Les prions sont des protéines qui suite à des changements de conformation acquièrent un caractère infectieux. Ils sont à l’origine de traits dominants, héritables de façon non-Mendélienne, chez les mammifères, les champignons filamenteux et les levures. Le mauvais repliement et l’agrégation des protéines sont à l’origine de plus de 40 maladies, parmi lesquelles on retrouve des maladies neurodégénératives telles que les maladies d’Alzheimer, de Parkinson et de Huntington. Il a été montré que les formes agrégées des protéines supposées responsables de ces maladies (i.e. peptide amyloïde-β, tau, α-synucléine, huntingtine) se propagent de cellule en cellule à la manière des prions. La levure Saccharomyces cerevisiae possède plusieurs prions qui sont autant d’excellents modèles biologiques pour la compréhension des mécanismes de formation et de propagation des prions.[PSI+] et [URE3], issus respectivement de la conversion sous forme prion du terminateur de la traduction Sup35p et d’un régulateur du métabolisme azoté Ure2p, sont à ce jour les deux prions les mieux documentés chez la levure. Les chaperons moléculaires et leurs co-chaperons modulent la formation, la réplication et la propagation des prions chez la levure. Cependant, l’élimination ou la dégradation de ces prions sont encore mal connus. Notre laboratoire a montré que le protéasome 26S est capable de dégrader les formes soluble et fibrillaire de Sup35p. Dans la première partie de ma thèse, nous avons étudié le rôle du protéasome 26S dans la dégradation des formes soluble et fibrillaire d’Ure2p. Nous avons montré que, comme pour Sup35p, le protéasome 26S dégrade Ure2p soluble en générant des peptides amyloïdes issus du domaine prion N-terminal ainsi qu’un fragment C-terminal résistant à la protéolyse. Nous avons montré que le domaine prion déstructuré est nécessaire pour la reconnaissance et la dégradation par le protéasome. Contrairement à ce qui avait été observé pour Sup35p, Ure2p sous sa forme fibrillaire est totalement résistante à la dégradation protéasomale. Nous suggérons que la variabilité structurale aux seins des particules de prions dans un contexte cellulaire dicte leurs interactions avec les machineries protéolytiques, et plus particulièrement avec le protéasome.Les prions de levure ont principalement été étudiés dans un contexte de cellules en division active. Cependant, dans la nature, la plupart des cellules sont retrouvées dans un état quiescent post-mitotique. Nous n’avons que très peu d’informations sur le devenir des particules de prions lorsque les cellules entrent dans un état quiescent. De même les conséquences physiologiques des prions sur la survie à long terme des levures sont très peu documentées. Dans la seconde partie de ma thèse, nous avons utilisé le prion [PSI+] comme modèle pour répondre à ces questions. Différentes conformations des agrégats de Sup35p conduisent à des souches phénotypiquement distinctes du prion [PSI+]. Nous avons constaté que les agrégats de Sup35p subissent des changements ultra-structuraux et fonctionnels au cours des différentes phases de croissance cellulaire. Ainsi, nous avons observés des changements importants dans la distribution de taille et dans l’infectiosité des polymères de Sup35p résistants au SDS formant les briques élémentaires du prion [PSI+]. Ces changements interviennent sans affecter les informations structurales spécifiques à chaque souche de prion [PSI+]. De façon remarquable, bien que [PSI+] n’affecte pas le taux de croissance des levures, ce prion semble prolonger significativement la durée de vie des levures. Cet effet bénéfique semble pouvoir se fixer de façon efficace et permanente dans les cellules et persister même après élimination de [PSI+]. La fixation génétique de caractéristiques épigénétiques induites par [PSI+] ont été déjà observées et l’ensemble de ces résultats suggère que [PSI+] (et éventuellement d’autres prions) peut jouer le rôle de capaciteurs évolutifs transitoires. / “Proteinaceous infectious particles”, or prions, are self-perpetuating alternate conformations of proteins that are responsible for heritable non-Mendelian traits in mammals, filamentous fungi and yeast. On a more general note, protein misfolding and aggregation is at the origin of over forty protein folding disorders including devastating neurodegenerative diseases such as Alzheimer’s, Parkinson’s or Huntington’s diseases. The aggregated proteins responsible for these diseases (i.e. amyloid-β peptide/tau, α-synuclein and huntingtin) were shown to propagate from cell to cell in a prion-like manner. The yeast Saccharomyces cerevisiae hosts many prion or prion-like proteins, unrelated in sequence and function, which proved to be excellent models for understanding the dynamics of prion aggregation and distribution upon cell division.Sup35p and Ure2p which cause the [PSI+] and [URE3] heritable traits, respectively, stand out as the most studied and best characterized yeast prions to date. A plethora of cellular factors, mostly belonging to various molecular chaperone families, were shown to affect yeast prion formation and propagation. Clearance of protein aggregates and prion particles is however poorly understood and documented. Our laboratory showed that the 26S proteasome degrades both the soluble and prion-associated fibrillar forms of Sup35p. In the first part of my thesis, we investigated the role of the 26S proteasome in the degradation of the soluble and fibrillar forms of Ure2p. We found that, as with Sup35p, the 26S proteasome is able to degrade the soluble native Ure2p, generating an array of amyloidogenic N-terminal peptides and a C-terminal fragment which is resistant to proteolysis. The N-terminal prion domain was shown to act as a degron required for proteasomal engagement and degradation. In contrast to Sup35p, fibrillar Ure2p resisted proteasomal degradation. We expect the structural variability within prion assemblies in a cellular context to dictate their interaction with proteolytic machineries in general and the proteasome in particular.The biology of yeast prions has been mostly explored in the context of logarithmically dividing cells. In nature however, most cells are generally in a post-mitotic non-dividing quiescent state. Yet little is known about the fate and properties of prion particles upon yeast cells entry into the stationary or quiescent states and the physiological consequences of harboring these prions throughout the lifespan of yeast cells. In the second part of my thesis, we addressed this issue using the [PSI+] prion as a model. Structurally different conformers of Sup35p aggregates can lead to distinct [PSI+] strains with different prion phenotypes. We found that Sup35p prion particles undergo growth phase-dependent ultrastructural and functional changes. Indeed, the size distributions of SDS-resistant core-prion particles significantly change during growth without affecting the structural information specific to each prion strain. The infectious properties of Sup35p prion particles undergo dramatic growth phase-dependent changes. Importantly, we found that while [PSI+] has little to no effects on the growth rates of yeasts, it robustly prolongs their chronological lifespan. Furthermore, this beneficial effect can then be permanently and efficiently fixed in the cells even when [PSI+] is subsequently lost. Similar genetic fixation of [PSI+]-induced epigenetic characteristics were previously observed and suggested [PSI+] (and possibly other prions) can act as transient evolutionary capacitators.

The pathology of Alzheimer's disease (AD) remains elusive. Competing evidence links amylois \U+fffd\-peptide (A\U+fffd\) amyloid formation to the phenotype of AD (1). The mechanism of amyloid fibril formation has been an ongoing investigation for many years. A\U+fffd\10-23 peptide, a fragment of A\U+fffd\1-42 peptide, contained crucial hydrophobic core residues (2). In this study, an investigation was launched to study the aggreagation process of A\U+fffd\1023 peptide and its ability to form amyloid fibrils. Furthermore, the presence of its hydrophobic core showed importance for its ability to aggregate and form amyloid fibrils. Thereafter, the inhibition of A\U+fffd\1-42 peptide aggregation was studied by using pyrimidine-based compounds. A\U+fffd\1-42 peptides, known to be neurotoxic, aggregate to form amyloid fibrils (3). This investigation may provide insight into the development of novel small molecular candidates to treat AD. / by Ahmad Alex Hijazi. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Les maladies à prions et d’Alzheimer appartiennent à un groupe de maladies neurodégénératives caractérisées par l’accumulation dans le système nerveux central (SNC) de protéines amyloïdes, respectivement la PrPSc et les peptides Aβ. Même si ces maladies ont des étiologies et des manifestations physiopathologiques distinctes, il est suspecté que des mécanismes communs de neurodégénérescence puissent être mobilisés dans ces différentes affections du SNC. Les maladies à prions s’imposent comme un paradigme qui permet l’étude des maladies neurodégénératives amyloïdes. Disposer d’un agent infectieux, la protéine prion scrapie, PrPSc, présente l’avantage de pouvoir initier un processus neurodégénératif et de cerner la nature et la séquence des événements menant à la perte d’homéostasie neuronale. Les mécanismes mis en évidence grâce à l’infection à prions peuvent être objectivés dans d’autres maladies neurodégénératives. Dans les maladies à prions, il est clairement établi que la PrPSc exerce sa toxicité dans les neurones en déviant la/les fonction(s) de la forme non pathologique des prions, la protéine prion cellulaire, PrPC. Les travaux du laboratoire ont permis d’assigner une fonction de signalisation à la PrPC et d’identifier plusieurs intermédiaires de signalisation contrôlés par la PrPC, ce qui a conduit à proposer plusieurs rôles pour la PrPC dans les neurones : régulation de l’équilibre d’oxydoréduction, adhérence, neuritogenèse, survie, contrôle des fonctions associées au neuromédiateur. Une partie de mes travaux de thèse a permis d’illustrer une nouvelle facette de la PrPC dans le contrôle des fonctions neuronales. Au travers d’un couplage à la kinase Lyn et d’une interaction avec la protéine LRP1 et le cuivre, la PrPC du corps cellulaire gouverne l’état d’activation de la kinase GSK3β, qui à son tour, contrôle le trafic et l’activité d’un autorécepteur sérotoninergique, le récepteur 5HT1B. En modulant l’activité de ce récepteur, la PrPC favorise la neurotransmission. A partir de l’infection à prions, mes travaux dévoilent des mécanismes de neurodégénérescence communs aux maladies à prions et à la maladie d’Alzheimer (AD). Dans les neurones infectés par les prions, comme les neurones dérivés de souris modèles pour AD, la suractivation de la kinase PDK1 provoque la phosphorylation et l’internalisation de l’αsécrétase TACE, ce qui annule l’activité neuroprotectrice de TACE à la membrane plasmique. TACE internalisée est découplée de trois de ses substrats, (i) la PrPC, ce qui favorise sa conversion en PrPSc, (ii) la protéine précurseur des peptides amyloïdes APP, ce qui augmente la production des peptides neurotoxiques Aβ et (iii) les récepteurs au TNFα, ce qui rend les neurones malades vulnérables au stress inflammatoire. In vitro comme in vivo, l’inhibition de PDK1 permet de rétablir l’activité neuroprotectrice de TACE et de contrecarrer les effets neurotoxiques de la PrPSc ou de Aβ. Mes travaux établissent également que les Rho kinases (ROCK) sont des régulateurs positifs de l’activité de PDK1. Dans un contexte physiologique, les ROCK interagissent avec PDK1 et phosphorylent PDK1, contribuant à son activité basale. Dans un contexte infectieux, le gain d’activité des ROCK augmente le « pool » de molécules de PDK1 qui interagissent avec et sont phosphorylées par les ROCK, à l’origine de la suractivation de PDK1. Inhiber les ROCK exerce un double effet protecteur dans les neurones infectés par les prions en abaissant le niveau de PrPSc via le module de signalisation PDK1TACE et en préservant la polarité et la connectivité des neurones par action sur le cytosquelette d’actine. Le module ROCKPDK1 émerge comme une cible thérapeutique potentielle pour les maladies à prions et autres maladies neurodégénératives amyloïdes. / Pas de résumé en anglais

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 183-205).

To protect public health, detection methods have been developed to monitor drinking water for pathogens. The goal of this dissertation is to evaluate and utilize novel methods that enhances detection and further reduces the risk of waterborne pathogens. The study in Appendix A developed a method to monitor the microbial quality of treated drinking water at the tap utilizing point-of-use (POU) filter. Tap water supplies were monitored in vending machines throughout Southern Arizona using solid block carbon (SBC) filters as a monitoring tool. Out of 48 SBC filters 54.2% were positive for at least one organism. The number of filters positive for total coliforms, E. coli, Enterococci, and enterovirus was 13, 5, 19, and 3, respectively, corresponding to 27.1%, 10.4%, 39.6%, and 6.3% of the total filters. These results suggest that the SBC filter can be used to monitor large volumes of treated drinking water and detect the incidence of indicators and pathogens. The study in Appendix B evaluated the fate of infectious prions in multiple water sources quantitatively utilizing a method that only detects infectious prions. A reduction of PrPˢᶜ was observed at 25°C and 37°C ranging between 0.41-log₁₀ and 1.4-log₁₀ after 1 week. After 8 weeks at 25°C and 37°C, inactivation ranged between 1.65-log₁₀ and 2.15-log₁₀. A maximum rate of inactivation in water occurred at 50°C, ranging from 2.0-log₁₀ and 2.51-log₁₀ after one week. The results from all types of water suggest that dissolved organic matter and temperature influence PrPˢᶜ infectivity. The study in Appendix C evaluated real-time sensors for monitoring microbial contaminants. Most sensor parameters evaluated exhibited an increase in sensor response to an increase in E. coli concentrations. Responses to E. coli concentrations at or below 10³ cfu/mL were very low due to near background levels, and responses to concentrations above 10⁶ cfu/mL exceeded threshold levels for sensors that use light scattering methods due to saturation in the flow cell. The data produced effectively shows that the sensors evaluated could be used to monitor microbial intrusion events in water distribution systems.

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

No description available.

No description available.

Stanley Prusiner’s proposal that a protein, the prion, was the infectious agent of transmissible spongiform encephalopathies (TSEs) created a great deal of interest, and discussion was not limited to the TSE research field. In fact, Prusiner actively appealed to individuals outside the TSE research field, particularly science writers, corporate funders, and scientists in other research areas. These individuals, in turn, provided Prusiner with the resources, publicity, and expertise to undertake his innovative research program. Prusiner’s emphasis on the revolutionary nature of prion and his claim that prion research could shed light on more common diseases fostered media interest and corporate support. Early newspaper reports stressed Prusiner’s interpretation of experimental results and helped to keep the idea of a protein agent alive until more evidence could be amassed. Corporate funders, with the aid of non-expert scientists, provided Prusiner with the means to carry out expensive, novel experiments and with the opportunity to collaborate with renowned researchers. Prusiner and his supporters were also adept at communicating the protein-only theory and were able to mitigate the impact of specialist criticism. They conceptualized prion protein conversion by using metaphors and analogies and by arguing that parallel phenomena already occurred in Nature. This shifted the discussion away from the problem of replication and recast the search for the TSE agent as a biochemical puzzle. These conceptualizations also enabled Prusiner to engage scientists in other fields, thereby turning his research program into an interdisciplinary enterprise. Moreover, the BSE crisis and Prusiner’s Nobel Prize provided opportunities to further discussion of prions to a wider audience. The engagement of non-experts ultimately created a vast and stable network of interested parties and supporters that was crucial to Prusiner’s success and to the acceptance of the protein-only theory.

Stanley Prusiner’s proposal that a protein, the prion, was the infectious agent of transmissible spongiform encephalopathies (TSEs) created a great deal of interest, and discussion was not limited to the TSE research field. In fact, Prusiner actively appealed to individuals outside the TSE research field, particularly science writers, corporate funders, and scientists in other research areas. These individuals, in turn, provided Prusiner with the resources, publicity, and expertise to undertake his innovative research program. Prusiner’s emphasis on the revolutionary nature of prion and his claim that prion research could shed light on more common diseases fostered media interest and corporate support. Early newspaper reports stressed Prusiner’s interpretation of experimental results and helped to keep the idea of a protein agent alive until more evidence could be amassed. Corporate funders, with the aid of non-expert scientists, provided Prusiner with the means to carry out expensive, novel experiments and with the opportunity to collaborate with renowned researchers. Prusiner and his supporters were also adept at communicating the protein-only theory and were able to mitigate the impact of specialist criticism. They conceptualized prion protein conversion by using metaphors and analogies and by arguing that parallel phenomena already occurred in Nature. This shifted the discussion away from the problem of replication and recast the search for the TSE agent as a biochemical puzzle. These conceptualizations also enabled Prusiner to engage scientists in other fields, thereby turning his research program into an interdisciplinary enterprise. Moreover, the BSE crisis and Prusiner’s Nobel Prize provided opportunities to further discussion of prions to a wider audience. The engagement of non-experts ultimately created a vast and stable network of interested parties and supporters that was crucial to Prusiner’s success and to the acceptance of the protein-only theory.