Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...

Die Parkinson-Krankheit (PA) ist durch Fehlfaltung und Aggregation von Alpha-Synuklein (a-Syn) charakterisiert. Dieses zeigt Ähnlichkeiten mit dem Verhalten von Prionen bei transmissiblen spongiformen Enzephalopathien. Pathologische a-Syn Aggregate propagieren, wie Prione, über selbsttempliertes Seeding (Keimung). Aufgrund von Studien, die eine Übertragbarkeit der a-Syn Seeding-Aktivität nachgewiesen haben, war das Ziel dieser Dissertation die Risikobewertung potenzieller Übertragungen von a-Syn Seeds zu verbessern. Die Menge an Seeding-aktiven a-Syn in Hirn- und Magengewebe von PA-Patienten wurde durch Real-Time Quaking-Induced Conversion (RT-QuIC) Assays quantifiziert. Die Ergebnisse zeigten in beiden Geweben hohe Seeding-Titer, was die Notwendigkeit für effektive Inaktivierungsprotokolle unterstreicht. Die Quantifizierung des Effekts der Dampfsterilisation bei 134 °C ergab eine unerwartet hohe, Prion-übersteigende, Resistenz von PA-assoziiertem a-Syn gegen Dampfsterilisation. Die Verlängerung der Sterilisationsdauer führte zu einem identischen Reduktionsfaktor des Seeding-Titers. Fourier-Transform-Infrarot (FT-IR) Spektroskopie wurde genutzt, um eine Stammdifferenzierung zwischen a-Syn Aggregaten aus PA-assoziierten transgenen Mausmodellen mit den a-Syn-Mutationen A30P und A53T vorzunehmen, die für familiäre PA beim Menschen prädisponieren. Etablierte Protokolle zur Untersuchung von Prionstämmen (PS) wurden verfeinert um 5 hamsteradaptierte PS zu vergleichen: 263K, 22A-H, BSE-H, CWD-H, und ME7-H. Hier führte die Zugabe des Farbstoffs Kongo Rot zu spektralen Veränderungen in einigen PS und resultierte in der erfolgreichen Unterscheidung aller untersuchten PS. Mittels PMCA wurden die winzigen Mengen an a-Syn Aggregaten aus Maus-Hirngeweben für die anschließende FT-IR Spektroskopie vervielfältigt. Die Spektren der A30P und A53T a-Syn Aggregate wiesen allerdings keine Unterschiede auf, und der Zusatz von Kongo Rot hatte keinen Effekt auf die Spektren. / Parkinson’s Disease (PD) is characterized by misfolding and aggregation of Alpha-Synuclein (a-Syn), which shows similarities to the behavior of prions in transmissible spongiform encephalopathies (TSEs). Pathological a-Syn aggregates, like prions, propagate by self-templated seeding. Due to reports of transmissible seeding activity of a-Syn, the goal of this thesis was to improve risk assessment of potential a-Syn seed transmissions. Real-Time Quaking-Induced Conversion (RT-QuIC) assays were used to quantify seeding active a-Syn in brain and stomach tissue of PD patients. The results show high seeding titers in both tissues, highlighting the need for effective inactivation protocols. Quantification of the effects of steam sterilization at 134 °C on the seeding activity of cerebral PD-associated a-Syn revealed a surprisingly high, prion-exceeding, resistance to steam sterilization. Extended duration of steam sterilization has no further inactivation effect and led to an identical reduction factor of the seeding activity titer. Fourier-Transform Infrared (FT-IR) spectroscopy was used to attempt strain differentiation between a-Syn aggregates from PD-associated transgenic mouse models with the a-Syn A30P and A53T mutations which predispose for familial forms of PD in humans. Established protocols for prion strain examination were refined to compare five hamster-adapted prion strains: 263K, 22A-H, BSE-H, CWD-H, and ME7-H. Here, the addition of a dye (Congo Red) led to spectral changes of some prion strains and resulted in the successful distinction of all investigated prion strains. To increase the tiny amounts of a-Syn aggregates in the brain tissues of mice, PMCA was used to multiply these aggregates for subsequent FT-IR spectroscopy. However, the spectra of the A30P and A53T a-Syn aggregates were undistinguishable, and the addition of CR had no significant effect on the spectra.

<p>The copper-binding ability of the prion protein may be closely connected to its function. Identifying the exact function of the prion protein can clarify the underlying mechanism in prion diseases. In this work, the copper-binding octapeptide region in the human prion protein has been studied. The structural characteristics of the binding site are examined by quantum chemical structural optimization. The calculations aim at identifying a substitute for copper(II) to be used in NMR-spectroscopic studies of the copper-binding region. The dynamical and structural features of the peptide region are investigated in molecular dynamics simulations. Aspects of importance in the development of model systems in molecular dynamics simulation are addressed.</p>

La tremblante atypique a été découverte suite à l'intensification du dépistage des encéphalopathies spongiformes transmissibles des petits ruminants. Les premiers éléments disponibles suggéraient que cette forme pourrait ne pas être d'origine infectieuse, à la différence de la forme classique de tremblante. Cette nouvelle forme a soulevé des questions sur sa dangerosité pour l'homme et l'animal et ses conséquences sur l'efficacité des programmes de lutte contre la tremblante. L'objectif global de la thèse, était de décrire la situation épidémiologique de la maladie chez les ovins et d'en explorer les facteurs de risque. Nous avons entrepris une étude descriptive des résultats de la surveillance de la tremblante en France et en Europe, une exploration des facteurs de risque liés aux pratiques d'élevage et à la génétique ainsi qu'une étude de l'agrégation des cas sur le plan géographique et dans les troupeaux atteints. Dans la plupart des cas, nous avons comparé nos résultats à ceux obtenus pour la tremblante classique. Ces études ont montré que la tremblante atypique avait une prévalence assez homogène entre les différentes sous-populations comparées de l'ordre de six cas pour dix mille animaux testés, ce qui contrastait avec les résultats pour la tremblante classique, nettement plus variables. Les facteurs de risque génétique étaient marqués et aucun facteur de risque évoquant une origine infectieuse de la maladie n'a été identifié. De plus, la prévalenve de la tremblante atypique n'est pas différente dans les troupeaux atteints et dans la population générale et les cas n'avaient pas tendance à s'agréger. Ces résultats confortent l'idée que la tremblante atypique est peu ou pas contagieuse, ce qui est compatible avec une origine non infectieuse.

The human prion protein (PrP) gene (PRNP) codon 129 (M/V) polymorphism is a susceptibility factor for variant Creutzfeldt-Jakob Disease (vCJD) and a major determinant of clinico-pathological phenotype in sporadic CJD. The role of codon 129 in defining susceptibility and strain transmission properties has been investigated in three lines of transgenic mice that express human PrP. The human PRNP gene has directly replaced the murine version, by gene targeting, and variation at codon 129 has given the three genotype lines (HuMM, HuMV, and HuVV). The genetics of these three mouse lines are otherwise identical, and therefore differences in transmission properties can be directly attributable to the codon 129 genotype. vCJD inoculation has shown that all three codon 129 genotype mice are susceptible with a ranking of transmission efficiency of HuMM>HuMV>HuVV. HuMM mice develop the most widespread neuropathology with features similar to human vCJD. Subclinical infection was noted in each mouse line. These data suggest that the vCJD strain is transmissible to humans of each of the three codon 129 genotypes, implying that non-MM cases of human infection with bovine spongiform encephalopathy (BSE) may exist but with long subclinical incubation periods. Inoculation of material from blood transfusion associated vCJD showed no change in transmission properties suggesting that the threat of a future epidemic of human-to-human vCJD infection has not been increased by adaptation of the vCJD strain. However the route of infection, for example via blood transfusion or surgery, may be more efficient that the original oral route of BSE infection. sCJD is classified into six subgroups according to clinico-pathological features, and defined by codon 129 genotype and electrophoretic mobility type (1 or 2) of disease associated PrPSc (MM1, MM2, MV1, MV2, VV1, VV2). Typical cases from each subgroup have shown specific transmission properties suggesting that the subgrouping is defining separate disease strains. The commonest subgroup (MM1) was the most transmissible and the HuVV mouse line the most susceptible host. These data outline the transmission risk from all sCJD types to recipients of each codon 129 genotype should an infection event occur, and show the significant role of recipient codon 129 genotype in defining the clinical or subclinical state and the success or failure of transmission. This is important for determining individual risk following known exposure, and for modelling the potential of iatrogenic infection from sCJD patients.

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.

Le rôle du domaine N-terminal (N-ter) de la protéine PrP dans le processus de conversion de la PrPc en un isoforme infectieux PrPSc est mal connu. L'objectif de ma de thèse a été de créer un prion synthétique composé de la partie N-ter de PrP et de la protéine Doppel (Dpl) pour mieux appréhender le rôle de cette région dans les mécanismes d'agrégation et de pathogenèse des maladies à prions. Trois protéines chimériques PrP/Dpl recombinantes ont été exprimées, purifiées et agrégées in vitro à en oligomères. Ces oligomères solubles présentent des caractéristiques biochimiques et structurales similaires à celles de la PrPSc: riches en feuillets β, résistants à la protéolyse, et formant des structures protofibrillaires. Les propriétés de ces agrégats nous ont conduit à explorer la relation qui existe entre la PrP et la protéine C1q du complément. In vitro, C1q participe de façon coopérative dans le processus d'agrégation et forme un complexe avec des oligomères de petite taille (12-15 mers) qui active la voie classique du complément. L'interaction de C1q avec les oligomères de chimères n'a pas de conséquence fonctionnelle. En parallèle, nous avons créé 10 lignées de souris transgéniques exprimant ces protéines PrP/Dpl, et établies sur fond PrP0/0. Ces protéines chimériques sont exprimées au niveau des rafts, comme la PrP. Les études fonctionnelles et infectieuses sont en cours. L'ensemble de nos données tend à montrer que le domaine N-ter de PrP ne suffit pas à transformer Dpl en un prion synthétique. Par ailleurs, une partie de nos travaux apporte une vision nouvelle quant au rôle de la protéine C1q vis-à-vis d'intermédiaires oligomériques dans les amyloïdoses.

<p>This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.</p><p>The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.</p><p>mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.</p><p>Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.</p>

Le tropisme cellulaire des prions infectieux diffère selon lespèce animale, celui-ci est corrélé à la souche infectieuse et à des facteurs spécifiques de lhôte. Par exemple, certains prions infectieux sont lymphotropiques, notamment en cas de scrapie chez les moutons et de variant de la maladie de Creutzfeldt-Jakob (vMCJ) chez lhomme. Par opposition, certains prions se caractérisent par un neurotropisme comme observé chez des patients Creutzfeldt-Jakob atteints de la forme sporadique ou chez des bovins atteints dencéphalopathies spongiformes bovines (ESB). Lhypothèse de notre travail repose sur les observations suivantes : dans le cas du variant de la maladie de Creutzfeldt-Jakob et des encéphalopathies spongiformes bovines, lagent responsable est identique, la voie dinoculation et les lésions neurologiques le sont également, seul le tropisme de cette souche pour les organes lymphoïdes diffère. En effet, les amygdales, la rate et lappendice sont infectieux chez lhomme. Par contre, linfectiosité est surtout confinée au niveau du système nerveux chez le bovin. Lors dune inoculation expérimentale par voie orale de lagent responsable de lESB chez les bovins, les plaques de Peyer iléales sont les seuls tissus lymphoïdes infectieux. Notre hypothèse de travail est que des propriétés de lhôte interviennent dans le tropisme de lagent infectieux. Deux axes de recherche ont été envisagés afin de vérifier cette hypothèse :  Lanalyse de la distribution des fibres nerveuses au sein des tissus lymphoïdes associés aux muqueuses (MALT) des espèces bovine et humaine  Létude de lexpression de PrPc et de ses isoformes au sein des tissus lymphoïdes et nerveux des espèces bovine et humaine. Pour atteindre au mieux nos objectifs, il nous manquait un outil essentiel permettant la caractérisation spécifique des FDC bovines. En effet, aucun marqueur spécifique de ces cellules nétait commercialisé. Nous avons donc produit, en collaboration avec le Centre dEconomie Rural de Marloie, un anticorps monoclonal spécifiquement dirigé contre les cellules folliculaires dendritiques (FDC) bovines. Cet anticorps nous a permis détudier la distribution des FDC au sein des organes lymphoïdes bovins. Une attention particulière a été portée aux FDC isolées à partir des plaques de Peyer jéjunales (PPJ) et iléales (PPI). Lapparente différence dinfectivité de ces tissus lymphoïdes chez des bovins atteints expérimentalement dESB nous a conduit à comparer les capacités fonctionnelles des FDC isolées à partir de PPJ et de PPI. Ces observations sont décrites et discutées dans le chapitre 1. Dans le chapitre 2, nous avons établi une cartographie des fibres nerveuses au sein des amygdales, des plaques de Peyer iléales et jéjunales bovines de plusieurs catégories dâge et ensuite comparé ce pattern dinnervation à celui des amygdales humaines; ceci permettra de pister les voies potentielles de neuro-invasion. Une attention particulière a été portée à linterface cellules folliculaires dendritiques fibres nerveuses. En effet, les FDC matures jouent un rôle prépondérant dans la pathogenèse des maladies à prion puisquen leur absence, une infection périphérique na pas lieu. De plus, la proximité entre fibres nerveuses et FDC est un paramètre intervenant dans la neuro-invasion; nous avons dès lors aussi analysé les contacts entre les FDC et les éléments nerveux. Lexpression de la PrPc est une condition sine qua non pour la formation de PrPres. Cette protéine cellulaire sert probablement de récepteur pour son homologue infectieux mais sert surtout de substrat pour lamplification de PrPres ; toute modification au niveau de sa synthèse pourrait entraîner un changement de la cinétique dinfection et pourrait expliquer lapparente absence dinfectivité constatée au niveau du système immunitaire chez les bovins. Lexpression tissulaire et cellulaire spécifique disoformes de la PrPc représente un facteur de lhôte potentiellement capable dinfluencer le tropisme cellulaire de lagent infectieux chez lhumain et le bovin. Cette expression a été étudiée dans les systèmes MALT bovins et humains. Pour affiner notre étude, nous avons analysé, par des techniques de western-blotting, le glycopattern de la PrPc ainsi que lexpression de ses formes tronquées dans les tissus lymphoïdes humains et bovins mais également dans des populations cellulaires spécifiques, les lymphocytes et les FDC. Afin de vérifier si les isoformes de PrPc sont spécifiques aux tissus lymphoïdes, nous avons effectué une étude comparative du pattern de glycosylation et du ratio des formes clivées de PrPc, exprimés au sein de différentes régions du système nerveux central bovin et humain. Les résultats de ces travaux sont repris dans le chapitre 3.