La contamination des ressources en eau potable et des terres agricoles par les pesticides qui entrent dans la chaine alimentaire est un problème environnemental et sanitaire majeur. Des études épidémiologiques ont montré un lien entre l’exposition aux pesticides et la maladie de Parkinson, mais peu d’études sont disponibles sur les autres pathologies neurodégénératives. Notre hypothèse est que les pesticides pourraient être des facteurs déclencheurs ou aggravants, communs aux maladies neurodégénératives.Au laboratoire, nous avons identifié par criblage sur la protéine prion, PrP, un composé nommé A6 décrit pour ses propriétés herbicides. A6 est un dérivé de l’α-terthiényl, molécule naturelle extraite des œillets d’inde. Le composé A6 a la capacité de promouvoir des formes oligomériques de la protéine prion sur une lignée cellulaire infectée par des prions. Mon projet de recherche visait à étudier les effets du bio-herbicide A6 sur l’agrégation et la propagation des prions in vivo. Des souris infectées par les prions ont été traitées avec différentes doses de cette molécule (5, 10 et 20 mg/kg). A la dose de 5 mg/kg, le composé A6 diminue le temps de survie des animaux avec une augmentation de la charge amyloïde. Tandis qu’aux plus fortes doses le traitement par A6 augmente la survie des souris avec une diminution de la charge amyloïde. Par un test de centrifugation rapide (RCA), nous avons montré que les faibles concentrations d’A6 favorisent la formation d’oligomères solubles de PrPSc SDS résistants (rSDS-PrPSc), alors que les plus fortes concentrations induisent des formes insolubles. L’analyse des cerveaux montre que seules les souris traitées avec 10 et 20 mg/kg d’A6 présentent des formes dimériques de rSDS-PrPSc. Cette étude montre une dualité d’effet du composé A6 : à de faibles doses, il favorise la pathologie probablement via des formes oligomériques solubles favorisant la réplication des prions. A de plus fortes doses, le composé A6 piègerait une partie de l’infectiosité des prions sous forme d’agrégats amorphes SDS résistants, bloquant la réplication des prions, et ayant un effet « protecteur ».Une recherche d’analogues structurels au composé A6 a permis l’identification d’une classe d’antifongiques commercialisés : les anilinopyrimidines. Cette famille est composée de 3 molécules : cyprodinil, mépanipyrim et pyriméthanil, qui luttent contre des champignons responsables de la pourriture grise des fruits. L’analyse de nombreux rapports a révélé que nous sommes chroniquement exposés à des résidus d’anilinopyrimidines. Dans cette deuxième étude, nous avons étudié l’effet de ces 3 fongicides dans plusieurs modèles de la maladie d’Alzheimer (MA) et les conséquences sur les marqueurs de la pathologie. Des études d’incubation ex vivo associées à des expériences de cinétique d’agrégation de peptides Aβ1-42 ont montré que ces composés interagissent directement avec les peptides et accélèrent sa cinétique d’agrégation. Afin de déterminer leurs effets in vivo, nous avons exposé chroniquement des souris J20 à un cocktail des 3 fongicides via l’eau de boisson. Les souris ont été traitées à 0,1 μg/L (0,44 nM) de chaque composé, correspondant à la concentration maximale autorisée dans l’eau potable. Après 9 mois de traitement, l’analyse des souris J20 a montré une augmentation du nombre et de la surface des plaques amyloïdes au niveau de l’hippocampe et du cortex. Pour déterminer à quel moment se produit l’effet proagrégant du cocktail, une étude longitudinale d’apparition des agrégats à 3, 6 et 9 mois par microscopie biphotonique, a montré que les plaques amyloïdes augmentent entre 6 et 9 mois et que les pesticides exacerbent les agrégats vasculaires. Les anilinopyrimidines modifient la production et la clairance des peptides Aβ en augmentant l’expression de BACE1 et en diminuant l’expression de la néprilysine. Nos travaux montrent que les antifongiques aggravent la MA. / Contamination of the drinking water and agricultural lands by the use of pesticides entering into the food chain is a major environmental and health problem. Epidemiological studies have shown a link between pesticide exposure and Parkinson's disease, but few studies are available on other neurodegenerative disorders. Our hypothesis is that pesticides may be triggering or aggravating factors common to neurodegenerative diseases.In the laboratory, we identified by a screening on the prion protein, PrP, a compound named A6 described for its herbicidal properties. A6 is a derivative of α-terthienyl, a natural molecule extracted from marigolds. A6 compound has the ability to promote oligomeric forms of the prion protein on a prion-infected cell line. My research project aimed to study the effects of the bioherbicide A6 on prion aggregation and propagation in vivo. Mice infected with prions were treated with different doses of this molecule (5, 10 and 20 mg/kg). At the dose of 5 mg/kg, A6 compound decreases the survival time of animals with an increase of the amyloid load. While at the higher doses, A6 treatment increases survival of mice with a decreased amyloid burden. Using a rapid centrifugation assay (RCA), we have shown that low concentrations of A6 promote soluble SDS-resistant oligomers of PrPSc (rSDS-PrPSc), while higher concentrations favour insoluble forms. Brain analysis shows that only mice treated with 10 and 20 mg/kg of A6 exhibit dimeric forms of rSDS-PrPSc. This study shows a dual effect of A6 compound: at low doses, it strenghtens the pathology probably via soluble oligomeric forms favouring the replication of prions. At higher doses, A6 compound would trap part of the infectivity of prions as SDS resistant amorphous aggregates, blocking prion replication, and thus having a “protective” effectA search for structural analogs of A6 compound allowed the identification of a class of commercial antifungals: anilinopyrimidines. This family is composed of 3 molecules: cyprodinil, mepanipyrim and pyrimethanil, used to fight against fungi responsible for the gray mold of fruits. Analysis of many reports have revealed that we are chronically exposed to residues of anilinopyrimidines. In this second study, we evaluated the impact of the 3 fungicides in several models of Alzheimer’s disease (AD) and their consequences on pathological markers. Ex vivo incubation studies associated to kinetics of fibril formation of Aβ1-42 peptides have shown that these compounds interact directly with Aβ peptides and accelerate its kinetics of aggregation. To determine their effects in vivo, we chronically exposed J20 mice to a cocktail of the 3 fungicides through drinking water. Mice were treated with 0.1 μg/L (0.44 nM) of each compound, corresponding to the maximal concentration allowed in the tap water. After 9 months of treatment, analysis of J20 mice showed an increase in the number and surface of plaques in the hippocampus and cortex. To determine the moment when the pro-aggregative effect occurs, a longitudinal study of appearance of aggregates at 3, 6 and 9 months by 2-photon microscopy was done. Our results showed that amyloid plaques increase between 6 and 9 months, and exacerbate vascular amyloid aggregates. Anilinopyrimidines modified the production and clearance of Aβ peptides by increasing BACE1 expression and by decreasing neprilysin expression. Our researches show a role of fungicides in the aggravation of AD.

As doenças neurodegenerativas (DN) estão entre as principais causas de mortalidade e morbidade nos países ocidentais. Não há ainda um tratamento definitivo para estas neuropatias, mas os estudos têm indicado mecanismos comuns de toxicidade que incluem disfunção mitocondrial, estresse oxidativo e apoptose. Assim, as mitocôndrias constituem alvos importantes para futuras estratégias de neuroproteção a fim de tratar, prevenir ou até mesmo retardar a neurodegeneração. Neste contexto, o canabidiol (CBD), um constituinte não psicoativo da Cannabis sativa e cuja propriedade neuroprotetora tem sido sugerida por diferentes estudos, surge como uma alternativa bastante promissora. Diferentes mecanismos moleculares podem estar envolvidos na neuroproteção exercida pelo CBD. Embora o potencial efeito benéfico do canabidiol com relação às doenças neurodegenerativas já tenha sido sugerido, não há ainda estudos que abordem precisamente os mecanismos de proteção contra a toxicidade mitocondrial cerebral, evento chave no processo neurodegenerativo. O presente estudo teve como objetivo investigar os efeitos do CBD em mitocôndrias cerebrais de rato, bem como possíveis mecanismos de neuroproteção. Foram avaliados os seguintes parâmetros: função mitocondrial, estresse oxidativo mitocondrial e transição de permeabilidade de membrana mitocondrial (TPMM). Os resultados obtidos sugerem que o canabidiol é capaz de proteger as mitocôndrias cerebrais contra o intumescimento osmótico induzido por cálcio/fosfato, contra a produção de H2O2 induzida por terc-butil hidroperóxido e contra a peroxidação lipídica induzida por Fe2+ e citrato. A captação mitocondrial de cálcio e a capacidade fosforilativa não foram afetadas. / Neurodegenerative diseases (ND) are among the leading causes of mortality and morbidity in Western countries. There is not a definitive treatment for these neuropathies, but studies have indicated mechanisms of toxicity which include mitochondrial dysfunction, oxidative stress and apoptosis. Therefore, mitochondria are important targets for future neuroprotective strategies to treat, prevent or even slow the neurodegeneration. In this context, cannabidiol (CBD), a constituent of non-psychoactive Cannabis sativa and whose neuroprotective property has been suggested by different studies, emerges as a promising alternative. Different molecular mechanisms may be involved in the neuroprotection exerted by CBD. Although the potential beneficial effects of cannabidiol in relation to neurodegenerative diseases has already been suggested, there are no studies addressing specifically the mechanisms of protection against mitochondrial toxicity brain, a key event in the neurodegenerative process. This study aimed to investigate the effects of CBD on rat brain mitochondria, as well as the mechanisms of neuroprotection. The following parameters were evaluated: mitochondrial function, mitochondrial oxidative stress and permeability transition of the mitochondrial membrane (MPT). The results suggest that cannabidiol can protect brain mitochondria against: the osmotic swelling induced by calcium/phosphate, the production of H2O2 induced by tert-butyl hydroperoxide and the lipid peroxidation induced by Fe2+ and citrate. The mitochondrial calcium uptake and phosphorylative capacity were not affected.

No presente trabalho foram avaliados os efeitos da estimulação em ambiente enriquecido (AE) para a memória e para os marcadores neuropatológicos da Doença de Alzheimer (DA) em camundongos transgênicos (TG) que superexpressam a proteína precursora de amilóide humana. Nos animais TG ou C57Bl/6, estimulados dos 3 aos 7 meses de idade, o AE promoveu manutenção da memória relacionada a estímulo aversivo por até 2 meses. Em animais mais velhos (8 a 12 meses de idade), a manutenção da memória foi verificada, independente do estímulo. Em ambas as idades, a memória espacial não foi alterada com o AE. Não houve alteração do número de placas senis nos animais mais jovens. Porém, em TG de 12 meses estimulados, houve redução de 85% na densidade de placas no hipocampo quando comparados ao grupo TG controle. Nessa idade, porém, não foi possível identificar a formação de enovelamentos neurofibrilares. Em conclusão, a estimulação crônica em ambiente enriquecido contribuiu para a formação de reservas cognitivas, protetoras no caso de doenças neurodegenerativas como a DA. / In the present work the effects of chronic stimulation in enriched envitonment (EE) for memory and neuropathological hallmarks of the Alzheimer\'s disease (AD) were evaluated in transgenic mice (TG) that overexpress the human amyloid precursor protein. In TG or C57Bl/6 animals, stimulated from 3 to 7 months of age, submition in EE kept fear conditioning memory for as long as two months. In older mice (8 to 12 months of age) memory keeping was verified, independent of the stimulus. In both ages, spatial memory was not altered with EE. There was no difference in senile plaques density in younger animals. However, in stimulated 12-months-old animals, there was a reduction of 85% in the density of senile plaques in hippocampus, when compared to TG control animals. In this age, however, it was not possible to identify neurofibrilary tangles. In conclusion, chronic stimulation in enriched environment contributed to formation of cognitive reserves, protective in the presence of neurodegenerative diseases, as AD.

Le système nerveux consiste en plusieurs types cellulaires quels interagissent avec eux-mêmes pour conduire des potentiels d’action du soma au travers l’axone vers la synapse. Dans les nerfs périphériques, les cellules de Schwann interagissent avec les neurones par enrouler autour l’axone et créer une gaine de myéline. Cette gaine de myéline permit une conduction rapide des potentiels d’action de nœud de Ranvier vers nœud de Ranvier, quels sont petits régions non-myélinisés de l’axone. En plus, les cellules de Schwann transportent du lactate vers le neurone et le neurone utilise ce lactate pour la production d’énergie sous la forme d’ATP. Cette production est nécessaire, parce que beaucoup de processus dans des cellules, comme la conduction des potentiels d’action, ont besoin d’ATP. Trois mécanismes sont impliqués à la production d’ATP : la glycolyse dans le cytosol et le cycle de Krebs et la chaîne de transport d'électrons dans les mitochondries. Par contre, la production d’ATP par des mitochondries résulte en la production du dérivés réactifs de l'oxygène (DRO), quels provoquent du stress oxydative. DRO peuvent être présent en plusieurs formes et ces formes différentes ont des traits spécifiques, mais tous les formes peuvent endommager la cellule. Par exemple, l’ion superoxyde est très réactif donc ils agissent extrêmement vite avec les molécules dans son environnement. Par contre, peroxyde d'hydrogène est un type de DRO quel est moins réactif, mais peut diffuser à plus longue distances et peut oxyder des cibles plus distales. Heureusement, les cellules sont équipées avec un système antioxydant compétant en consistent une groupe d’enzymes antioxydant, quels réduisent du DRO à l’eau. Si les mitochondries dysfonctionnent ou si l’équilibre entre DRO et antioxydants devient en disbalance, des neuropathies pourraient se développer, comme sclérose latérale amyotrophique (SLA), sclérose en plaques (SEP), maladie d'Alzheimer et maladie de Parkinson. Au SNP, des neuropathies périphériques comme la maladie Charcot-Marie-Tooth (CMT) peuvent se développer en raison d’excès du DRODans cette thèse, je présente un modèle élaboré pour la production d’ATP et DRO par les mitochondries in vivo. Je montrerai comment les cellules de Schwann utilisent l’effet Warburg, la transition de métabolisme de phosphorylation oxydative à glycolyse, pour la production de lactate, quel est transporté vers le neurone pour la production d’énergie. Je montrerai aussi que sans l’effet Warburg dans des cellules de Schwann, le métabolisme neuronal est détérioré menant à la production d’ATP diminué, déficits neuronales et des problèmes moteurs. Suite à l’activité nerveuse, la production d’ATP par des mitochondries augmente, mais aussi la production de DRO. Pourtant les deux productions ne sont pas en parallèle. En plus, je montre que la physiologie mitochondriale est affectée par des neuropathies. Aux souris avec défectueux mitofusin2, un modèle pour CMT2A, le contact entre le réticulum endoplasmique et les mitochondries est diminué, à côté d’un changement de morphologie et fonction mitochondriale. En plus, démyélinisation résulte dans des déficits de la production ATP et DRO, montrent une dissociation pathologique entre la production d’ATP et DRO.Pour obtenir ces résultats, des techniques d'imagerie avancées étaient utilisé pour imager les nerfs périphériques des souris transgéniques. Ces transgènes sont introduit dans les souris par injection des vecteurs virales, quels induisent l’expression des sondes fluorescents dans les cellules neuronales. Ces sondes fluorescentes étaient détectées par microscopie multiphotonique. En plus du modèle de la production d’ATP et DRO dans des nerfs périphériques, je présente un protocole pour introduire des vecteurs viraux dans le nerf sciatique. / The nervous system consists of several cell types that interact with each other in order to conduct action potentials from the neuronal soma through axons to the synapse. In peripheral nerves, Schwann cells interact with neurons by wrapping around the axon and creating a myelin sheath. This myelin sheath allows for fast conduction of action potentials from node of Ranvier to node of Ranvier, which are small unmyelinated areas of the axon. In addition, Schwann cells transfer lactate to the neuron, which the axonal mitochondria use to produce energy in the form of ATP. This is necessary, because many cellular processes, such as the conduction of action potentials use ATP. The production of ATP involves three mechanisms: anaerobic glycolysis in the cytosol and the Kreb’s cycle and electron transport chain within mitochondria. However, the production of ATP by mitochondria also results in the production of reactive oxygen species (ROS), which cause cell damage. ROS can be present in several different forms and these different forms have specific properties. For example, superoxide anions are highly reactive and subsequently react rapidly with the molecules in their environment. Hydrogen peroxide on the other hand is less reactive but hence can diffuse over longer distances and react with their targets more distally. Fortunately, the cell contains a competent antioxidant system, which can reduce ROS to water. When mitochondria malfunction or when the equilibrium between ROS and antioxidants becomes in disbalance, neuropathies can develop, such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Alzheimer’s disease or Parkinson’s disease. In the PNS, peripheral neuropathies can develop such as Charcot-Marie-Tooth disease as a result from an excess of ROS.In this thesis, I will provide an elaborate model for ATP and ROS production by axonal mitochondria in vivo. I will show how Schwann cells use the Warburg effect, the shift in metabolism from oxidative phosphorylation to anaerobic glycolysis, to produce lactate, which is then transported to the neuron for energy production. I also demonstrate that without the Warburg effect in Schwann cells neuronal metabolism would be impaired, leading to impaired ATP production, neuronal deficits and motor problems. Following action potential firing, not only ATP is produced by mitochondria, but also ROS, although with independent dynamics. In addition, I show that mitochondrial physiology is affected by several neuropathologies. In mitofusin2 deficient mice, a model for CMT2A, contact between the endoplasmic reticulum and mitochondria is impaired next to affected mitochondrial morphology and function. Also demyelination causes deficits in mitochondrial ATP and ROS production, showing a pathologic decoupling between ATP and ROS.To obtain these results, advanced imaging techniques were used to image peripheral nerves of transgenic mice. These transgenes were introduced in mice via injection of viral vectors which induce expression of fluorescent probes in neuronal cells. These fluorescent probes were detected via multiphoton microscopy. Next to the model for ATP and ROS production in peripheral nerves, I provide a protocol for introducing viral vectors into mouse sciatic nerves.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is an early onset neurodegenerative disorder resulting from mutations in the SACS gene that encodes the protein sacsin. Sacsin is a 520kDa multi-domain protein localised at the cytosolic face of the outer mitochondrial membrane with suggested roles in proteostasis and most recently in the regulation of mitochondrial morphology. An excessively interconnected mitochondrial network was observed as a consequence of reduced levels of sacsin protein following SACS knockdown in neuroblastoma cells as well as in an ARSACS patient carrying the common Quebec homozygous SACS mutation 8844delT. Moreover, it was suggested that sacsin has a role in mitochondrial fission as it was found to interact with mitochondrial fission protein Dynamin related protein 1 (Drp1). The aim of this thesis was to explore sacsin’s role in the regulation of mitochondrial morphology and dynamics in non-Quebec ARSACS patients and sacsin knockdown fibroblasts. This study shows that loss of sacsin function promotes a more interconnected mitochondrial network in non-Quebec ARSACS patients and in sacsin knockdown fibroblasts. Moreover, recruitment of the essential mitochondrial fission protein Drp1 to the mitochondria was significantly reduced in ARSACS patient cells and in sacsin knockdown fibroblasts. This reduced recruitment of Drp1 to mitochondria also occurred when cells were treated to induce mitochondrial fission. Furthermore, both the size and intensity of Drp1 foci localised to the mitochondria were significantly reduced in both sacsin knockdown and patient fibroblasts. Finally, reduced ATP production, decreased respiratory capacity of mitochondria and an increase in mitochondrial reactive oxygen species demonstrated impaired mitochondrial function in ARSACS patient and sacsin knockdown fibroblasts. These results suggest a role for sacsin in the stabilisation or recruitment of cytoplasmic Drp1 to prospective sites of mitochondrial fission similar to that observed by other mitochondrial fission accessory proteins.

多聚谷氨酰胺疾病 (Polyglutamine diseases) 是一類在各自的致病基因編碼區的CAG重複編碼擴張造成的顯性遺傳神經退退化疾病。已擴大的CAG訊息核醣核酸 (Expanded CAG transcripts) 在多聚谷氨酰胺蛋白疾病作出細胞毒性作用。從基因減弱篩查中，我發現U2AF50能修飾已擴大的CAG訊息核醣核酸的毒性。並發現U2AF50能與已擴大的CAG訊息核醣核酸作實體互動，能參與已擴大的CAG訊息核醣核酸的核出口 (Nuclear export)。U2AF50的基因減弱增強已擴大CAG訊息核醣核酸在細胞核的累積和毒性。這突出核醣核酸的核出口在多聚谷氨酰胺疾病的重要性，並暗示細胞核是已擴大的CAG訊息核醣核酸毒性的起源地。此外，我鑑定已擴大的CAG訊息核醣核酸在亞細胞的分佈，並發現它們特別累積在核仁 (Nucleolus) 內。核仁是核糖體核醣核酸（rRNA）的轉錄場所。我發現已擴大的CAG訊息核醣核酸減弱rRNA基因啟動子 (rRNA promoter) 的活性並且抑制核糖體核醣核酸的轉錄。 核糖體核醣核酸基因轉錄的抑制，促進核糖體蛋白RpL23和E3連接酶MDM2蛋白作實體互動，從而增強p53的穩定性導。穩定的p53能夠轉移至線粒體 (Mitochondria)。我還發現，線粒體內的p53能打亂Bcl-xL與 Bak的實體互動，導致細胞色素C釋放到細胞質，這導致凋亡蛋白酶 (Caspases) 的活化和細胞凋亡。我的研究，首次證明核仁參與在多聚谷氨酰胺疾病的發病機制中，揭示了在多聚谷氨酰胺疾病中的新致病機制。 / Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of CAG-repeat encoding glutamine within the coding region of the respective disease genes. Expanded CAG transcripts have been reported to contribute to cytotoxicity in polyQ diseases. From a candidate gene knockdown screen, I identified U2AF50 as a modifier of RNA toxicity. U2AF50 has been reported to be involved in RNA nuclear export, and I showed that it interacted specifically with expanded CAG transcripts. Knockdown of U2AF50 expression enhanced nuclear accumulation of expanded CAG transcripts and neurotoxicity. This part of my work highlights the role of RNA nuclear export in polyQ degeneration and implies that the nucleus is a major site for RNA toxicity. In addition, I determined the subcellular distribution of expanded CAG transcripts and found that they particularly localized in the nucleolus. The nucleolus is a critical sub-nuclear compartment for ribosomal RNA (rRNA) transcription. I discovered that expanded CAG transcripts in nucleolus inhibited rRNA transcription by inactivating the rRNA gene promoter activity. Inhibition of rRNA transcription promoted the interaction between ribosomal protein L23 and the ubiquitin E3 ligase MDM2, which led to the stabilization of p53 and its accumulation in mitochondria. I also found that mitochondrial p53 disrupted the interaction between the anti-apoptotic protein, Bcl-xL, and pro-apoptotic protein, Bak, subsequently causing Cytochrome c release, caspase activation, and apoptosis. In summary, my study first describes the involvement of nucleolar function in polyQ pathogenesis and uncovers a new pathogenic mechanism in polyQ diseases. / Detailed summary in vernacular field only. / Tsoi, Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 220-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis Committee --- p.ii / Declaration --- p.iii / Acknowledgement --- p.iv / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Abbreviations --- p.viii / List of Figures --- p.x / List of Tables --- p.xvi / Table of Contents --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Introduction to Polyglutamine Diseases --- p.1 / Chapter 1.1.1 --- Etiology of Polyglutamine Diseases --- p.1 / Chapter 1.1.2 --- Common Features of Different Types of Polyglutamine Disease --- p.1 / Chapter 1.2 --- Pathogenic Mechanisms of Expanded Polyglutamine Proteins --- p.4 / Chapter 1.2.1 --- Pathogenesis of Polyglutamine Diseases --- p.4 / Chapter 1.2.1.1 --- Loss-of-function toxicity --- p.4 / Chapter 1.2.1.2 --- Gain-of-function toxicity --- p.4 / Chapter 1.3 --- Expanded CAG Transcript-mediated Pathogenic Mechanism --- p.6 / Chapter 1.3.1 --- Identification of the Toxic Role of Expanded CAG Transcripts --- p.6 / Chapter 1.3.2 --- Nuclear Foci Formation of Expanded CAG Transcripts and Polyglutamine Pathogenesis --- p.8 / Chapter 1.4 --- Receptor-mediated RNA nuclear export Transport --- p.9 / Chapter 1.4.1 --- Introduction to RNA Nuclear Export --- p.9 / Chapter 1.4.2 --- Regulation of RNA Nucleocytoplasmic Transport and Human Diseases --- p.11 / Chapter 1.5 --- Function of Nucleolus --- p.12 / Chapter 1.5.1 --- Ribosomal RNA Transcription --- p.12 / Chapter 1.5.2 --- Nucleolar Stress and Apoptosis --- p.15 / Chapter 1.6 --- Research Plan --- p.17 / Chapter 1.6.1 --- Project Objective --- p.17 / Chapter 1.6.2 --- Experimental Model --- p.17 / Chapter 1.6.2.1 --- In vivo Drosophila Model --- p.17 / Chapter 1.6.2.2 --- In vitro Cell Culture Model --- p.19 / Chapter 1.6.2.3 --- Transgenic Mouse Model --- p.20 / Chapter 1.6.3 --- Significance of the Present Study --- p.21 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Molecular Cloning --- p.22 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.22 / Chapter 2.1.2 --- Primers Used for PCR --- p.29 / Chapter 2.1.3 --- Restriction Digestion --- p.31 / Chapter 2.1.4 --- Agarose Gel Electrophoresis --- p.32 / Chapter 2.1.5 --- Preparation of genomic DNA from A Single Adult Fly --- p.34 / Chapter 2.1.6 --- Removal of 5' Phosphate Groups on Linearized Plasmids --- p.35 / Chapter 2.1.7 --- Addition of 5' Phosphate Group to Linker Oligonucleotides --- p.35 / Chapter 2.1.8 --- Ligation Reaction --- p.37 / Chapter 2.1.9 --- Bacterial Transformation --- p.37 / Chapter 2.2 --- Mammalian Cell Culture --- p.40 / Chapter 2.3 --- Drosophila Culture --- p.44 / Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.48 / Chapter 2.5 --- Microscopy --- p.51 / Chapter 2.6 --- Protein Sample Preparation and Concentration Measurement --- p.53 / Chapter 2.7 --- Co-immunoprecipitation --- p.57 / Chapter 2.8 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting --- p.62 / Chapter 2.9 --- Bacterial Protein Purification --- p.65 / Chapter 2.1 --- DNA Methylation Assay --- p.68 / Chapter 2.11 --- Mitochondrial Fraction Isolation --- p.79 / Chapter 3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Polyglutamine Diseases Toxicity by Altering the Subcellular Localization of Expanded CAG Transcripts in vivo / Chapter 3.1 --- The Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in vivo --- p.72 / Chapter 3.1.1 --- Expanded CAG Transcripts Predominantly Localize in the Nucleus in Drosophila Model of Machado-Joseph Disease --- p.72 / Chapter 3.1.2 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in an Inducible Model of Machado-Joseph Disease --- p.73 / Chapter 3.1.3 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in Inducible DsRed[subscript CAG100] Model. --- p.76 / Chapter 3.1.3.1 --- Expanded CAG Transcripts Induce the Expression of Pro-apoptotic Genes --- p.77 / Chapter 3.1.3.2 --- Co-expression of p35 Suppresses the Toxicity Induced by the Expanded CAG Transcripts --- p.80 / Chapter 3.2 --- A Candidate-gene RNA Interference Approach was Employed to Identify Genetic Factors Involved in Nuclear Export of Expanded CAG Transcripts --- p.80 / Chapter 3.3 --- Confirmation of the Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Machado-Joseph Disease in vivo --- p.84 / Chapter 3.4 --- The Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Different Drosophila Models of Polygultamine Diseases --- p.84 / Chapter 3.5 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Specifically Modulates Expanded CAG Transcript-induced Toxicity in vivo --- p.87 / Chapter 3.5.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enhances Expanded CAG Transcript-induced Toxicity --- p.87 / Chapter 3.5.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate Expanded PolyQ Protein-induced Toxicity --- p.89 / Chapter 3.5.3 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Alter the Expression Level of Expanded CAG Transcripts in vivo --- p.89 / Chapter 3.5.4 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate the Toxicity in Fragile X syndrome in vivo --- p.91 / Chapter 3.6 --- Over-expression of Human U2 Small Nuclear Riboprotein Auxiliary Factor 65 Does Not Modulate Expanded CAG Transcript-induced Toxicity in Drosophila --- p.91 / Chapter 3.7 --- Expanded CAG Transcripts Does Not Compromise Endogenous Function of U2 Small Nuclear Riboprotein Auxiliary Factor 50 --- p.94 / Chapter 3.8 --- A Correlation between Nucleocytoplasmic Localization of Expanded CAG Transcripts and Its Induced Toxicity --- p.97 / Chapter 3.8.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched DsRedCAG100 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.8.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched MJDCAG78 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.9 --- Expanded CAG-repeat on the Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 in vivo and in vitro --- p.102 / Chapter 3.9.1 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50 in vivo --- p.102 / Chapter 3.9.2 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.9.3 --- Expanded CAG Transcripts Directly Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.10 --- Identification of Expanded CAG Transcripts Interacting Domain on U2 Small Nuclear Riboprotein Auxiliary Factor 65 --- p.107 / Chapter 3.10.1 --- Generation of Different Myc-tagged U2 Small Nuclear Riboprotein Auxiliary Factor 65 Expression Constructs --- p.107 / Chapter 3.10.2 --- RNA Recognition Motif 3 on U2 Small Nuclear Riboprotein Auxiliary Factor 65 Is Essential for the Interaction with Expanded CAG Transcripts --- p.109 / Chapter 3.11 --- Nuclear RNA Export Factor 1 is Involved in U2 Small Nuclear Riboprotein Auxiliary Factor 65-mediated Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.1 --- The Effect of Full Length U2 Small Nuclear Riboprotein Auxiliary Factor 65 and its Corresponding Deletion Mutants on Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.2 --- Formation of Complexes Composed of Nuclear RNA Export Factor 1/U2 Small Nuclear Riboprotein Auxiliary Factor 65/Expanded CAG Transcripts in HEK293 Cells --- p.115 / Chapter 3.12 --- The Nuclear Export of Expanded CAG Transcripts is Mediated by U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 --- p.120 / Chapter 3.13 --- Aging Compromises the Nuclear Export of Expanded CAG Transcripts in Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.1 --- Expanded CAG Transcripts Accumulate in the Nucleus of Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.2 --- Expression Level of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Declines with Age in Mice --- p.124 / Chapter 3.14 --- Discussion --- p.127 / Chapter 3.14.1 --- Expanded CAG Transcripts Induce Nuclear Toxicity through a Mechanism Independent on Pathogenic Mechanism Mediated by Other Trinucleotide Repeats Expansion --- p.127 / Chapter 3.14.2 --- Nuclear Accumulation of Expanded CAG Transcripts Leads to Neurodegeneration --- p.128 / Chapter 3.14.3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Expanded CAG Transcript-induced Toxicity by Mediating the Subcellular Localization of Expanded CAG Transcripts --- p.129 / Chapter 3.14.4 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 Regulate the Nuclear Export of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.1 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 Interacts with Expanded CAG Transcripts and Mediates the Subcellular localization of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.2 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 Requires Nuclear RNA Export Factor 1 to Mediate the Nuclear Export of Expanded CAG Transcripts --- p.131 / Chapter 3.14.4.3 --- Developmental Decline of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Protein Level is a Factor That Leads to Progressive Neurodegeneration in Polyglutamine Diseases --- p.134 / Chapter 4 --- Expanded CAG Transcripts Induce Nucleolar Stress / Chapter 4.1 --- Expanded CAG-repeat Sequence Mediates the Nucleolar Localization of RNA Transcripts in vitro --- p.135 / Chapter 4.1.1 --- Machado-Joseph Disease Cell Model --- p.135 / Chapter 4.1.2 --- EGFPCAG Cell Model --- p.137 / Chapter 4.2 --- Expanded CAG Transcripts Suppress Nucleolar Function in vitro and in vivo --- p.140 / Chapter 4.2.1 --- Expanded CAG Transcripts Suppress Ribosomal RNA Transcription in vivo --- p.140 / Chapter 4.2.1.1 --- Drosophila Model of Machado-Joseph Disease --- p.140 / Chapter 4.2.1.2 --- Drosophila Model of DsRedCAG --- p.142 / Chapter 4.2.1.3 --- Transgenic Mouse Model of PolyQ Disease --- p.142 / Chapter 4.2.2 --- Expanded CAG Transcripts Suppress rRNA Transcription in vitro --- p.145 / Chapter 4.2.2.1 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.145 / Chapter 4.2.2.2 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.145 / Chapter 4.3 --- Expanded CAG Transcripts Disrupt the Interaction between RNA Polymerase I and rRNA Promoter in vitro --- p.148 / Chapter 4.4 --- Expanded CAG Transcripts Disrupt the Interaction between Upstream Binding Factor and Upstream Control Element in vitro and in vivo --- p.149 / Chapter 4.4.1 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.149 / Chapter 4.4.2 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vivo --- p.151 / Chapter 4.5 --- Expanded CAG Transcripts Induce DNA Hyper-methylation on Upstream Control Element in vitro and in vivo --- p.151 / Chapter 4.5.1 --- The HpaII-PCR Assay for DNA Methylation --- p.154 / Chapter 4.5.2 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vitro --- p.154 / Chapter 4.5.2.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.154 / Chapter 4.5.2.2 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.156 / Chapter 4.5.3 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vivo --- p.156 / Chapter 4.5.4 --- Expanded CAG Transcripts Disrupt the Regulatory Mechanism of Upstream Control Element Methylation in vitro --- p.159 / Chapter 4.6 --- Expanded CAG Transcripts Induce Nucleolar Stress and Apoptosis --- p.161 / Chapter 4.6.1 --- Expanded CAG Transcripts Induce Nucleolar Stress in vitro and in vivo --- p.162 / Chapter 4.6.1.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.162 / Chapter 4.6.1.2 --- Transgenic Mouse Model of PolyQ Disease --- p.162 / Chapter 4.6.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro and in vivo --- p.165 / Chapter 4.6.2.1 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro --- p.165 / Chapter 4.6.2.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vivo --- p.167 / Chapter 4.6.3 --- Expanded CAG Transcripts Enrich p53 in Mitochondria in vitro --- p.167 / Chapter 4.6.4 --- Expanded CAG Transcripts Lead to Disruption of interaction between Bcl-xL and Bak by p53 in mitochondria in vitro --- p.169 / Chapter 4.6.5 --- Expanded CAG Transcripts Lead to Release of Cytochrome c in vitro --- p.171 / Chapter 4.6.6 --- Expanded CAG Transcripts Lead to Activation of Caspase 3 in vitro --- p.173 / Chapter 4.7 --- Discussion --- p.176 / Chapter 4.7.1 --- Expanded CAG Transcripts Compromise Nucleolar Function --- p.176 / Chapter 4.7.2 --- Expanded CAG Transcripts Induce Apoptosis via Nucleolar Stress --- p.176 / Chapter 4.7.3 --- The Origin of Nucleolar Stress Induced by Expanded CAG Transcripts --- p.178 / Chapter 5 --- Expanded CAG Transcripts Interact with Nucleolin and Deplete It from Upstream Control Element to Suppress Ribosomal RNA Transcription / Chapter 5.1 --- Nucleolin is an Interacting Partner of Expanded CAG Transcripts --- p.180 / Chapter 5.1.1 --- Nucleolin is Pulled down by S1-tagged Expanded CAG Transcripts in vitro --- p.180 / Chapter 5.1.2 --- Expanded CAG Transcripts Interact with Endogenous Nucleolin in vitro --- p.181 / Chapter 5.1.3 --- Expanded CAG Transcripts Directly Interact with Nucleolin in vitro --- p.184 / Chapter 5.2 --- RNA Recognition Motifs 2 and 3 on Nucleolin Interact with Expanded CAG Transcripts --- p.184 / Chapter 5.2.1 --- Generation of Expression Constructs Carrying Full Length Nucleolin and its Deletion Mutants --- p.184 / Chapter 5.2.2 --- Mapping of Domains on Nucleolin Required for Interacting with Expanded CAG Transcripts --- p.187 / Chapter 5.3 --- Nucleolin Regulates Ribosomal RNA Transcription by Mediating the DNA Methylation of Upstream Control Element in HEK293 Cells --- p.187 / Chapter 5.3.1 --- Nucleolin is involved in Regulating the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.191 / Chapter 5.3.2 --- Nucleolin is Involved in Regulating DNA Methylation Level of Upstream Control Element in vitro --- p.191 / Chapter 5.3.3 --- Nucleolin Associates with Upstream Control Element in vitro --- p.194 / Chapter 5.4 --- Expanded CAG Transcripts Deplete Nucleolin from Upstream Control Element in vitro and in vivo --- p.194 / Chapter 5.4.1 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vitro --- p.197 / Chapter 5.4.2 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vivo --- p.197 / Chapter 5.4.3 --- Expanded Polyglutamine Proteins does not Interact with Nucleolin in vitro --- p.200 / Chapter 5.5 --- Over-expression of Nucleolin Counteracts the Effect of Expanded CAG Transcripts on Ribosomal RNA Transcription in vitro --- p.200 / Chapter 5.5.1 --- Over-expression of Nucleolin Restores the Methylation Level of Upstream Control Element in Dose-dependent Manner in vitro --- p.200 / Chapter 5.5.1.1 --- The Dosage Effect of Nucleolin on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts in vitro --- p.202 / Chapter 5.5.1.2 --- Does-dependent Expression of Nucleolin in vitro --- p.202 / Chapter 5.5.1.3 --- The Effect of Nucleolin Over-expression on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts is Dose-dependent in HEK293 cells --- p.205 / Chapter 5.5.2 --- Over-expression of Nucleolin Does Not Alter the Expression Level of Expanded CAG Transcripts in vitro --- p.205 / Chapter 5.5.3 --- Over-expression of Nucleolin Relieves the Nucleolar Stress induced by Expanded CAG Transcripts in vitro --- p.208 / Chapter 5.6 --- Discussion --- p.212 / Chapter 5.6.1 --- The Physical Interaction between Expanded CAG Transcripts and Nucleolin Leads to Suppression of Ribosomal RNA Transcription --- p.212 / Chapter 5.6.2 --- Expanded CAG Transcripts Deprive Upstream Control Element of Nucleolin to Induce Toxicity --- p.212 / Chapter 5.6.3 --- Nucleolin Suppresses Expanded CAG Transcript-induced Cell Death --- p.213 / Chapter 5.6.4 --- Expanded CAG Transcripts Employ both p53-dependent and p53-independent pathways to Induce Cell Death --- p.214 / Chapter 6 --- Concluding Remarks --- p.216 / References --- p.220

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Diseases which affect the central nervous system present a huge burden to sufferers and caregivers. In tandem with longevity, prevalences of age-related neurodegenerative diseases are increasing. However, despite the evident necessity for pharmaceutical interventions, there has been a distinct lack of drug development to combat these disorders. This is largely attributed to high financial costs of using rodent models. Thus the validation of a more cost-effective in vivo system would facilitate pharmaceutical screening. The work presented in this thesis addresses this issue by assessing the utility of zebra fish in two costly areas of translational neurobiology { lead identi cation and safety pharmacology. An aversive classical conditioning assay was developed and automated as a behavioural screening method. This robust assay allows fast assessment of cognition and cognitive decline. The effect of neurotoxin treatment on aversive learning was then assessed using this assay, demonstrating its efficacy as a screening tool for neurodegeneration research. Subsequently, a transgenic zebra fish line - expressing a mutated form of the Alzheimer's-associated human amyloid precursor protein - was assessed, demonstrating an age-related cognitive impairment. Additionally new genetic zebra fish lines were generated, which over-express genes (both endogenous and transgenic) related to Alzheimer's-like pathologies. Whilst these were not assessed within this thesis, they present promising tools for possible future investigations. Regarding safety pharmacology, regulatory bodies require all CNS-penetrant drugs be assessed for abuse potential. Zebra fish display reward responses to several common drugs of abuse (e.g. amphetamine, cocaine, morphine). Thus, the latter sections of this thesis evaluated the utility of zebra fish for assessing human abuse potential. A CPP paradigm was utilised to test a range of drugs, with the sensitivity and specificity of zebra fish compared to previous reports using rodent. Additionally, the development of a zebra fish drug discrimination assay was attempted. However the paradigms utilised failed to develop an efficacious assay.

L’évaluation du comportement animal est devenue un outil fondamental dans les champs de recherche des neurosciences translationnelles. Elle est en effet utile pour l’étude des mécanismes physiologiques entraînant les maladies neurodégénératives mais aussi pour la compréhension des modifications fonctionnelles induites par des manipulations génétiques ou traitement chimique et pour tester l’efficacité de composés ou molécules. Chez l’Homme, on utilise des batteries de tests cognitifs et moteurs pour qualifier et quantifier les atteintes des maladies neurodégénératives telles que les maladies d’Alzheimer et de Parkinson. Le travail présenté ici s’inscrit dans l’optique de promouvoir Microcebus murinus comme modèle pour l’étude des maladies neurodégénératives liées à l’âge. Le microcèbe est un petit primate lémurien, proche phylogénétiquement de l’Homme, présentant des particularités prometteuses susceptibles d’accélérer la découverte de nouveaux traitements curatifs et prophylactiques pour les pathologies liées à l’âge. La validation du modèle nécessite l’utilisation de tests comportementaux discriminant l’atteinte neurodégénérative. Nous avons mis au point un ensemble de tests comportementaux évaluant l’apprentissage, la mémoire et les fonctions motrices. Des animaux sains ont été évalués et ont permis de définir des protocoles spécifiques au microcèbe. Ces protocoles ont ensuite été appliqués à des animaux ayant subi un traitement pour induire des pathologies de type Parkinson ou Alzheimer afin de détecter l’apparition d’éventuels déficits. / Animal behavior has become a fundamental tool in translational neuroscience area and is useful for studying physiological mechanisms underlying neurological diseases and also for understanding the functional modifications induced by genetic manipulation or chemical treatment. The experiment of new treatments requires animal models miming the human pathology. In Humans batteries of cognitive and motor tests are used to qualify and quantify the impairment due to neurodegenerative disease as Alzheimer's disease and Parkinson’s disease. The aim of this work is to promote Microcebus murinus as a model of age related neurodegenerative pathologies. The grey mouse lemur, a small prosimian primate, phylogenetically close to Human, presents specific characteristics susceptible to provide important information on the validity and efficacy of new. The validation of a model requires the use of behavioral tests to discriminate neurodegenerative impairment. A set of behavioral tests were worked out to evaluate learning memory and motor functions. Healthy animals were evaluated and allowed to define protocols species specific. These protocols were then applied on animals treated to induce pathology such as Parkinson or Alzheimer in order to detect cognitive or motor impairments.

Polyglutamine (polyQ) diseases describe a group of late-onset progressive neurodegenerative disorders which are caused by the CAG triplet repeat expansion in the coding region of disease genes. Such expansions result in expanded polyQ tracts in the disease proteins which confer neurotoxicity. To date, nine such diseases are reported including Huntington's disease and several types of spinocerebellar ataxias. Misfolding of polyQ proteins and formation of intracellular SDS-insoluble protein aggregates are closely associated with the toxicity of these diseases. In particular, impairment of the ubiquitin-proteasome system (UPS) which is responsible for protein degradation has been observed in polyQ diseases. Recently, ubiquitin ligases, which govern substrate specificity of the UPS, have gained huge attention in polyQ disease pathogenesis studies. In humans, cullin (Cul) proteins, including Cul1, 2, 3, 4 & 5, are integral components of a group of ubiquitin ligases called cullin- RING ubiquitin ligases (CRLs). Each CRL displays distinct substrate specificity through specific substrate receptors. Cullin proteins are evolutionarily conserved and Cul orthologues are found in the Drosophila genome. In the present study, it was found that individual Culs displayed distinct effects on polyQ pathogenesis in Drosophila polyQ models. Particularly, it was found that Cul1 modulated polyQ-induced toxic phenotype. This modification was accompanied with an alteration in the ubiquitination level and SDS-solubility properties of expanded polyQ protein. Through genetic interaction studies and biochemical analyses, it is suggested that Cul1-based CRL specifically targets SDS-insoluble species of expanded polyQ protein for ubiquitination via selective recognition by CG2010 substrate receptor. On the other hand, it was found that expanded polyQ protein induced accumulation of CRL substrates in cells. Current data support a hypothesis that polyQ protein would impair the ubiquitin ligase activity of CRLs upon expansion of the polyQ domain, through interfering with neddylation of cullin and other uncharacterized mechanisms. Taken together, the present study identifies Cul1-CRL as a novel E3 ligase that modifies polyQ toxicity through modulating ubiquitination of expanded polyQ protein, and demonstrates a pathological mechanism by expanded polyQ protein through impairing CRL activity. These findings would lead to a better understanding of polyQ pathogenesis and give insights on developing treatments against polyQ diseases. / Wong, Kam Yan. / Adviser: Ho-Yin Edwin Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 260-273). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.