The host encoded cellular prion protein (PrPC) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrPC can undergo conversion into a conformationally-altered isoform (PrPSc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Thus, tissues expressing PrPC are potential sites for conversion of PrPSc during TSE pathogenesis. Although much is known about the role of PrPSc in prion diseases, the normal function of PrPC is poorly understood. Lines of mice and cattle in which PrPC has been ablated by gene knockout show no major phenotypical alterations other than resistance to TSE infection. However, recent reports using Prnp-null mouse models have suggested the participation of PrPC in neural stem/progenitor cell proliferation and differentiation. The first objective in our study was to map the expression of PrPC in twenty six somatic and reproductive tissues in ruminants. Our second objective was to characterize the ontogeny of PrPC expression during bovine embryonic and early fetal development. Finally, we used a mouse embryonic stem cell (mESC) model to study the potential role of PrPC during neurogenesis. In adult tissues, intense expression of PrPC was detected in the central nervous system (CNS), thymus and testes, whereas the liver, striated muscle and female reproductive tissues showed the lowest expression. We observed that PrPC was associated with tissues undergoing cellular differentiation including spermatogenesis, lymphocyte activation and hair follicle regeneration. Analyses in bovine embryos and fetuses indicated peaks in expression of PrPC at days 4 and 18 post-fertilization, stages associated with the maternal-zygote transition and the maternal recognition of pregnancy and initiation of placental attachment, respectively. Later in development, PrPC was expressed in the CNS where it was localized in mature neurons of the neuroepithelium and emerging neural trunks. Based on these observations, we hypothesized that PrPC was involved in neurogenesis. We tested this hypothesis in a murine embryonic stem cell model (mESC). mESC were induced to form embryoid bodies (EBs) by placing them in suspension culture under differentiating conditions and allowed to differentiate in vitro for 20 days. We detected increasing levels of PrPC starting on day 12 (8.21- fold higher vs. day 0; P < 0.05) and continuing until day 20 (20.77-fold higher vs. day 0; P < 0.05). PrPC expression was negatively correlated with pluripotency marker Oct-4 (r= -0.85) confirming that mESC had indeed differentiated. To provide a more robust system for assessing the role of PrPC in neural differentiation, mESC were cultured with or without retinoic acid (RA) to encourage differentiation into neural lineages. Induction of EBs with retinoic acid (RA) resulted in an earlier up-regulation of PrPC and nestin (day 12 vs. day 16; P < 0.05). In addition, immunofluorescence studies indicated co-expression of PrPC and nestin in the same cells. The results of these experiments suggested a temporal link between PrPC expression and expression of nestin, a marker of neural progenitor cells. We next tested whether PrPC was involved in RA-enhanced neural differentiation from mESC using a PrPC knockdown model. Plasmid vectors designed to express either a PrP-targeted shRNA or scrambled, control shRNA were transfected into mESC. Stable transfectants were selected under G418 and cloned. PrP-targeted and control shRNA clones, as well as wild-type mESC, were differentiated in presence of RA and sampled as above. PrPC expression was knocked down in PrP-targeted shRNA cultures between days 12 and 20 (62.2 % average reduction vs. scrambled shRNA controls). Nestin expression was reduced at days 16 and 20 in PrPC knockdown cells (61.3% and 70.7%, respectively vs. scrambled shRNA controls). These results provide evidence that PrPC plays a role in the neural differentiation at a point up-stream from the stages at which nestin is expressed. In conclusion, the widely distributed expression of PrPC in ruminant tissues suggests an important biological role for this protein. In the present work we have provided evidence for the participation of PrPC in the differentiation of mESC along the neurogenic pathway. / Ph. D.

Prionkrankheiten sind übertragbare, tödliche neurodegenerative Erkrankungen beim Menschen und bei Tieren. Sie basieren auf der Konversion des zellulären Prion-Proteins (PrPC) in seine pathogene Form (PrPSc). Durch diese Konversion sind Prionkrankheiten pathogen und übertragbar. Bis heute ist weder der dem zugrunde liegende Mechanismus verstanden noch eine Behandlung gefunden worden. Die sichere Diagnose einer sporadischen Prionkrankheit ist ausschließlich mittels Gehirnbiopsie möglich, weswegen zu Lebzeiten des Patienten häufig nur die Diagnose einer wahrscheinlichen Prionkrankheit erfolgt. Zusammen mit der klinischen Heterogenität der Prionkrankheiten weisen neueste Erkenntnisse auf das Vorhandensein mehrerer humaner Prionstämme hin. Für die Suche nach Medikamenten fehlt ein geeigneter Wirkstoff-Suchtest, der auf der humanen Pathogenese basiert, für einen hohen Durchsatz geeignet und gut reproduzierbar ist. Die real-time quaking-induced conversion (RT-QuIC), eine neu entwickelte in vitro-Methode, erlaubt den Nachweis von bisher nicht messbaren Mengen an PrPSc in humanem Liquor cerebrospinalis (Liquor). Dazu werden die selbst-replizierenden Eigenschaften des PrPSc genutzt. Erste Untersuchungen weisen auf distinkte Eigenschaften humaner Prionkrankheiten in der RT-QuIC hin. Zum Einsatz in Diagnostik und Forschung bedarf es jedoch einer umfassenden Validierung der Methode für die Anwendung mit humanem Liquor. In dieser Arbeit beträgt die Sensitivität der RT-QuIC 85,5 % und die Spezifität 99,5 % für humane Prionkrankheiten. Die Reproduzierbarkeit im Ringversuch ist gut bis exzellent. Die Kurzzeitlagerungen der Liquorproben bei Raumtemperatur und +4°C sowie die Langzeitlagerung bei −80°C und das wiederholte Einfrieren und Auftauen haben keinen Einfluss auf die Testergebnisse. Jedoch führt die Kontamination mit Blut zu falsch-negativen Resultaten. Diese Ergebnisse weisen auf eine Eignung der RT-QuIC zur sicheren Diagnose von Prionkrankheiten zu Lebzeiten der Patienten hin. Zur Charakterisierung des Reaktionspotentials möglicher humaner Prionstämme wurden Liquorproben von verschiedenen humanen Prionkrankheiten wie bspw. der sporadischen und der genetischen Form mittels RT-QuIC untersucht. Die Auswertung der Daten zeigt distinkte Eigenschaften des PrPSc im Liquor, die moduliert werden durch die Form der Prionkrankheit, den Prnp Codon 129-Genotyp und die Krankheitsdauer. Diese Ergebnisse zeigen das Potential der RT-QuIC, die selbst-replizierenden Eigenschaften des PrPSc im Liquor zu untersuchen, womit erstmals eine Methode zur Verfügung steht, um diese Effekte in Patienten während der symptomatischen Phase zu studieren. Zur Nutzung der RT-QuIC als neuartige Methode zur Wirkstoffsuche wurde die Wirkung mehrerer Stoffe auf die RT-QuIC-Reaktion untersucht. Doxyzyklin inhibiert diese Reaktion sowohl in Korrelation mit der Dosis als auch mit dem Zeitpunkt der Zugabe. Diese Ergebnisse weisen auf eine Eignung der RT-QuIC zur Suche von Stoffen hin, die den PrP-Konversionsprozess inhibieren und zeigen die inhibierende Wirkung von Doxyzyklin auf die in-vitro-Amplifikation von PrPSc.

Prionen sind das infektiöse Agens transmissibler spongiformer Enzephalopathien von Tieren und Menschen. Prionen bestehen hauptsächlich aus einer abnormal gefalteten und aggregierten Isoform des zellulären Prionproteins (PrP). Die Replikation von Prionen findet mutmaßlich durch keiminduzierte Polymerisation des Prionproteins statt. Es existieren verschiedene Prionstämme, die unterschiedliche Eigenschaften aufweisen, aber vom selben zellulären Prionprotein abstammen können. Neben PrP scheinen Kofaktormoleküle an der Prionreplikation beteiligt zu sein. Weiterhin wird angenommen, dass Kofaktoren bei der Definition von Stammeigenschaften beteiligt sind, sowie ein Einfluss auf die Infektiosität von Prionen besteht. In dieser Arbeit wurden die Auswirkungen verschiedener Kofaktoren auf die Replikation von vier Hamster-adaptierten Prionstämmen in vitro mittels der Methode der „Protein Misfolding Cyclic Amplification“ (PMCA) untersucht. Es wurden stammabhängige Unterschiede bezüglich der Anforderungen an die Replikationsbedingungen in der PMCA, sowie Kofaktor-Selektivitäten festgestellt. Der Einfluss von Kofaktoren wurde durch den Vergleich ausgewählter biologischer, biochemischer und biophysikalischer Eigenschaften von in vitro erzeugten PMCA Produkten (PrPres) mit denen nativer Prionkeime untersucht. Es zeigte sich, dass Kofaktoren Stammeigenschaften, wie die biologische Keimaktivität in primären Gliazellkulturen und biochemische Eigenschaften, wie die Migration in SDS-Gelen, beeinflussen können. Um festzustellen, ob unterschiedliche Kofaktorbedingungen während der PMCA messbare Veränderungen der Proteinkonformation hervorrufen, wurde PMCA generiertes PrPres mittels FT-IR Spektroskopie in einer Pilotstudie charakterisiert. Erste Befunde zeigten spektrale Unterschiede zwischen den Proteinkeimen und deren PMCA Produkten bei allen Stämmen, unabhängig von den Kofaktorbedingungen. / Prions are the causative agent of transmissible spongiform encephalopathies in animals and humans such as scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). Prions are thought to be composed essentially of a misfolded and aberrantly aggregated isoform of the cellular prion protein (PrP) and to replicate by seeded PrP polymerization. Prions may exist in the form of distinct strains that differ in their phenotypic characteristics although they are derived from the same cellular prion protein. Cofactor molecules other than PrP may be involved in prion replication and may be a determinant of strain properties. Furthermore, cofactors may also be required for conveying infectivity. The present study examined the effects of different cofactor molecules on the replication efficacy of four hamster adapted prion agents using the method of serial protein misfolding cyclic amplification (PMCA) as in vitro assay for PrP misfolding and aggregation. The study revealed strain dependent differences of PMCA conditions and cofactors required for efficient in vitro replication. The impact of cofactors was assessed by comparative analyses of selected biological, biochemical and biophysical properties of PMCA products (PrPres) and native prion seeds. The biological seeding activity as monitored in a primary hamster glial cell assay, and biochemical properties such as electrophoretic migration in SDS-gels, were affected differently by different cofactors. In order to define the impact of putative cofactors on the molecular conversion of PrP in more detail, changes in the spatial structure associated with different cofactor molecule conditions during amplification of PrPres in PMCA was monitored by Fourier transform-infrared (FT-IR) spectroscopic analysis. Largely preliminary data revealed spectral differences between native prion seeds and progeny PMCA generated PrPres for all prion strains, but no variations due to different cofactor conditions.

No description available.

La maladie sporadique de Creutzfeldt-Jakob (sCJD) est une encéphalopathie spongiforme transmissible mortelle caractérisée par une large gamme de manifestations cliniques et pathologiques. Les caractéristiques pathologiques de la SDMC dépendent en grande partie de la présence d'une forme mal repliée de protéine prion cellulaire (PrPC); Connu sous le nom de PrPSC et polymorphisme (méthionine et valine) au codon 129 du gène PRNP qui code pour PrPC. Les facteurs étiologiques exacts de la MCSJ sont encore inconnus. Le génotype Codon 129 du gène PRNP et le type de PrPSC (type 1 ou type 2) influent sur l'hétérogénéité de la maladie telle que définie par des caractéristiques pathologiques spécifiques de la région qui peuvent réguler les voies moléculaires qui conduisent au développement de phénotypes pathogènes dépendants des sous-types. Dans cette étude, nous avons étudié l'ensemble de la réglementation protéomique dans la région cerebrale du cerveau des deux sous-types les plus répandus (MM1 et VV2) des patients atteints de SDMC utilisant une électrophorèse sur gel bidimensionnelle (2DE) et une spectrométrie de masse. L'analyse de toutes les taches de protéines sur les gels 2DE avec le logiciel DECODON Delta2D a révélé vingt-cinq taches de protéines différenciées et l'identification de ces taches avec MALDI-TOF MS / MS a révélé quatre-vingts trois protéines différentiellement réglementées dans les deux sous-types dans la région du cervebelle du cerveau par les patients atteints de sCJD. Quarante protéines dans le sous-type MM1 et quarante-trois protéines dans le sous-type VV2 ont été classées. Douze protéines étaient communément réglementées dans les deux sous-types, dont cinq d'entre elles présentaient une régulation expresnelle inverse et le repos sept avait une régulation expresnelle similaire dans les deux sous-types. Les trois principaux mécanismes moléculaires cellulaires réglementés dans les deux sous-types comprennent: i) le cycle cellulaire; L'expression des gènes et la mort cellulaire, ii) - la réponse au stress cellulaire / le stress oxydatif et iii) - la transduction du signal et les fonctions synaptiques La plupart des protéines sous la classification des réponses au stress cellulaire étaient associées aux fonctions moléculaires cellulaires liées au stress oxydatif. DJ-1 qui est un capteur bien connu de stress oxydatif, a également été jugé réglementé dans la catégorie des réponses au stress cellulaire. Le DJ-1 protège les cellules contre le stress oxydatif directement en translocant au noyau pour l'activation de gènes antioxydants et indirectement en activant la voie Nrf2 / ARE. Nos résultats expérimentaux ont démontré l'activation de la voie Nrf2 / ARE dans les sous-types MM1 et VV2 de sCJD. Le DJ-1 a également montré une régulation positive significative dans son expression de l'ARNm dans les sous-types MM1 et VV2 mais l'expression des protéines uniquement dans le sous-type VV2 dans le cervelet du cerveau des patients atteints de sCJD. En outre, l'expression de la protéine DJ-1 a également été augmentée au cours des stades pré-symptomatiques et symptomatiques dans le cervelet du cerveau des modèles de souris de sCJD (MM1 et VV2) et pendant le stade clinique dans les échantillons de CSF des patients atteints de SDMC. Ces résultats suggèrent l'implication du stress oxydatif lors de la pathophysiologie de la SDMC et l'utilisation de DJ-1 comme capteur potentiel de stress oxydatif pendant la phase clinique de la sCJD.

O GBM é o tipo mais agressivo de glioma, apresentando células indiferenciadas (CTGs), responsáveis pela proliferação, invasão e recidiva tumoral. Avaliamos o papel da proteína PrPC e seu ligante HOP na proliferação e autorrenovação de CTGs. Cultivamos linhagens de GBM humano em neuroesferas e geramos populações knockdown para PrPC e HOP. Observamos co-localização de PrPC e CD133 na superfície e sua internalização conjunta estimulada por cobre, sugerindo recrutamento de CD133 mediado por PrPC. O silenciamento de PrPC reduz a expressão de marcadores de células-tronco e autorrenovação, diminui a expressão de proteínas de adesão e afeta a migração celular. O silenciamento de HOP reduz a proliferação, recuperada com o tratamento com HOP em células PrPC+. A capacidade tumorigênica e proliferativa de neuroesferas knockdown para PrPC e/ou HOP in vivo é reduzida. Finalmente, um peptídeo de HOP que bloqueia a interação com PrPC inibe a proliferação e autorrenovação em células PrPC+, indicando potencial do complexo PrPC-HOP como alvo para terapias contra o GBM. / GBM is the most aggressive type of glioma, presenting undifferentiated cells (GSCs), responsible for proliferation, invasion and tumor recurrence. We evaluated the role of the PrPC and its ligand HOP in the proliferation and self-renewal of GSCs. We cultured human GBM lineages in neurospheres and generated knockdown populations for PrPC and HOP. We observed co-localization of PrPC and CD133 on the surface and their co-stimulated copper internalization, suggesting PrPC-mediated recruitment of CD133. PrPC silencing reduces the expression of stem cell markers and self-renewal, decreases adhesion proteins expression, and affects cell migration. HOP silencing reduces proliferation, recovered with HOP treatment in PrPC+ cells. The tumorigenic and proliferative capacity of neurospheres PrPC and/or HOP knockdown in vivo is decreased. Finally, a HOP peptide which blocks PrPC-HOP interaction inhibits proliferation and self-renewal in PrPC+ cells, indicating PrPC-HOP complex potential as a target for therapies against GBM.

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 &#947;1 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 &#947;-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.

Diese Arbeit befasst sich mit der Kinetik der Fehlfaltung und Aggregation von Proteinen. Anhand dreier Beispiele, der Phosphoglyceratkinase (PGK) aus Hefe, einer Variante von Barstar und des Prion-Proteins des Syrischen Hamsters (SHaPrP(90-232)) wurde insbesondere die Kinetik der Bildung von Amyloidfibrillen und deren kinetischer Vorläuferstrukturen mittels dynamischer und statischer Lichtstreuung, Circulardichroismus, Infrarotspektroskopie, Elektronenmikroskopie und teilweise analytischer Chromatographie untersucht. Die Kinetiken wurden mit Konzepten der Aggregationstheorie von Kolloiden und der chemischen Kinetik beschrieben. Die Modellierung der Kinetiken weist ausgehend von der monomeren PGK bei pH 2 und 190 mM NaCl auf eine zweistufige Reaktionskaskade, bestehend aus irreversiblen, bimolekularen Elementarschritten hin. Während der ersten Stufe wird ein engverteiltes Ensemble von Oligomeren mit einer mittleren Masse von 10 Monomeren und wesentlichen Anteilen an beta-Faltblattstrukturen gebildet. Die Protofibrillen entstehen durch die Vereinigung der strukturell polaren Oligomere, die durch die erste Reaktionsstufe bereitgestellt werden und als kritische Oligomere bezeichnet werden. Die gefundene Kopplung des Wachstums der intermediären Zustände und die Zunahme der beta-Faltblattstruktur kann innerhalb eines verallgemeinerten Diffusions-Kollisions-Modells interpretiert werden, bei dem die beta-Stränge durch intermolekulare Wechselwirkungen stabilisiert werden. Die Fehlfaltung und Aggregation des SHaPrP(90-232) bei pH 4.2 und 1 M GuHCl und geeigneten Zusätzen an Salz zeigt einen augenscheinlichen Zweizustandsübergang mit hoher Reaktionsordnung ( >2.5) zwischen dem monomeren, alpha-helikalen Ausgangszustand und einem beta-faltblattreichen, ringförmigen Oktamer. Die Progresskurven der Umwandlung der Sekundärstruktur lassen sich mit dem Zeitverlauf einer bimolekularen Reaktion anpassen. Das Oktamer bildet bei hohen eingesetzten Proteinkonzentrationen Multimere. Auf sehr langen Zeitskalen setzt die Bildung von Protofibrillen ein. Das kritische Oktamer stellt die Vorstufe der nachgeschalteten Wachstumsphänomene dar. Unter geeigneten Umgebungsbedingungen kann der nicht-nativ, partiell gefaltete Zustand von Barstar bei niedrigem pH (A-Zustand) in einem zweistufigen Prozess erst in Protofibrillen und anschlie"send in reife Amyloidfibrillen konvertiert werden. Zur Aktivierung der Konversion des oligomeren A-Zustandes (mittlere Masse von 16 Monomeren) sind moderate Ionenstärken ([NaCl]>0) und erhöhte Temperaturen (T>50°C) notwendig. Die Bildung der Protofibrillen ist unabhängig von der eingesetzten Proteinkonzentration. Bei Raumtemperatur und entsprechender Ionenstärke bilden sich amorphe Aggregate. Dagegen führt die Erhöhung der Temperatur in Abwesenheit von Salz zur Dissoziation des oligomeren A-Zustandes. Alle drei Proteine müssen zur Ausbildung protofibrillärer Strukturen und gegebenenfalls reifer Fibrillen oligomere Zustände mit partiell gefalteter Konformation einnehmen. Diese kritischen Oligomere sind langlebige Intermediate, die den Dreh- und Angelpunkt für die Bildung nachgeordneter Strukturen darstellen. Die Bildung von Amyloidfibrillen ist somit ein mehrstufiger hierarchischer Strukturbildungsprozess. Die in der Literatur bekannten Modelle der nukleierten Polymerisierung und der nukleierten Konformationskonversion werden dem höchstens in gewissen Teilaspekten gerecht. Die Annahme einer universellen Kinetik der Amyloidbildung kann im Lichte der Ergebnisse dieser Arbeit nicht aufrechterhalten werden. Dagegen scheinen die Zustände des kritischen Oligomers und der Protofibrille als Hierarchiestufen der Amyloidbildung generische Bestandteile des Prozesses zu sein. Die Kinetik der Bildung der verschiedenen Hierarchiestufen weist keine nennenswerten Gemeinsamkeiten zwischen den drei untersuchten Proteinen auf. / This thesis deals with the kinetics of misfolding and aggregation of proteins. The kinetics of amyloid formation and precursors of three proteins, phosphoglcerate kinase (PGK), a barstar variante and the Syrian hamster Prion protein (SHaPrP(90-232)) were investigated by the use of dynamic and static light scattering, infrared spectroscopy, circular dichroism, electron microscopy and in part by analytical chromatography. The kinetics were described with concepts from the theory of colloidal aggregation and chemical kinetics. The modelling of the kinetics starting from the monomeric PGK at pH 2 and 190 mM NaCl points to a two stage reaction cascade built up by irreversible, bimolecuar elementary reaction steps. During the first stage a narrow distributed ensemble of oligomeric states with an average mass of ten monomers and essentially ordered amounts of beta-sheet structure is built up. Protofibrils are formed by coalescence of the structural polar oligomers provided by the first stage which are termed critical oligomers. The found coupling between growth and acquisition of beta-sheet structure is interpreted in terms of a generalized diffusion-collision model, where stabilization takes place by intermolecular interactions. The misfolding and aggregation of SHaPrP(90-232) shows an apparent two-state transition between the initial monomeric, alpha-helical state and an beta-sheet rich, annular octamer with high reaction order (>2.5) at pH 4.2 and 1 M GuHCl with appropriate amounts of salt added. Progress curves monitoring the secondary structure transition can be fitted by the time-course of bimolecular reactions. The octamer forms multimers at high protein concentrations. Formation of protofibrils sets up on very long time-scales. The critical octamer is a precursor for all subsequent growth processes. The non-native, partially folded state of barstar at low pH (A-state) can be converted in a two-stage process first to protofibrils and then to mature amyloid fibrils under appropriate environmental conditions. Conversion of the oligomeric A-state (average mass of 16 monomers) can be activated by elevated temperatures (T>50°C) in the presence of moderate amounts of salt ([NaCl]>0). Formation of protofibrils is independent of protein concentration. Amorphous aggregates are formed at room temperature with sufficient amounts of salt added. In contrast elevated temperatures in absence of salt lead to dissociation of the oligomeric A-state. All three proteins have to populate an oligomeric, partially folded state to form protofibrils and eventually mature fibrils. These critical oligomers are long-lived intermediates which are the pivotal point from which all other structures arise. Formation of amyloid fibrils is a hierarchical assembly process where structures are built up by several stages. Models known from the literature, in particular nucleation polymerization and nucleated conformational conversion, only master partial aspects of amyloid formation. The wide-spread assumption of a universal kinetics of amyloid formation turns out to be unjustified. In contrast, the states of critical oligomer and protofibril seem to be generic parts of the hierarchical assembly process. Comparison of the kinetics of each hierarchical level amoung the three investigated proteins shows no considerable similarities.

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.

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 &#946;-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 &#946;-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.