Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Objetivos: testar a validade da relaÃÃo diÃmetro transverso do cerebelo (DTC) /circunferÃncia abdominal (CA) como mÃtodo diagnÃstico ultra-sonogrÃfico da restriÃÃo de crescimento fetal (RCF). Determinar, atravÃs de curva ROC (receiver operator characteristic), o melhor ponto de corte da relaÃÃo DTC/CA. Verificar se a relaÃÃo DTC/CA tem sua acurÃcia modificada na dependÃncia do tipo de RCF (simÃtrica ou assimÃtrica) ou do tempo entre a ultra-sonografia e o parto. Comparar DTC/CA, no ponto de corte obtido, com a relaÃÃo comprimento do fÃmur (CF) /circunferÃncia abdominal (CA). MÃtodo: estudo prospectivo, seccional, envolvendo 250 gestantes com gravidez Ãnica, idade gestacional precisa, feto vivo. Foram realizadas ultra-sonografias obstÃtricas atà a resoluÃÃo da gestaÃÃo, mas somente a Ãltima foi considerada para anÃlise. Os neonatos cujas relaÃÃes DTC/CA estiveram maiores do que o ponto de corte determinado pela curva ROC foram considerados acometidos por RCF. Idem para a relaÃÃo CF/CA. Considerou-se como padrÃo-ouro para o diagnÃstico de RCF os recÃm-nascidos cujos pesos situaram-se abaixo do percentil 10 para a idade gestacional nas curvas de Lubchenco et al. (1963), corrigidas para sexo. Definiu-se RCF simÃtrica neonatos com Ãndice ponderal de Rohrer situado entre 2,2 e 3.0. Aqueles com RCF cujos Ãndices fossem < 2,2 foram classificados como RCF assimÃtrica. Resultados: a prevalÃncia da RCF foi de 12,4%. O ponto de corte da relaÃÃo DTC/CA determinado pela curva ROC foi 16,15. A sensibilidade, especificidade, valores preditivos positivo e negativo, acurÃcia, razÃes de verossimilhanÃa positiva e negativa foram de 77,4%, 82,6%, 38,7%, 96,3%, 82%, 4,5 e 3,7, respectivamente. Na RCF simÃtrica a sensibilidade e especificidade foram de 80,8% e 81,7%, respectivamente. Na assimÃtrica a sensibilidade e especificidade foram 60% e 75%, respectivamente. Resultados menores do que na simÃtrica, porÃm, nÃo estatisticamente significantes (p > 0,05). No intervalo de zero a sete dias entre a Ãltima ultra-sonografia e o parto, a sensibilidade e especificidade foram de 81,5% e 82,1%, respectivamente. No intervalo de oito a 14 dias, a sensibilidade e especificidade foram de 50% e 84,3%, respectivamente, sem diferenÃa estatisticamente significante entre os dois intervalos (p > 0,05). O ponto de corte da relaÃÃo CF/CA foi de 22,65, com sensibilidade, especificidade, valores preditivos positivo e negativo, acurÃcia, razÃes de verossimilhanÃa positiva e negativa de 67,7%, 81,7%, 34,4%, 94,7%, 80%, 3,7 e 2,5, respectivamente. ConclusÃes: a relaÃÃo DTC/CA no ponto de corte 16,15 mostrou-se mÃtodo eficaz no diagnÃstico de RCF, tanto simÃtrica quanto assimÃtrica, nÃo sendo influenciada pelo tempo entre a Ãltima ultra-sonografia e o parto. Sendo mÃtodo independente da idade gestacional, à especialmente Ãtil nos casos em que este dado à ignorado. A relaÃÃo CF/CA mostrou-se menos eficaz do que a DTC/CA no diagnÃstico da RCF. / Objectives: to evaluate the validity of transverse cerebellar diameter (TCD)/abdominal circumference (AC) ratio as an ultrasonographic diagnosis method of fetal growth restriction (FGR). To calculate by receiver operator characteristic (ROC) curve the best cut-off value of TCD/AC ratio. To verify whether TCD/AC has its accuracy modified according to the dependence of type of FGR (symmetric and asymmetric) or according to the time between ultrasonography and deliverance. To compare TCD/AC ratio at its cut-off with the femur length (FL)/ abdominal circumference (AC) ratio. Method: a prospective cross-sectional study, carried out in 250 pregnant women with singleton pregnancies between 20 and 42 weeks of gestation, known accurate gestational age with ultrasound confirmation, living fetuses. Obstetrics sonographic examinations were accomplished until gestation resolution, but only the last one, within 14 days of the deliverance, was used for analysis. Neonates with TCD/AC ratio greater than the cut-off, established by ROC curve were diagnosed as FGR. The same was considered for FL/AC ratio. We classified as gold standard for FGR in new-born infants, who presented birth weight bellow 10th percentile of gestational age according to the growth curves of Lubchenco et al. (1963), corrected according to their sex. Neonates showing FGR and Rohrer ponderal index between 2,2 and 3,0 were labeled as symmetric FGR. Those showing FGR and ponderal index below 2,2 were labeled as asymmetric FGR. Results: prevalence of FGR among the study group was 12,4%. The best cut-off value calculated by ROC curve for TCD/AC ratio was 16,15. The sensitivity, specificity, accuracy, positive predictive values and negative predictive values, likelihood ratio for positive and negative tests were 77,4%, 82,6%, 38,7%, 96,3%, 82%, 4,5 and 3,7, respectively. In the symmetric FGR, sensitivity and specificity were 80,8% and 81,7%, respectively. In the asymmetric FGR, sensitivity and specificity were 60% and 75%, respectively. Results lower than in the symmetric FGR, but not statistically significant (p > 0,05). In the interval zero to seven days between sonographic examination and deliverance, sensitivity and specificity were 81,5% and 82,1%, respectively. In the interval of eight to 14 days, sensitivity and specificity were 50% and 84,3%, respectively, with no statistically significant difference (p > 0,05). The best cut-off value calculated by ROC curve for FL/AC ratio was 22,65, showing sensitivity, specificity, accuracy, positive predictive values and negative predictive values, likelihood ratio for positive and negative tests of 67,7%, 81,7%, 34,4%, 94,7%, 80%, 3,7 and 2,5, respectively. Conclusions: TCD/AC ratio at cut-off 16,15 proved to be an effective method in antenatal diagnosis of FGR, both symmetric as asymmetric, with no influence of interval between ultrasonography examination and deliverance. As a gestational age-independent method, it is useful enough in the occurrence of cases where these data are unknown. FL/AC ratio proved is not so effective as TCD/AC ratio in diagnosis of FGR.

Diversos neurônios apresentam uma atividade constitutiva de NF-?B, o qual desempenha múltiplas funções fisiológicas, além da modulação de respostas patológicas. A melatonina, hormônio produzido ritmicamente pela glândula pineal na fase de escuro, é também um fator autócrino e parácrino envolvido em múltiplos processos biológicos, sendo que a citoproteção é uma ação de destaque dessa molécula. A melatonina inibe a translocação nuclear do NF-?B e a expressão do seu produto iNOS em modelos de danos celular. No presente trabalho avaliamos se o efeito citoprotetor da melatonina depende do estado de ativação do NF-?B em cultura de células granulares de cerebelo, tendo em vista que essas células apresentam uma atividade basal deste fator de transcrição fundamental para a sobrevivência das células. Além disso, questionamos se essas células em cultura produziriam melatonina e se esta teria algum papel citoprotetor. Testamos a viabilidade da cultura de células granulares de cerebelo de rato (Wistar 7-8 dias de idade) após 24 horas de incubação com melatonina na presença ou ausência de LPS. Em condição basal a melatonina diminuiu a sobrevivência das células e inibiu a morte celular induzida pelo LPS. Este efeito foi compatível com os resultados da ativação do NF-?B e da expressão da iNOS. Na presença do LPS a melatonina bloqueia a indução da translocação nuclear do NF-?B, a expressão da iNOS e a produção de NO. Quando apenas a melatonina foi incubada, observamos uma inibição transiente (15 min.) do NF-?B, seguida por um aumento do conteúdo nuclear do fator de transcrição (60 min.). A expressão da iNOS seguiu o mesmo perfil, ou seja, sofreu uma inibição transiente (30 min.) seguida de um aumento acima do nível basal após 120 minutos de incubação. Portanto, demonstramos que a melatonina afeta de forma diferente a viabilidade de células granulares de cerebelo dependo do contexto em que as células se encontram. Além disso, obtivemos evidências de que essas células expressam a enzima a AA-NAT, e produzem melatonina, que exerce função protetora para a cultura. Desta forma, nossos dados proporcionam uma base mecanicista para a compreensão da influência do contexto celular na resposta à melatonina / Several neurons constitutively express NF-?B, which plays some physiological roles, besides the well-known control of pathological responses. Melatonin, the hormone produced by the pineal gland rhythmically in the dark phase is also an autocrine and paracrine factor of immune competent cells, involved in multiple biological processes and the cytoprotective action is a highlight of this molecule. Melatonin inhibits the nuclear translocation of NF-?B and the expression of iNOS in models of cell damage. The present study evaluated whether the cytoprotective effect of melatonin depends on the state of activation of NF-?B in cultured cerebellar granule cells, given that these cells have a basal activity of this transcription factor essential for cell survival. Moreover, we questioned whether these cells in culture produce melatonin and whether it would have a cytoprotective role. We tested the viability of the rat (7-8 days old Wistar) cerebellar granule cell culture after 24 h incubation with melatonin in the presence or absence of LPS. In basal condition melatonin decreased cell survival while inhibited cell death induced by LPS. These effects were consistent with the results from the activation of NF-?B and the expression of iNOS. In the presence of LPS melatonin blocked the activation of the NF-?B , the expression of iNOS and the production of NO. When only melatonin was incubated, we observed a transient reduction (15 min) of NF-?B nuclear content, followed by an increase of its nuclear content (60 min). The iNOS expression followed the same profile, i.e. undergone a transient inhibition (30 min), followed by an increase above baseline after 120 min of incubation. Therefore, we have demonstrated that melatonin affects differently the viability of cerebellar granule cells depending on the context. Furthermore, we founded evidences that the granule cells in culture express the key enzyme in the synthesis of melatonin, AA-NAT and produce melatonin, which carries protective function for the culture. Our data provide a mechanistic basis for understanding the influence of cell context on the final output response to melatonin

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

La cellule de Purkinje constitue la seule sortie du cortex cérébelleux. En étudiant les caractéristiques de sa décharge spontanée sur l’animal éveillé, nous pouvons avoir un aperçu de l’intégration par le cortex cérébelleux de ses deux entrées excitatrices, les fibres moussues et les fibres grimpantes. Les souris transgéniques constituent une opportunité réelle de mieux comprendre le fonctionnement et les dysfonctionnements du cortex cérébelleux. Dans ce travail, nous décrivons la décharge spontanée des cellules de Purkinje chez l’animal normal et dans différents modèles de souris ataxiques. Nous avons ainsi mis en évidence différents patterns d’activité correspondant à différents degrés d’ataxie. Ainsi, sur les souris consommant chroniquement de l’éthanol et sur les souris SCA1, qui présentent un très léger trouble de la coordination motrice, nous avons trouvé une fréquence de décharge diminuée des spikes simples et des spikes complexes, sans augmentation de la rythmicité ni émergence d’oscillation du potentiel de champ de local. Les souris déficientes en calbindine, en calrétinine, en parvalbumine, en Ube 3A maternelle, ou ayant subi in utero un syndrome d’alcoolisme fœtal présentent un trouble de la coordination plus net, mais nécessitant toujours des tests adaptés pour être mis en évidence. Ces souris présentent une oscillation rapide soutenue par la décharge rythmique et synchrone des cellules de Purkinje. Cette oscillation est synchronisée dans l’axe des fibres parallèles, et est inhibée par les inhibiteurs des gap junctions, des récepteurs GABAA et NMDA. Par contre, les souris BK-/- dont l’ataxie est évidente même en conditions standards, présentent une oscillation lente synchronisée dans les axes sagittal et frontal, en phase avec les bursts des cellules de Purkinje et avec la décharge des cellules de Golgi. L’existence de ces différents patterns qui regroupent des conditions physiopathologiques qui n’ont pas d’autre point commun qu’un même niveau de déficit de la coordination motrice suggère que les troubles cérébelleux puissent être classifiés en un nombre limité de catégories permettant ainsi une approche thérapeutique plus ciblée. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Les ataxies (HCA) et paraparésies spastiques héréditaires constituent les deux extrémités du spectre des entités neurodégénératives spinocérébelleuses (SCE). Elles sont marquées par une forte hétérogénéité clinique, avec des signes associés variés, et génétique. Elles peuvent se transmettre sur tous les modes d'hérédité, et des mutations ont été décrites dans une myriade de gènes. Les SCE sont donc une entité qui bénéficie particulièrement des avancées technologiques de la Nouvelle Génération de Séquençage. Ce travail décrit des résultats obtenus sur de grandes cohortes, par séquençage de panel de gènes ciblés ou de l'exome entier, ainsi que des études de familles. Celles-ci nous ont permis de décrire de nouveaux modes de transmission de mutations dans des gènes déjà connus en pathologie humaine, avec un dans un cas une dysfonction similaire, dans l'autre un gain versus une perte de fonction. Nous rapportons aussi deux gènes nouvellement impliqués, dans une forme autosomique dominante de HCA (CACNA1A), et dans un sous-type autosomique récessif de dystonie avec atrophie cérébelleuse (TOR1AIP1). Nos résultats illustrent bien la refonte nosologique en marche dans les maladies génétiques complexes, qui remettent en permanence les corrélations génotype-phénotype en question. Nous discutons du pourquoi et du comment du diagnostic moléculaire dans cette nouvelle ère du séquençage. / Hereditary cerebellar ataxias (HCA) and spastic paraplegias constitute both ends of the neurodegenerative spectrum of spinocerebellar entities (SCE). Theses diseases are marked by a pronounced heterogeneity, both clinically, with various additional neurological or extraneurological signs, and genetically. They can indeed follow all transmission modes, and mutations in a myriad of genes have been described. SCE is hence a group of diseases that benefit greatly from Next-Generation Sequencing technologies. This work reports both screenings of large cohorts of patients with either panel or whole exome sequencing, as well as family studies. The latter allowed us to describe new modes of transmission for genes previously involved in human pathology, with either similar protein dysfunction, or loss- versus gain-of-function. We also describe two new genes implicated in a form of autosomal dominant HCA (CACNA1A), and an autosomal recessive subtype of dystonia and cerebellar atrophy (TOR1AIP1). Our results are illustrative of the genetic remodelling underway in complex genetic diseases, with permanent questioning of genotype-phenotype correlations. We discuss the how and the why of molecular diagnosis in this new era of sequencing.

Le contrôle postural fait intervenir l’intégration cérébelleuse de différentes entrées sensorielles (le vestibule, la vision, la somesthésie). Nous avons évalué le contrôle postural à l’aide de différents dispositifs : la plateforme Techno Concept®, le Multitest Framiral® et les mouvements oculaires avec l’oculomètre : le Mobile e(ye)BRAIN T2® chez des participants contrôles et des enfants présentant des déficits neuro-développementaux, troubles appartenant au spectre autistique et dyslexie. Au cours de nos travaux, nous avons décrit un aspect développemental physiologique des indices posturaux et oculomoteurs et également un déficit de ces indicateurs auprès d’enfants avec retards d’apprentissage. En effet, chez ces enfants, la stabilité posturale est déficitaire et les stratégies d’exploration sont différentes par rapport aux contrôles. Nous avons basé nos hypothèses sur le fait que ce déficit serait du à un défaut d’utilisation des informations sensorielles ainsi qu’à une mauvaise intégration cérébelleuse. Les résultats de nos études nous permettent de mieux préciser les caractéristiques neuropsychologiques impliquant les fonctions cérébelleuses chez ces enfants afin de pouvoir suggérer des prises en charge thérapeutiques multimodales capables d’entrainer simultanément plusieurs fonctions. Notre objectif final est de pouvoir identifier une médiation thérapeutique spécifique pour ces enfants. / Postural control involves cerebellar integration of several sensory inputs (vestibular, visual and somesthesic). We evaluated postural control with force plateform: Techno Concept®, Multitest Framiral® and the eye movements with the Mobile e(ye)BRAIN T2® in healthy children population as well as in children with developmental disorders (autistic spectrum desorders and dyslexia). The results of our studies showed a developmental aspect of postural and oculomotor factors in healthy children and a deficit of both postural and oculomotor control in children with autistic spectrum desorders and with dyslexia. Indeed, in these children postural stability is poor and visual strategy is different with respect to healthy children. Our hypothesis is that these deficits could be due to a lack in using appropriately sensory inputs and of their integration via cerebellar activity. The importance of these studies is to better understand the involvement of cerebellar function in such developmental disorders. Our final goal is to suggest new training tecniques to use sensory inputs more efficiently in these children with developmental disorders.

Résumé : Introduction : L’ataxie récessive spastique de Charlevoix-Saguenay (ARSCS) est une maladie neuromusculaire héréditaire rare qui affecte notamment les voies spinocérébelleuses. Elle est caractérisée par une triple composante de signes et symptômes incluant l’ataxie et la dysarthrie (atteintes cérébelleuses), la spasticité aux membres inférieurs (atteintes pyramidales) et une faiblesse distale qui engendre des difficultés de préhension (atteintes neuropathiques). Des avancées récentes permettent de croire que des essais thérapeutiques seront bientôt possibles. Dans ce contexte, il est nécessaire de développer une échelle de gravité de la maladie pour permettre la sélection des patients et la documentation de l’histoire naturelle. But : L’objectif de ce projet était de développer les items de la section cérébelleuse du Disease Severity Index for Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (DSI-ARSACS) et de documenter ses qualités métrologiques. Méthode : La section cérébelleuse a été élaborée à l’aide du modèle de développement de Streiner et Norman (2008) qui comprend trois grandes étapes : planification, construction et validation. La planification et la construction ont été réalisées à l’aide de consultations d’experts par la méthode Delphi et d’une recension systématique des écrits. La validité de construit (convergente et discriminatoire) et la fidélité (intra-évaluateur et inter-évaluateur) ont été documentées. Vingt-huit participants ont été recrutés selon un échantillonnage stratifié pour l’âge et le sexe. Ils devaient avoir un diagnostic confirmé génétiquement et être âgés entre 18 ans et 59 ans. La validité convergente a été documentée avec des outils évaluant les aptitudes motrices des membres supérieurs (9HPT, PPT, TDNS), la gravité de l’ataxie (SARA), la mobilité (6MWT, 10mWT, échelle de Berg), le fonctionnement dans les activités quotidiennes (Index de Barthel), la participation sociale (MHAVIE) et la qualité de vie (SF-12v2). La validité discriminante a été documentée selon le sexe, le groupe d’âge et le stade de la maladie. La section cérébelleuse a été appliquée à trois reprises par deux physiothérapeutes à deux semaines d’intervalle pour évaluer la fidélité intra et inter-évaluateurs. Résultats : La section cérébelleuse comporte 6 items d’évaluation liés aux fonctions motrices du cervelet. Le sous total de la section cérébelleuse est fortement corrélé avec la majorité des outils d’évaluation (r ≥ 0,69, p = 0,00), à l’exception du SF-12v2 (r ≤ 0,36, p ≥ 0,06). Une différence significative (p < 0,00) a été démontrée entre chaque groupe d’âge pour la majorité des items et le sous-total de la section cérébelleuse de l’échelle. Le résultat de chaque item et le sous-total de la section cérébelleuse augmentent significativement avec le stade de la maladie (p < 0,00). La fidélité intra et inter-évaluateurs correspond à un accord fort (κ ≥ 0,69) pour la majorité des items et le sous-total. Conclusion : Ce projet a permis de développer la section cérébelleuse de l’échelle de gravité DSI-ARSACS avec de bonnes qualités psychométriques (validité et fidélité). L’échelle pourra être utilisée dans le processus de catégorisation et d’évaluation des participants pour de futurs essais thérapeutiques. / Abstract : Introduction: Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is a rare hereditary neuromuscular disease that mainly affects the spinocerebellar tract. It is characterized by a triad of signs and symptoms, including ataxia and dysarthria (cerebellum impairment), spasticity in the lower limbs (pyramidal impairment) and a distal weakness leading to prehension difficulties (neuropathic impairment). Recent research advances suggest that clinical trials may soon become possible. In this context, it is necessary to develop a disease severity index in order to select patients and document natural history of disease. Objective: The project aimed to develop the items of the cerebellar section of the Disease Severity Index for Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay DSI-ARSACS and document their metrological properties. Method: The cerebellar section was developed using the Streiner and Norman (2008) model, which involves three stages: planning, construction and validation. The planning and construction stages were conducted by carrying out a literature review, obtaining expert opinions and completing a Delphi process. Construct validity (convergent and discriminant) and reliability (intra- and inter-raters) were documented. Twenty-eight participants between the ages of 18 and 59 were recruited using a stratified sampling method based on age and gender. All of them had a genetically confirmed diagnosis of ARSACS. Convergent validity was documented with measurements of upper limbs motor abilities (9HPT, PPT, TDNS), ataxia severity (SARA), mobility (6MWT, 10mWT, Berg scale), functional status in activities of daily living (Barthel index), social participation (MHAVIE) and quality of life (SF-12v2). Discriminant validity was documented according to gender, age group and disease stage. The cerebellar section was administered three times, two weeks apart, by two physiotherapists to assess intra- and inter-rater reliability. Results: The cerebellar section includes 6 assessment items linked to cerebellum motor functions. The cerebellar section subscore was strongly correlated with the majority of assessment tools (r ≥ 0.69, p = 0.00), excluding SF 12v2 (r ≤ 0,36, p ≥ 0,06). A significant difference (p < 0.00) was observed between each age group for most items and the subscore of the cerebellar section. Results on each item and the subscore significantly increase with disease stage (p < 0.00). Intra- and inter-rater reliability reflects a strong level of agreement (κ ≥ 0.69) on the majority of items and the subscore. Conclusion: The cerebellar section of the DSI-ARSACS shows good psychometric properties (validity and reliability). The index can be used to categorize and assess future participants in clinical trials.

La maladie de Parkinson (MP) est la deuxième maladie neurodégénérative la plus commune. Les symptômes principalement observés chez les patients atteints de la MP sont la rigidité, les tremblements, la bradykinésie et une instabilité posturale. Leur sévérité est souvent asymétrique. La cause principale de ces symptômes moteurs est la dégénérescence du circuit dopaminergique nigro-striatal qui mène à un débalancement d’activité du circuit cortico-striatal. Ce débalancement de circuits est le point essentiel de cette thèse. Dans les protocoles de recherche décrits ici, des patients atteints de la MP (avant et après une dose de levodopa) et des participants contrôles sains ont effectué des mouvements auto-initiés ou en réponse à des stimulis externes pendant que l’on mesurait leur activité cérébrale en imagerie par résonance magnétique fonctionnelle (IRMf). Dans cette thèse, nous abordons et mettons en évidence quatre (4) points principaux. En première partie (chapitre 2), nous présentons un recensement de la littérature sur les cicruits cortico-striataux et cortico-cérébelleux dans la MP. En utilisant des méthodes de neuroimagerie, des changements d’activité cérébrale et cérébelleuse ont été observés chez les patients atteints de la MP comparés aux participants sains. Même si les augmentations d’activité du cervelet ont souvent été attribuées à des mécanismes compensatoires, nos résultats suggèrent qu’elles sont plus probablement liées aux changements pathophysiologiques de la MP et à la perturbation du circuit cortico-cérébelleux. En général, nous suggérons (1) que le circuit cortico-cérébelleux est perturbé chez les patients atteints de la MP, et que les changements d’activité du cervelet sont liés à la pathophysiologie de la MP plutôt qu’à des mécanismes compensatoires. En deuxième partie (chapitre 3), nous discutons des effets de la levodopa sur les hausses et baisses d’activité observés chez les patients atteints de la MP, ainsi que sur l’activité du putamen pendant les mouvements d’origine interne et externe. De nombreuses études en neuroimagerie ont montré une baisse d’activité (hypo-activité) préfrontale liée à la déplétion de dopamine. En revanche, l’utilisation de tâches cognitives a montré des augmentations d’activité (hyper-activité) corticale chez les patients atteints de la MP comparés aux participants sains. Nous avons suggéré précédemment que ces hypo- et hyper-activités des régions préfrontales dépendent de l’implication du striatum. Dans cette thèse nous suggérons de plus (2) que la levodopa ne rétablit pas ces hyper-activations, mais plutôt qu’elles sont liées à la perturbation du circuit méso-cortical, et aussi possiblement associées à l’administration de médication dopaminergique à long terme. Nous montrons aussi (3) que la levodopa a un effet non-spécifique à la tâche sur l’activité du circuit cortico-striatal moteur, et qu’elle n’a pas d’effet sur l’activité du circuit cortico-striatal cognitif. Nous montrons enfin (chapitre 4) que la levodopa a un effet asymétrique sur les mouvements de la main droite et gauche. À peu près 50% des patients atteints de la MP démontrent une asymétrie des symptômes moteurs, et ceci persiste à travers la durée de la maladie. Nos résultats suggèrent (4) que la levodopa pourrait avoir un plus grand effet sur les patrons d’activations des mouvements de la main la plus affectée. / Parkinson’s disease (PD) is the second most common neurodegenerative disease, mainly manifested by tremor, rigidity, bradykinesia and postural instability, and often an asymmetry of symptom severity of the left and right sides of the body. The depletion of dopamine of the nigrostriatal pathway is the primary cause of the motor symptoms observed in patients with PD, leading to an imbalance in basal-ganglia prefrontal circuits. In the protocols described here, patients with PD before and after levodopa administration and healthy participants performed self-initiated (SI) and externally triggered (ET) movements with the left and right hand during functional magnetic resonance imaging (fMRI). In the chapters of this thesis, we argue and provide evidence for four main points. The first portion (chapter 2) provides a literature review on cortico-striatal and cortico-cerebellar circuit disruption in PD. Using neuroimaging techniques, changes in cerebral and cerebellar activity have been observed in patients with PD compared with healthy participants. Although increases in activity in the cerebellum have often been interpreted as compensatory mechanisms, we provide evidence that they are more likely to be related to pathophysiological changes of the disease, and the disruption of the cortico- cerebellar circuit. In general, we argue (1) is that activity in the cerebellum is linked to the pathophysiology of PD. In the second section (chapter 3) we discuss the effect of levodopa on the patterns of cortical hypo- and hyper-activity in PD, as well as the activity of the putamen in SI and ET movements. Many studies have shown cortical hypo-activity in relation to nigrostriatal dopamine depletion. In contrast, some cognitive studies have also identified increases in cortical activity in patients with PD as compared with healthy control participants. We have previously suggested that cortical hypo- and hyper-activations depend on striatal recruitment. In this thesis, we further show that hyper-activations in the prefrontal cortex are not reestablished with levodopa administration. We suggest (2) that they are rather associated with mesocortical dopamine circuit dysfunction, and perhaps linked with long- term dopaminergic medication administration. Furthermore, we show (3) that levodopa has a non-task specific effect on the motor cortico-striatal loop, but does not affect the cognitive cortico-striatal circuit. Finally (chapter 4), we show that the effect of levodopa on movements of the left and right hands is not symmetrical. Previous studies have shown that in about 50% of patients, one side of the body is more severely affected, and this asymmetry persists throughout the duration of the disease. Our results suggest (4) that levodopa may have stronger effects on the cerebral hemodynamic patterns related to the movements of the more affected hand than on those of the less affected hand.

La maladie de Parkinson (MP) est la deuxième maladie neurodégénérative la plus commune. Les symptômes principalement observés chez les patients atteints de la MP sont la rigidité, les tremblements, la bradykinésie et une instabilité posturale. Leur sévérité est souvent asymétrique. La cause principale de ces symptômes moteurs est la dégénérescence du circuit dopaminergique nigro-striatal qui mène à un débalancement d’activité du circuit cortico-striatal. Ce débalancement de circuits est le point essentiel de cette thèse. Dans les protocoles de recherche décrits ici, des patients atteints de la MP (avant et après une dose de levodopa) et des participants contrôles sains ont effectué des mouvements auto-initiés ou en réponse à des stimulis externes pendant que l’on mesurait leur activité cérébrale en imagerie par résonance magnétique fonctionnelle (IRMf). Dans cette thèse, nous abordons et mettons en évidence quatre (4) points principaux. En première partie (chapitre 2), nous présentons un recensement de la littérature sur les cicruits cortico-striataux et cortico-cérébelleux dans la MP. En utilisant des méthodes de neuroimagerie, des changements d’activité cérébrale et cérébelleuse ont été observés chez les patients atteints de la MP comparés aux participants sains. Même si les augmentations d’activité du cervelet ont souvent été attribuées à des mécanismes compensatoires, nos résultats suggèrent qu’elles sont plus probablement liées aux changements pathophysiologiques de la MP et à la perturbation du circuit cortico-cérébelleux. En général, nous suggérons (1) que le circuit cortico-cérébelleux est perturbé chez les patients atteints de la MP, et que les changements d’activité du cervelet sont liés à la pathophysiologie de la MP plutôt qu’à des mécanismes compensatoires. En deuxième partie (chapitre 3), nous discutons des effets de la levodopa sur les hausses et baisses d’activité observés chez les patients atteints de la MP, ainsi que sur l’activité du putamen pendant les mouvements d’origine interne et externe. De nombreuses études en neuroimagerie ont montré une baisse d’activité (hypo-activité) préfrontale liée à la déplétion de dopamine. En revanche, l’utilisation de tâches cognitives a montré des augmentations d’activité (hyper-activité) corticale chez les patients atteints de la MP comparés aux participants sains. Nous avons suggéré précédemment que ces hypo- et hyper-activités des régions préfrontales dépendent de l’implication du striatum. Dans cette thèse nous suggérons de plus (2) que la levodopa ne rétablit pas ces hyper-activations, mais plutôt qu’elles sont liées à la perturbation du circuit méso-cortical, et aussi possiblement associées à l’administration de médication dopaminergique à long terme. Nous montrons aussi (3) que la levodopa a un effet non-spécifique à la tâche sur l’activité du circuit cortico-striatal moteur, et qu’elle n’a pas d’effet sur l’activité du circuit cortico-striatal cognitif. Nous montrons enfin (chapitre 4) que la levodopa a un effet asymétrique sur les mouvements de la main droite et gauche. À peu près 50% des patients atteints de la MP démontrent une asymétrie des symptômes moteurs, et ceci persiste à travers la durée de la maladie. Nos résultats suggèrent (4) que la levodopa pourrait avoir un plus grand effet sur les patrons d’activations des mouvements de la main la plus affectée. / Parkinson’s disease (PD) is the second most common neurodegenerative disease, mainly manifested by tremor, rigidity, bradykinesia and postural instability, and often an asymmetry of symptom severity of the left and right sides of the body. The depletion of dopamine of the nigrostriatal pathway is the primary cause of the motor symptoms observed in patients with PD, leading to an imbalance in basal-ganglia prefrontal circuits. In the protocols described here, patients with PD before and after levodopa administration and healthy participants performed self-initiated (SI) and externally triggered (ET) movements with the left and right hand during functional magnetic resonance imaging (fMRI). In the chapters of this thesis, we argue and provide evidence for four main points. The first portion (chapter 2) provides a literature review on cortico-striatal and cortico-cerebellar circuit disruption in PD. Using neuroimaging techniques, changes in cerebral and cerebellar activity have been observed in patients with PD compared with healthy participants. Although increases in activity in the cerebellum have often been interpreted as compensatory mechanisms, we provide evidence that they are more likely to be related to pathophysiological changes of the disease, and the disruption of the cortico- cerebellar circuit. In general, we argue (1) is that activity in the cerebellum is linked to the pathophysiology of PD. In the second section (chapter 3) we discuss the effect of levodopa on the patterns of cortical hypo- and hyper-activity in PD, as well as the activity of the putamen in SI and ET movements. Many studies have shown cortical hypo-activity in relation to nigrostriatal dopamine depletion. In contrast, some cognitive studies have also identified increases in cortical activity in patients with PD as compared with healthy control participants. We have previously suggested that cortical hypo- and hyper-activations depend on striatal recruitment. In this thesis, we further show that hyper-activations in the prefrontal cortex are not reestablished with levodopa administration. We suggest (2) that they are rather associated with mesocortical dopamine circuit dysfunction, and perhaps linked with long- term dopaminergic medication administration. Furthermore, we show (3) that levodopa has a non-task specific effect on the motor cortico-striatal loop, but does not affect the cognitive cortico-striatal circuit. Finally (chapter 4), we show that the effect of levodopa on movements of the left and right hands is not symmetrical. Previous studies have shown that in about 50% of patients, one side of the body is more severely affected, and this asymmetry persists throughout the duration of the disease. Our results suggest (4) that levodopa may have stronger effects on the cerebral hemodynamic patterns related to the movements of the more affected hand than on those of the less affected hand.

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.