O prion celular (PrPc) é uma glicoproteína ligada à membrana plasmática por uma âncora de GPI (glycosylphosphatidylinositol). A sua isoforma anormal (PrPsc) é uma molécula infecciosa que causa várias doenças neurodegenerativas em mamíferos. A etiologia dessas doenças está associada a uma mudança conformacional pós-traducional de PrPc que ocorre após sua internalização (Prusiner, 1998). Na tentativa de desvendar as funções fisiológicas de PrPc, nosso grupo tem identificado e caracterizado as interações celulares que PrPc participa. A primeira delas é a interação entre PrPc e STI1 (Stress Inducible Protein 1). Essa interação transduz sinalização por cAMP e PKA levando a neuroproteção contra morte celular programada (Chiarini e cols, 2002; Zanata e cols, 2002). A segunda é a interação específica que existe entre PrPc e as proteínas da matriz extracelular, laminina e vitronectina, contribuindo para os processos neuronais, tais como crescimento, manutenção (Graner e cols., 2000 a e b) e regeneração dos neuritos (Hajj e cols., submetido), além da formação de memória de curta e longa duração (Coitinho e cols., submetido). Na primeira parte deste trabalho, procuramos investigar os genes regulados pelos sinais resultantes dessas interações e também pela remoção de PrPc usando a técnica de \"differential display\'\' RT-PCR. Na segunda parte do trabalho, caracterizamos que a interação PrPc - laminina é capaz de induzir uma sinalização transitória de cálcio, a qual ocorre mesmo na ausência de cálcio do meio extracelular. PrPc é uma molécula que cicla continuamente entre a membrana plasmática e os compartimentos intracelulares. Estudos recentes têm correlacionado o processo de internalização de PrPc com alguns dos seus papeis fisiológicos, tais como, homeostase de Cu2 + (Brown, 2001 ), interação com receptor de laminina (Gauczynski e cols, 2001) e até na conversão de PrPc para PrPsc (McKinley e cols, 1991; Arnold e cols, 1995). Portanto, na terceira parte deste trabalho, caracterizamos a localização e o tráfego celular de PrPc mostrando que PrPc está localizado na membrana plasmática e em compartimentos intracelulares e que trafega pelo Golgi, membrana plasmática, endossomos iniciais e de reciclagem. Foram mapeados ainda domínios na região amino-terminal responsáveis pela internalização de PrPc e na região carboxi-terminal como participantes da via secretora. Este trabalho contribuiu para o esclarecimento de alguns eventos biológicos relacionados à sinalização e ao tráfego de PrPc. Estes achados são de grande importância para a determinação das funções celulares de PrPc e ainda dos mecanismos envolvidos com as doenças relacionadas com esta molécula. / The cellular prion protein (PrPc) is a glycoprotein anchored to the plasma membrane by GPI (Glycosyl-phosphatidylinositol). Its abnormal isoform (PrPsc) is the infectious protein responsible for several neurodegenerative diseases. The main etiology of the prion diseases is related to conformational changes in the PrPc molecule, which occur after its internalization (Prusiner, 1998). In order to elucidate the physiological functions of PrPc, our group identified and characterized interactions between PrPc and other cellular molecules. The first is the interaction between PrPc and STI 1 (Stress Inducible Protein 1). This interaction has an important role in the neuroprotection against apoptosis through cAMP and PKA signaling (Chiarini et al., 2002; Zanata et al., 2002). PrPc also interacts with proteins of the extracellular matrix such as laminin and vitronetin. These interactions contribute for neurite outgrowth, maintenance and regeneration (Graner et al., 2000 a and b; Hajj et al., submitted) and also in memory formation (Coitinho et al., submitted). In the first part of this work we have applied the differential dysplay RTPCR technique in order to identify genes that are regulated by PrPc - STI 1 interaction and also by the deletion of PrPc. In the second part we have demonstrated that PrPc-laminin interaction induces transient calcium signaling in neuronal cells, which occurs even in the absence of extracellular calcium. PrPc cycles continuously between the plasma membrane and intracellular compartments. This mechanism is associated with some of the physiological function of PrPc, such as Cu2+ homeostasis (Brown, 2001 ), interaction with laminin receptor (Gauczynski et al., 2001 ), and PrPc conversion into PrPsc (McKinley et al., 1991; Arnold et al., 1995). Thus, in the third part of this project, we have characterized the PrPc localization at the cell surface and in intracellular compartments. The protein trafficking through Golgi apparatus, plasma membrane, early and recycling endosomes was also defined. Moreover, we have determinated that N-terminus PrPc domain is responsible for its internalization while C-terminus participates in PrPc delivery. Therefore, this work has contributed to elucidate biological events related to the cell signaling and trafficking of PrPc, which are important for the characterization of PrPc physiological functions and to understand the pathological mechanisms related to this molecule.

La pathogénèse de la plupart des maladies neurodégénératives incluant les maladies transmissibles comme les encéphalopathies à prion, les maladies génétiques de type maladie de Huntington et les maladies sporadiques comme les maladies d’Alzheimer et de Parkinson est directement liée à la formation d’agrégats protéiques fibrillaires. Pendant de nombreuses années, le concept de dissémination et d’infectivité de ces agrégats a été réservé aux maladies à prion. Cependant, de récents résultats montrent que ces protéines amyloidiques extracellulaires (β-amyloïde) comme intracellulaires (α-synucléine, tau, huntingtin) sont capables de bouger (et possiblement de se répliquer) d’une zone à l’autre du cerveau à la façon des prions (Brundin et al., 2010; Jucker and Walker, 2011; Aguzzi and Rajendran, 2009). Récemment une nouveau lien a été établie entre prions et différentes protéinopathies à agrégats. Il a été suggéré que le prion cellulaire, PrPC, dont la forme pathologique (PrPSc) est responsable des maladies à prion, pourrait servir de médiateur dans la toxicité de la protéine β-amyloïde impliquée dans la maladie d’Alzheimer comme dans d’autres conformations-β, indépendamment de la propagation des prions infectieux (revue de Biasini et al., 2012). Malgré une intense recherche sur les maladies neurodégénératives à prion ou non, de nombreuses questions restent ouvertes à la fois au niveau du mécanisme de dissémination des agrégats protéiques que du mécanisme de toxicité. Dans la première partie de ma thèse, j’ai contribué à étudier le rôle de cellules dendritiques (DCs) dans la dissémination de l’infection à prion aux neurones. J’ai démontré que le transfert de PrPSc des cellules dendritiques infectées par un homogénat de cerveau infecté par du prion vers les neurones était dû à contact direct entre ces cellules et a pour résultat la transmission de l’infectivité aux neurones en co-culture. Ces résultats confirment le possible rôle des cellules dendritiques dans la propagation du prion de la périphérie vers le système nerveux central. J’ai aussi trouvé un potentiel mécanisme de transfert de PrPSc des cellules dendritiques aux neurones via des nanotubes (TNTs) et exclu l’implication de la sécrétion de PrPSc dans notre système. Dans la seconde partie de ma thèse, j’ai étudié les mécanismes de dissémination et de toxicité des agrégats protéiques huntingtin et le possible rôle de PrPC dans ces évènements. J’ai démontré que les agrégats Htt sont transférés entre les lignées de cellules neuronales et les neurones primaires et qu’un contact direct cellule à cellule est requis. De même, j’ai montré l’implication des TNTs dans ce transfert et l’agrégation des Htt sauvages endogènes dans les neurones primaires, probablement en suivant le transfert des agrégats Htt. La dernière partie de mes résultats montre que PrPC est impliqué dans la propagation de la toxicité induite par les Htt mutants dans des neurones primaires en culture. / The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathies, inherited disorders like Huntington’s disease, and sporadic diseases like Alzheimer’s and Parkinson’s diseases, appear to be directly linked to the formation of fibrillar protein aggregates. For many years, the concept of aggregate spreading and infectivity has been confined to prion diseases. However, recent evidence indicate that both extracellular (e.g. amyloid-β) and intracellular (α- synuclein, tau, huntingtin) amyloidogenic protein are able to move (and possibly replicate) within the brains of affected individuals, thereby contributing to the spread of pathology in a prion-like manner (Brundin et al., 2010; Jucker and Walker, 2011; Aguzzi and Rajendran, 2009). Recently another intriguing connection has been made between prions and other aggregation proteinopathies, as it was suggested that the cellular prion protein, PrPC, whose pathological counterpart is responsible for prion diseases, possibly mediates the toxicity of Aβ, the pathogenic protein in Alzheimer’s disease, and of other β- conformers independently of the propagation of infectious prions (reviewed in Biasini et al., 2012). However, despite the intense research, many questions in prion and non-prion neurodegenerative diseases are still open regarding both the mechanism of protein aggregate spreading and the mechanism of toxicity. In the first part of my thesis, I contributed to investigate the role of DCs (dendritic cells) in the spreading of prion infection to neuronal cells. I demonstrated that the transfer of PrPSc from DCs (loaded with prion infected brain homogenate) to primary neurons was triggered by direct cell–cell contact and resulted in transmission of infectivity to the co-cultured neurons. These data confirm the possible role of DCs in prion spreading from the periphery to the nervous system. I also provided a plausible transfer mechanism of PrPSc through tunneling nanotubes (TNTs) shown to connect DCs to primary neurons and excluded the involvement of PrPSc secretion in our system. In the second part of my thesis, I investigated the mechanisms of the spreading and toxicity of Htt aggregates and the possible role of PrPC in these events. I demonstrated that Htt aggregates transfer between neuronal cells and primary neurons and that cell-cell contact is required. I also showed the involvement of TNTs in the transfer and reported the aggregation of endogenous wild-type Htt in primary neurons, possibly following the transfer of Htt aggregates. Finally, the last part of my results provides evidences that PrPC is involved in the spreading of the toxicity mediated by mutant Htt in primary neuronal cultures.

Les similitudes entre les mécanismes neurotoxiques responsables des encéphalopathies spongiformes Transmissibles (EST) et des synucléinoapthies, ainsi que la présence concomitante des formes pathologiques de la protéine prion et de l’α-synucléine au sein d’une même maladie neurodégénérative sont deux observations qui nous ont conduits à étudier les relations existant potentiellement entre les altérations moléculaires de l’α-synucléine et les maladies à prion. Après avoir développé des anticorps monoclonaux en immunisant avec de l’α-synucléine recombinante humaine des souris n’exprimant pas de façon endogène cet immunogène, nous avons caractérisé les altérations moléculaires de l’α-synucléine apparaissant conjointement à une symptomatologie motrice sévère lors du vieillissement de souris transgéniques (TgM83) surexprimant l’α-synucléine humaine mutée en A53T. Les essais d’inoculation intracérébrale de souris TgM83 par différentes souches de prion ont mis en évidence que la transmission de l’encéphalopathie spongiforme bovine de type H permet de déclencher chez ces animaux une maladie à prion de façon concomitante au développement d’altérations moléculaires de l’α-synucléine. Enfin, l’importante accélération de la pathologie liée a l’α-synucléine observée chez des souris TgM83 ayant été inoculées par des tissus contenant des formes altérées de l’α-synucléine, constitue un résultat soutenant le fait que la pathologie liée a l’α-synucléine serait capable de se propager expérimentalement de proche en proche, comme la protéine prion pathologique au cours des EST / The overlap of neurotoxic mecanisms involved in prion diseases and synucleinopathies, and the concomitant detection of pathological forms of prion and α-synuclein in a same neurodegenerative disease, raise questions about the existence of potential relationship between α‐synuclein molecular alteration and prion diseases. First, we developed monoclonal antibodies by immunizing mice presenting a spontaneous deletion of the α-synuclein gene with human recombinant α‐synuclein. Then, we characterized the molecular alterations appearing jointly to clinical signs during the aging of a transgenic mouse model of synucleinopathies (TgM83), overexpressing human A53T α‐synuclein. Then, an approach routinely done in the field of prion was used to trigger a synucleinopathy alongside a prion disease. For this purpose, TgM83 mice were inoculated intracerebrally by three different prion strains : transmission of H-type bovine spongiform encephalopathy allows the onset of a prion disease concomitantly to the α‐synuclein pathology developed by the TgM83 mouse model. Finally, intracerebral inoculation of TgM83 mice with brain homogenates from symptomatic mice affected by a synucleinopathy triggers an important acceleration of the α‐synuclein pathology, resulting in the early onset of motor clinical signs associated with molecular alterations of α-synuclein. These data suggest that α-synuclein alterations can be experimentally transmitted from one mouse to another, supporting the idea that, far from being confined to the transmissible spongiform encephalopathies, the « prion-like » propagation of misfolded neuronal proteins might occur in synucleinopathies

La détection de la protéine prion anormale dans les tissus lymphoïdes de patients britanniques suggère qu’après exposition à l’agent de la variante de la maladie de Creutzfeldt-Jakob (vMCJ) plus de 99% des contaminations pourraient demeurer cliniquement silencieuses. Ces données soulignent un risque de transmission secondaire par transfusion sanguine ce qui nous a conduit à une étude expérimentale. En parallèle des formes classiques de vMCJ, nos modèles murins et simiens de retransmission ont mis en evidence des phenotypes atypiques. Ces phénotypes échappent actuellement aux critères de diagnostic puisqu’aucune protéine prion anormale (PrPres) n’est détectée.Nos travaux ont eu pour but principal d’évaluer expérimentalement le risque sanguin au travers d’études de retransmission et de caractérisation de la replication des souches classiques et atypiques aux niveaux périphérique et central.Nous observons une très forte hétérogénéité dans la réplication de la PrP anormale dans les différents tissus lymphoïdes des macaques transfusés développant une vMCJ. Le niveau de contamination des tissus lymphoïdes apparait proportionnel à l’infectiosité sanguine de ces animaux et au risque de transmission de la maladie in vivo. Concernant les formes atypiques, la majorité des macaques transfusés n’ont pas de réplication dans les tissus lymphoïdes bien que ces phénotypes soient transmissibles expérimentalement à des modèles murins. Des transmissions à des souris immunodéficientes révèlent que les souches atypiques sont transmissibles par voie périphérique en l’absence d’un système immunitaire fonctionnel.Une alternative à l’expérimentation animale a été réalisée grâce aux « mini-brains » mimant la complexité du cerveau humain. Ces organoïdes cultivés en trois dimensions sont sensibles à au moins un isolat de prion associé aux formes sporadiques humaines. Les mini-brains pourraient ainsi constituer un nouvel outil d’étude des maladies à prions et permettre à termes la caractérisation des souches atypiques. / The detection of abnormal prion protein in the lymphoid tissues of UK patients suggests that after exposure to the agent of variant Creutzfeldt-Jakob disease (vCJD), more than 99% of contaminations may remain clinically silent. These data highlight a risk of secondary transmission through blood transfusion. In parallel to the classical vCJD forms, our experimental models in mice and macaques revealed another group which avoids the current diagnostic criteria, including the absence of abnormal prion protein (PrPres).The main goal of our work was to experimentally assess the risk of blood through retransmission studies and characterization of the abnormal replication of classical and atypical strains examined at peripheral and central levels.We observed a high heterogeneity of the distribution of the abnormal PrP in the lymphoid tissues of vCJD transfused macaques. The global level of contamination in lymphoid tissues seems proportional to the blood infectivity in these animals and to the risk of in vivo transmission of the disease. Regarding atypical forms, despite an absence of replication in lymphoid tissues, these phenotypes are experimentally transmissible. Transmissions to immunodeficient mice reveal that atypical strains are transmissible through peripheral routes in the absence of functional immune system.An alternative to animal testing has been achieved using to "mini-brains" mimicking the complexity of the human nervous system. These organoids cultured in three dimensions are sensitive to at least one prion isolate associated with human sporadic forms. Thus, mini-brains could constitute a new tool for studying prion diseases and improve the characterization of atypical strains.

No description available.

No description available.

A conversão da proteína prion celular normal (PrPc), cuja função ainda esta sob investigação, para a forma infecciosa (PrPsc) é a causa de algumas doenças neurodegenerativas em humanos e animais. Vários estudos têm sido realizados e mostram que PrPc pode participar de processos normais como memória, estresse oxidativo, neuritogênese e outros. Portanto, a elucidação dos processos de regulação de sua expressão é importante tanto para definir uma estratégia para controlar a infecção quanto para entender melhor a função fisiológica de PrPc. Este trabalho tem por objetivo avaliar a expressão do gene de PrPc, a partir da regulação da atividade de seu promotor frente a drogas que foram eleitas de acordo com a composição dos elementos de resposta a fatores de transcrição nele contidos. Para isto o promotor foi clonado em um vetor contendo o gene \"reporter\" de luciferase, células C6 e PC-12 foram transfectadas e clones com expressão estável de luciferase foram selecionados. Os resultados dos tratamentos dos clones celulares mostram que éster de forbol (TPA) e AMPc induzem a atividade do promotor de 1,5 a 3 vezes, ácido retinóico (RA) diminui esta atividade em cerca de 50% enquanto que NGF e Dexametasona não têm efeito. A dependência da conformação da cromatina na regulação deste gene também foi testada utilizando-se Tricostatina A (TSA), esta droga foi capaz de aumentar de 10 a 4.000 vezes a atividade do promotor, o que foi seguida tanto pela indução de expressão do RNAm quanto da proteína PrPc. Este efeito parece não ser generalizado a todos os promotores uma vez que esta droga não alterou expressão de GAPDH e de β-actina. Quando TPA e AMPc foram associados à TSA uma potencialização do efeito indutor destas drogas foi observada e a associação de RA e TSA mostrou que RA reduz a indução gerada por TSA. Estes novos dados indicam que a regulação de PrPc é extremamente dependente da conformação da cromatina. / Conversion of the cellular normal prion protein (PrPc), whose physiological function is still under investigation, to an infectious form called prion is the cause of some neurodegenerative diseases. Therefore, the elucidation of PrPc gene regulation is important both to define a strategy to control the infection and to better understand PrPc function. We cloned the rat PrPc gene promoter region into a luciferase reporter vector, transfected C6 and PC-12 cells and isolated clones with stable luciferase expression. The phorbol ester TPA and cAMP induced promoter activity by 1.5 to 3 times, retinoic acid decreased it by 50% while NGF and dexamethasone had no effect. We also tested the dependence of chromatin conformation for PrPc promoter activity using Trichostatin A (TSA), which was able to highly increase not only promoter activity but also PrPc rnRNA and protein leveIs. Moreover, the TSA effect seems to be restricted since any alteration was observed regarding GAPDH (Glyiceraldehyde 3-phosphate desydrogenase) and β-actin expression. When cAMP, TPA or retinoic acid were associated with TSA a potentiation of their primary effects was observed. These new data indicate that PrPc gene regulation is highly dependent on disruption of chromatin fiber assembly what permits assess of trascription factors.

Prions são definidos como partículas protéicas infecciosas compostas, quase que exclusivamente, por uma proteína conhecida como prion scrapie (PrPsc). O envolvimento dessas partículas na etiologia de doenças neurodegenerativas, tanto em homens como em animais, já está bem determinado. Acredita-se que o PrPsc seja sintetizado através de modificações pós-traducionais que ocorreriam na isoforma celular da proteína prion (PrPc), uma glicoproteína expressa constitutivamente na superfície de vários tipos celulares, principalmente de neurônios, ancorada na membrana plasmática por glicosil-fosfatidil inositol (GPI). Apesar de ser uma molécula conservada em várias espécies, a função de PrPc ainda permanece desconhecida. Interessados nos possíveis papéis fisiológicos desempenhados pelo PrPc, decidimos investigar certas interações que o PrPc poderia realizar com outras moléculas, na tentativa de se encontrar pistas sobre a função dessa proteína em células normais. Assim, nosso grupo identificou e vem caracterizando duas interações nas quais o PrPc está envolvido: com o seu receptor de 60-66 kDa e com a principal proteína não colagênica da matriz extracelular, a laminina (LN). Grande parte da caracterização dessas duas interações vem sendo desenvolvida graças ao uso de PrPc recombinante produzido em sistema heterólogo (em E.coli). O trabalho em questão trata principalmente da produção de PrPc recombinantes em sistema heterólogo e a utilização destes como importantes ferramentas para a melhor caracterização das interações identificadas. Alguns trabalhos na literatura vinham sugerindo a existência de um receptor para prions. Através da teoria da hidropaticidade complementar dos aminoácidos, nosso grupo foi capaz de identificar uma proteína de 60-66 kDa como sendo esse provável receptor. Ensaios de ligação in vitro utilizando PrPc recombinante, ou PrPc nativo (ancorado na superfície de células) confirmaram que a forma desse receptor presente na membrana (de 66 kDa) é capaz de se ligar ao PrPc. Com a ajuda de PrPc recombinante também foi possível verificar uma ligação específica, saturável e de alta afinidade (Kd da ordem de 10-8 M) entre PrPc e a LN. Através de ensaios de competição, utilizando peptídeos sintéticos correspondentes a domínios da LN já bem caracterizados e de funções estabelecidas, fomos capazes de mapear o sítio dessa molécula que se liga ao PrPc. A sequência identificada (RNIAEIIKDI) encontra-se na região C-terminal da cadeia γ1 da LN e, como demonstrado na literatura, esse domínio é responsável por estimular tanto a adesão celular, quanto o crescimento de neuritos em neurônios de cerebelo em cultura primária. De fato, resultados obtidos pelo nosso grupo indicam que a interação PrPc/LN participa no processo de neuritogênese. Experimentos de esquiva inibitória, realizados em colaboração com o grupo do Prof. Dr. Ivan Izquierdo (UFRGS) indicaram que a ligação PrPc/LN também desempenha um importante papel nos processos de memória e aprendizado. / Prions are defined as proteinaceous infectious particles that mediate the pathogenesis of certain neurodegenerative diseases, in humans and in animals. The prion particle is composed largely, if not entirely, by PrPsc (prion scrapie), a posttranslationaly modified isoform of the cellular host-encoded prion protein (PrPc). PrPc is a glycosylphosphatidylinositol anchored protein that is constitutively expressed by several cell types, mainly on neuronal cell surface. However, the physiological role of this conserved protein remains unclear. ínterested in this normal function of PrPc, our group decided to investigate some interactions that PrPc could be done with other molecules in order to find some clues about it. We have been identified and characterized two interactions that PrPc is involved: with its putative 60-66 kDa receptor and with laminin. In order to better characterize these interactions it was necessary to produce purified PrPc. In this work we will report the expression and the purification of mouse PrPc protein in heterologous system and its use as important tools to investigate the PrPc interactions. A specific cell surface receptor for PrPc has been predicted. Using the concept of complementary hydropathy, our group has identified a 60-66 kDa membrane protein in mouse brain, which seems to be a putative PrPc receptor. ln vitro binding assays using recombinant and native PrPc were able to confirm that the membrane receptor (66 kDa) binds PrPc. Recombinant PrPc was also useful to demonstrate a specific and high affinity (Kd around 10-8M) interaction between PrPc and laminin. In an attempt to map the PrPc binding site in this molecule, laminin peptides with established physiological functions were used in cornpetition binding assays. A peptide derived frorn C-terminal γ-1 chain of mouse laminin, RNIAEIIKDI, was the only one that was able to block the binding of laminin to PrPc, suggesting that this region comprises the PrPc binding site. It was reported in the literature that this peptide simulates the neurite outgrowth and cellular adhesion. In collaboration with Dr. Izquierdo\'s group (UFRGS) we demonstrated that the interaction characterized between this laminin\'s domain and PrPc is involved in the neuritogenesis process, as well as in learning and memory.

Ce mémoire rapporte les études de protéines amyloïdes en interaction avec des membranes modèle afin d’établir une relation structure toxicité. Nous avons choisi différents modèles membranaires (monocouches, bicouches) de composition lipidique et charges différentes et utilisé différentes méthodes physico-chimiques afin de caractériser les interactions des protéines amyloïdes avec les membranes.Nous avons montré l’importance de la contribution électrostatique dans les interactions de la protéine amyloïde HET-s (218-289) et ses mutants avec les membranes modèles.L’ellipsométrie a démontré que les mutants toxiques de HET-s (218-289) (M8, WT.Y1Y2V2) perturbentfortement les monocouches lipidiques à l’interface air-eau. La structure riche en feuillets β antiparallèles des protéines àl’interface air-eau et dans l’interaction avec les monocouches de lipides a été démontrée par la spectroscopie PMIRRAS (Polarization Modulation – Infrared Reflection Absorption Spectroscopy). Nous avons établie que l’interface air-eau peut modifier l’agrégation des protéines amyloïdes. A l’aide de la spectroscopie de fluorescence, la spectroscopie PWR (Plasmon-Waveguided Resonance) et la spectroscopie ATR-FTIR (Attenuated Total Reflection – Fourier Transform Infrared), nous avons mis en évidence que la protéine toxique M8 adopte une structure riche en feuillets β antiparallèles en altérant fortement l’intégrité des bicouches lipidiques. Au contraire, la protéine non toxique WT se structure en feuillets β parallèles dans ces interactions et elle ne perturbe pas l’homogénéité des membranes. La toxicité de la protéine M8 semble liée à son organisation différente et à sa capacité à réorganiser les membranes.Nos résultats confortent également l’hypothèse de la toxicité des oligomères amyloïdes.Une étude sur la fabrication d’une cellule microfluidique pour la séparation de différents types d’autoassemblage afin de les détecter et de les étudier en interaction avec des liposomes par spectroscopie infrarouge est présentée. Une cellule microfluidique de CaF2 de 8 μm d’épaisseur de canaux est obtenue et est utilisée pour la détection d’une protéine de test. / This manuscript reports the studies of amyloid proteins in interaction with membrane models in order to establish their structure-toxicity relationship.Membrane models (monolayer, bilayer) of different charge and lipid composition were used. We used various physico chemical methods to characterize the interaction of these amyloid proteins with membranes.We showed the importance of the electrostatic contribution in the interactions of the amyloid protein HET-s(218-289) and its mutants with model membranes.Ellipsometry showed that the toxic mutants of HET-s (218-289) (M8, WT.Y1Y2V2) strongly disturbed thelipid monolayers at the air-water interface. The structure rich in antiparallel β sheets of auto-assembled proteins at theair-water interface and in interaction with lipid monolayers at the air-water interface has been demonstrated by the PMIRRAS spectroscopy (Polarization Modulation - Infrared Reflection Absorption Spectroscopy). We established that theair-water interface can change the aggregation properties of amyloid proteins.By using fluorescence spectroscopy, PWR spectroscopy (Plasmon Resonance-Waveguided spectroscopy) and ATR-FTIR spectroscopy (Attenuated Total Reflection - Fourier Transform Infrared spectroscopy), we found that thetoxic protein (M8) adopted a structure rich in antiparallel β sheets greatly altered the integrity of lipid bilayers. Incontrast, the protein non-toxic (WT) organized in a structure rich in parallel β sheets in these interactions and it did notdisturb the homogeneity of the membranes. The toxicity of the protein M8 appears to be related to its differentorganization and its ability to rearrange membranes.Our results also support the hypothesis of the toxicity of amyloid oligomers.A study on the fabrication of a microfluidic cell for the separation of different aggregation states of amyloidproteins in order to detect these assemblies and to study their interaction with liposomes by infrared spectroscopy is presented. A CaF2 microfluidic cell with channels of 8 μm of thickness was obtained and was used for the detection of atested protein.

La RMN à l’état solide est une méthode de choix pour l’étude des protéines insolubles et des complexes protéiques de haut poids moléculaire. L’insolubilité intrinsèque des protéines fibrillaires, ainsi que leur architecture complexe, rendent difficile leur caractérisation structurale par la cristallographie et par la RMN en solution. La RMN à l‘état solide n’est pas limitée par le poids moléculaire et constitue donc un outil puissant pour l’étude des protéines fibrillaires. L’attribution des résonances RMN est le prérequis pour obtenir des informations structurales à résolution atomique. La première partie de ce travail de thèse décrit le développement de méthodes en RMN à l’état solide pour l’attribution des résonances. Nous avons appliqué ces méthodes afin d’attribuer le domaine C-terminal du prion Ure2 (33 kDa), qui est à ce jour la plus grande protéine attribuée par RMN à l’état solide. Nos résultats fournissent les bases pour l’étude de protéines à haut poids moléculaire à l’échelle atomique. Ceci est démontré dans la seconde partie de ce travail de thèse avec les premières études RMN à l’état solide des fibrilles des prions Ure2 et Sup35. Nous avons caractérisé la structure de ces prions pour les fibrilles entières ainsi que pour les domaines isolés. La troisième fibrille étudiée est l’α- synuclein, fibrille associée à la maladie de Parkinson, pour laquelle nous présentons l’attribution des résonances RMN ainsi que la structure secondaire d’un nouveau polymorphe. Les études présentées ici fournissent de nouvelles clés pour comprendre la diversité des architectures de fibrilles, en considérant les fibrilles comme entités individuelles d’un point de vue structural / Solid-state NMR is the method of choice for studies on insoluble proteins and other high molecular weight protein complexes. The inherent insolubility of fibrillar proteins, as well as their complex architecture, makes the application of x-ray crystallography and solution state NMR difficult. Solid-state NMR is not limited by the molecular weight or by the absence of long-range structural order, and is thus a powerful tool for the 3D structural investigation of fibrillar proteins. The assignment of the NMR resonances is a prerequisite to obtain structural information at atomic level. The first part of this thesis describes the development of solid-state NMR methods to assign the resonances in large proteins. We apply these methods to assign the 33 kDa C-terminal domain of the Ure2p prion which is up to now the largest protein assigned by solid-state NMR. Our results provide the basis to study high molecular weight proteins at atomic level. This is demonstrated in the second part with the first high-resolution solid-state NMR study of Ure2 and Sup35 prion fibrils. We describe the conformation of the functional domains and prion domains in the full-length fibrils and in isolation. The third fibrillar protein addressed in this work is the Parkinson’s disease related α-synuclein whereof we demonstrate the NMR resonance assignment and the secondary structure determination of a new polymorph. Thus, the studies described here provide new insights in the structural diversity of fibril architectures, and plead to view fibrils as individuals from a structural point of view, rather than a homogenous protein family