation of DNA damage response and repair in HD in vitro models. Fused in sarcoma (FUS) was the protein of our interest, as it has been confirmed to participate in DNA damage response and repair in multiple ways. Furthermore, FUS protein was implicated to have a relationship with neurodegenerative diseases, as it was found to play a role in the pathogenesis of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. FUS was also found to co-localize with mutant huntingtin protein in intracellular aggregates in HD mice models. In this project, donor/patient-specific induced pluripotent cells (iPSCs) and its derived striatal neurons were the main materials. By immunofluorescence staining approach of γH2AX and 53BP1, DNA double-strand breaks (DSBs) damage was investigated on iPSCs-derived in vitro striatal neurons. HD neurons showed an obvious and excessive accumulation of DNA DSBs damage. Then, in order to visualize FUS protein during DNA damage response procedure, eGFP tagged endogenous wild-type FUS iPSCs were generated, and later were differentiated into striatal neurons. UVA laser micro-irradiation was applied onto both hiPSCs and their differentiated striatal neurons in vitro models, simultaneously conducting with live-cell imaging approach. FUS was found to recruit to the DNA damage site induced by laser irradiation. For studying the kinetics of wild-type FUS protein during the response to laser irradiation, a novel and robust workflow was generated. By this workflow, the kinetics of FUS protein was characterized into four phases and a real-time scale of the kinetics was offered. After comparisons, a prominent change of FUS kinetics in HD at neuron-stage but not iPSC-stage was found. Furthermore, an intriguing different performance of FUS protein was found in different types of in vitro cellular models. In iPSCs, not all the laser-irradiated cells recruited FUS at the DNA damage site. The kinetics of the FUS protein also differed in different models. In conclusion, first, our in vitro striatal neuron model recapitulated the impaired DNA damage repair phenotype that published by other models. Second, new evidence was offered that wild-type FUS was involved in the pathogenesis of HD. Third, depending on cell-type, FUS performed differently during the response to the laser irradiation-induced DNA damage. Thus, these results suggest that the impaired DNA damage response and repair would be crucial to the mechanism of HD. Furthermore, the role of FUS protein playing especially the functional part in DNA damage response and repair might be a potential target for further investigation of neurodegenerative diseases including HD.

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites. The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD mice also exhibit worsened performance on the raised-beam test, a test for balance and coordination indicating that the PSA heterozygosity impairs the function of neurons with mutant huntingtin. In order to test whether increasing PSA expression ameliorates the HD phenotype in mice we created an adeno-associated virus (AAV) expressing the human form of PSA (AAV-hPSA). Unexpectedly, testing of AAV-hPSA in non-HD mice resulted in widespread toxicity at high doses. These findings suggest that overexpression of PSA is toxic to neurons in the conditions tested. In the second part of my dissertation work, I designed a model for following the silencing of huntingtin sequences in the brain. Firefly luciferase is a bioluminescent enzyme that is extensively used as a reporter molecule to follow biological processes in vivo using bioluminescent imaging (BLI). I created an AAV expressing the luciferase gene containing huntingtin sequences in the 3'-untranslated region (AAV-Luc-Htt). After co-injection of AAV-Luc-Htt with RNA-silencing molecules (RNAi) into the brain, we followed luciferase activity. Using this method, we tested cholesterol-conjugated siRNA, un-conjugated siRNA, and hairpin RNA targeting both luciferase and huntingtin sequences. Despite being able to detect silencing on isolated days, we were unable to detect sustained silencing, which had been reported in similar studies in tissues other than the brain. We observed an interesting finding that co-injection of cholesterol-conjugated siRNA with AAV-Luc-Htt increased luminescence, findings that were verified in cell culture to be independent of serotype, siRNA sequence, and cell type. That cc-siRNA affects the expression of AAV-Luc-Htt reveals an interesting interaction possibly resulting in increased delivery of AAV into cells or an increase in luciferase expression within the cell. My work presents a method to follow gene silencing of huntingtin targets in the brain, which needs further optimization in order to detect sustained silencing. Finally, in this dissertation I continue the study of bioluminescent imaging in the brain. We use mice that have been injected in the brain with AAV-Luciferase (AAV-Luc) to screen 34 luciferase substrate solutions to identify the greatest light-emitting substrate in the brain. We identify two substrates, CycLuc1 and iPr-amide as substrates with enhanced light-emitting properties compared with D-luciferin, the standard, commercially available substrate. CycLuc1 and iPr-amide were tested in transgenic mice expressing luciferase in dopaminergic neurons. These novel substrates produced luminescence unlike the standard substrate, D-luciferin which was undetectable. This demonstrates that CycLuc1 and iPr-amide improve the sensitivity of BLI in low expression models. We then used CycLuc1 to test silencing of luciferase in the brain using AAV-shRNA (AAV-shLuc). We were unable to detect silencing in treated mice, despite a 50% reduction of luciferase mRNA. The results from this experiment identify luciferase substrates that can be used to image transgenic mice expressing luciferase in dopaminergic neurons. My work contributes new data on the study of PSA as a modifier of Huntington’s disease in a knock-in mouse model of Huntington’s disease. My work also makes contributions to the field of bioluminescent imaging by identifying and testing luciferase substrates in the brain to detect low level of luciferase expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Huntington disease (HD) is an autosomal dominant disorder characterized by progressive decline of motor, cognitive, and behavioral function. Saccades (rapid, gaze-shifting eye movements) are affected before a clinical diagnosis of HD is certain (i.e. during the premanifest period of the disease). Fundamental questions remain regarding the neural substrates of abnormal saccades and the course of premanifest disease. This work addressed these questions using magnetic resonance imaging (MRI) and a longitudinal study of premanifest disease progression. Gray matter atrophy is a characteristic of HD that can be reliably detected during the premanifest period, but it is not known how such changes influence saccadic behavior. We evaluated antisaccades (AS) and memory guided saccades (MG) in premanifest and manifest HD, then tested for associations between impaired saccadic measures and gray matter atrophy in brain regions involved in these saccadic tasks. The results suggest that slowed vertical AS responses indicate cortical and subcortical atrophy and may be a noninvasive marker of atrophic changes in the brain. We also investigated the brain changes that underlie AS impairment using an event-related AS design with functional MRI (fMRI). We found that, in premanifest and manifest HD, blood oxygenation level dependent (BOLD) response was abnormally absent in the pre-supplementary motor area and dorsal anterior cingulate cortex following incorrect AS responses. These results are the first to suggest that abnormalities in an error-related response network underlie early disease-related saccadic changes, and they emphasize the important influence of regions outside the striatum and frontal cortex in disease manifestations. Though saccadic abnormalities have been repeatedly observed cross sectionally, they have not yet been studied longitudinally in premanifest HD. We found different patterns of decline; for some measures the rate of decline increased as individuals approached onset, while for others the rate was constant throughout the premanifest period. These results establish the effectiveness of saccadic measures in tracking premanifest disease progression, and argue for their use in clinical trials. Together, these studies establish the utility of saccade measures as a marker of HD neurodegeneration and suggest that they would be a valuable component of batteries evaluating the efficacy of neuroprotective therapies.

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