The Study of Two Strategies for Decreasing Mutant Huntingtin: Degradation by Puromycin Sensitive AminoPeptidase and RNA Interference: A Dissertation

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\u27-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

Implementation characteristics of collegial support systems for teachers in middle schools.

The purpose of this study was to determine whether or not components within middle school organizations promote and nurture efforts to implement collegial coaching programs. Three schools were selected on the basis of the following criteria: (1) the schools were middle schools, (2) coaching was in at least its third year of implementation, (3) the coaching concept encompassed a transaction among equals, and (4) the program was not related to staff evaluation. Data was collected at each site through interviews, direct observation and document review. Findings were reported using a cross-case analysis format in which cross case issues and information from the individual cases were dispersed throughout the data presentation in Chapter 4 and the data analysis in Chapter 5. The effects of teaming and common planning time on school climate and the enabling nature of enhanced levels of teacher empowerment were identified as powerful variables which contributed to the successful implementation of coaching programs at each site. Factors such as common planning time, supportive leadership, teacher accountability and empowerment in the areas of curriculum, scheduling, grouping, staff development, and staffing are analyzed. Ultimately the form of coaching in accepted use became much less formalized following the implementation period. As coaching becomes an accepted part of the fabric of the school, its practice becomes much more informal and focuses on the improvement of current instructional techniques. Time and the reluctance of teachers to assume quasi-administrative roles were found to be significant factors in this area. (Abstract shortened with permission of author.

Les premières écoles autochtones au Québec : progression, opposition et luttes de pouvoir, 1792-1853

Ce mémoire s'intéresse aux luttes de pouvoir qui ont entouré le développement des premières écoles pour les Autochtones de la vallée du Saint-Laurent, durant la première moitié du XIXe siècle. Il cherche principalement à analyser les enjeux politiques, religieux et éducatifs liés à la scolarisation des Amérindiens et à mettre en lumière la situation unique du Bas-Canada. La Conquête de la Nouvelle-France avait d'abord permis à des sociétés missionnaires protestantes de s'immiscer dans les villages des domiciliés de la vallée du Saint-Laurent, pour y promouvoir l'éducation et faire de nouveaux convertis. Plusieurs Autochtones furent formés dans des écoles américaines et retournèrent ensuite dans leur communauté, afin d'y fonder à leur tour des établissements scolaires. La situation politique au Bas-Canada, dans les années 1820 et 1830, provoqua toutefois une réaction particulière des missionnaires catholiques à l'égard des nouvelles écoles autochtones. Devant la montée d'une gestion laïque de l'éducation par l'État, et confrontés à certains Autochtones qui souhaitaient utiliser l'éducation, soit pour diffuser la religion protestante soit pour acquérir une plus grande autonomie politique, les missionnaires choisirent majoritairement de s'opposer à tous les nouveaux projets éducatifs. Cependant, l'échec de la rébellion patriote, la diminution du personnel du Département des Affaires indiennes et l'accroissement du pouvoir clérical dans l'éducation de la province créèrent un environnement favorable à un changement dans la politique des missionnaires catholiques à l'égard des écoles autochtones. À partir de 1845, ces missionnaires encouragèrent donc le développement de nouvelles institutions scolaires catholiques, afin de superviser et de contrôler l'enseignement, la morale et la religion des communautés amérindiennes. \ud ______________________________________________________________________________ \ud MOTS-CLÉS DE L’AUTEUR : Autochtones, Bas-Canada, histoire de l'éducation, missionnaires, Département des Affaires indiennes

On the need for soft dressing

In order to deal with IR divergences arising in QED or perturbative quantum gravity scattering processes, one can either calculate inclusive quantities or use dressed asymptotic states. We consider incoming superpositions of momentum eigenstates and show that in calculations of cross-sections these two approaches yield different answers: in the inclusive formalism no interference occurs for incoming finite superpositions and wavepackets do not scatter at all, while the dressed formalism yields the expected interference terms. This suggests that rather than Fock space states, one should use Faddeev-Kulish-type dressed states to correctly describe physical processes involving incoming superpositions. We interpret this in terms of selection rules due to large U(1) gauge symmetries and BMS supertranslations.Comment: 16+5 pages, 1 figure, v2: typos fixed, version submitted to JHE

La confiance groupale et l'efficacité des équipes de travail : le rôle de la coopération et de l'interdépendance à l'égard de la tâche

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Comment tuer Shakespeare

Depuis plus de vingt-cinq ans, Normand Chaurette écrit avec et contre Shakespeare. Dans Les Reines, la première pièce québécoise produite à la Comédie-Française, il a revu et corrigé Richard III du point de vue des personnages féminins. Entre un Othello inédit et sa récente traduction du Roi Lear, il a dû inventer une langue capable de rendre celle du dramaturge de Roméo et Juliette et du poète des Sonnets. Comment tuer Shakespeare est le récit de cet étonnant combat. Entre narration et essai, portraits et journal de création, ce livre est le regard singulier d'un homme de théâtre qui a l'audace de défier la présence à la fois concrète et fantomatique d'un increvable Shakespeare.Pour Alice Ronfar