Дисертації з теми "Prion conversion"

Les encephalopathies spongiformes transmissibles (est), encore appelees maladies a prions, sont un groupe d'affections neurodegeneratives dont les plus connues sont la maladie de creutzfeldt-jakob chez l'homme, et l'encephalopathie spongiforme bovine ou maladie de la vache folle chez l'animal. L'hypothese etiologique actuelle suggere que l'agent infectieux de ces affections est represente par une proteine appelee prp s c (pour forme scrapie de la proteine du prion). La prp s c correspond a une forme alteree d'une proteine normale appelee prp c (pour forme cellulaire de la proteine du prion) dont la fonction est a ce jour inconnue. Le passage de la prp c a la prp s c representait l'element essentiel du mecanisme pathogenique des est. Nos objectifs etaient d'etudier la conversion pathologique de la prp et ses consequences en culture cellulaire ainsi que les reponses therapeutiques envisageables. Ainsi, nous avons pu etudier le mecanisme d'action de l'amphotericine b et du rouge congo dans des modeles genetiques et infectieux des est en culture cellulaire. Par ailleurs, nous avons developpe une approche peptidique originale afin d'inhiber la conversion de la prp c en prp s c. Par la suite, notre travail nous a conduit a etudier le role de la prp et les consequences de sa conversion pathologique dans les mecanismes de defense contre le stress oxydant. Apres avoir montre que des cellules infectees par l'agent de la scrapie etaient plus sensibles au stress que des cellules temoins, nous avons poursuivi nos investigations sur une possible voie de transduction du signal impliquant la prp.

Les prions sont les pathogènes responsables de désordres neurodégénératifs d'issue fatale chez l'homme et de nombreuses espèces de mammifères. Ces agents sont composés d'une forme mal repliée (PrPse) d'une protéine de l'hôte, la protéine prion (PrPc). Durant l'infection, la PrPc adopte une conformation identique à celle de la PrPs, conduisant à une accumulation d'agrégats de PrPsc dans le cerveau, responsables des désordres neurologiques. Différentes souches de prions peuvent être propagées chez une même espèce. Peu d'informations sont disponibles sur le rôle de l'agencement de la PrPsc sous forme d'agrégats (structure quaternaire) dans divers aspects des maladies à prions (phénotype, propagation, transmission. . . ). Par des méthodes d'ultracentrifugation, d'approches physico-chimiques, de titrages d'infectiosité, de mesure d'activité de conversion in vitro, et de modèles de souris transgéniques, nous avons montré i) que des particules présentant le plus fort titre infectieux et l'activité de conversion la plus élevée, spécifiquement retrouvées chez les souches de prions tuant rapidement l'hôte, sont de taille réduite, ii) que la réaction de conversion in vitro de la PrP (PMCA) produit préférentiellement de petits oligomères au très fort pouvoir d'amplification par des cycles d'agrégation / fragmentation, et iii) que la structure quaternaire de la PrPS ne semble pas être un déterminant majeur dans l'aptitude des prions à franchir des barrières d'espèces. La structure quaternaire de cette protéine tient donc un rôle prépondérant dans divers aspects de la dynamique de propagation des prions allant du processus de conversion au phénotype de souche
Prion diseases are fatal neurodegenerative disorders that can affect human or many mammalian species. These infectious agents are composed of a misfolded state (PrPsc) of the endogenous form of the prion protein (PrP'). During the infection, PrPsc induces PrP' to convert into its pathological isoform, which forms aggregates in the brain causing the neurological disorders. Several prion strains are transmissible to a single host species. The implication of the aggregation state (quaternary structure) of PrPse in different aspects of prion diseases — phenotype, propagation, transmission. . . — is not well documented. Using ultracentrifugation methods, physicochemical approaches, infectivity assays, in vitro converting activity assays, and transgenic mouse models, we showed that : i) the most infectious and converting particles, specifically found in rapidly lethal priori strains, are actually of a small size, ii) that the in vitro conversion reaction of PrP (PMCA) mostly generates small particles which have a strong converting ability, via cycles of aggregation / fragmentation, and iii) that PrPsc quaternary structure does not seem to play a predominant role in prions potential to overcome species barriers. Ultimately, prion protein quaternary structure is strongly implicated in several aspects of prion propagation dynamics which goes from the conversion mechanisms to the disease phenotype

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of normal cellular prion protein (PrPC) to an abnormal disease-associated conformer (PrPSc). The aim of this thesis was to investigate the earliest event in prion infection using a novel cell system. Specifically, it aimed to assess the timescale that PrPC is converted to PrPSc following exposure to RML prions and identify the initial cellular site of PrPSc formation and propagation. The cell biology of the initial events of cellular prion infection are poorly understood since newly formed cellular PrPSc is immunologically indistinguishable from infectious prions in the inocula. As a solution to this problem, an epitope-tag was inserted into the sequence of endogenous PrPC to delineate the formation of de novo PrPSc. A PrP-knock down neuroblastoma cell line was reconstituted with mouse 3F4-, FLAG- and MYC-tagged PrPC. Following identification of cells expressing physiological levels of tagged PrPC, prion-susceptibility was determined by exposure to disease-associated prions. Cells expressing 3F4-tagged PrP, the MYC sequence at position 224 and the FLAG sequence at position 22 or 30 contained PrP resistant to formic acid and proteinase K digestion. A mouse bioassay demonstrated that the PrP-224AlaMYC cell line produce bona-fide infectious epitope-tagged PrPSc on exposure to RML prions. Investigation of de novo tagged PrPSc propagation in this novel cell system demonstrated that cellular prion infection is a dynamic process occurring within one minute of prion exposure and that the plasma membrane is the primary site of prion conversion. It was demonstrated that the late endosomes, lysosomes and endosomal recycling compartments do not appear to be key sites of PrP conversion and prion propagation, whilst the plasma membrane and early endocytic compartments are involved in this key process. The work in this thesis provides new insights into the cell biology of the initial stages of prion conversion and propagation and has implications for neurodegenerative diseases where prion-like mechanisms have been proposed.

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.

The research examines the development of women's prison writing through the journal of the Kingston Prison for Women, Tightwire. The journal enabled the prisoners to articulate their experience of prison for themselves as a specific subject-group, as women and as legal subjects. The research connects the prison writing to alterations in legal discourse which reflect the emergence of women as a specific group. The prison writings suggest that extra-legal discourse transforms legal discourse and practice. The appendix includes a selection of poems and comments from Tightwire .

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
This study concerns to a theoretical and field research to understand the symbolic and political strategies of religious conversion to evangelical churches in social subjects marked their stories of life in the practice, criminal careers and prospects. The social actors investigated here are prisoners of the Maximum Security Criminalist Penitentiary Geraldo Beltrão, located in the district of Mangabeira VI, in João Pessoa, State of Paraíba. Our goal is to demonstrate the impact of religious conversion in the process of regeneration, recovery and social re-inclusion of prisioners in the society of origin, before the current prison system. We try to identify the prison population as fertile ground for religious proselytism neopentecostal. Also we presented the report of a cult in prison, where trying to analyze how the celebrations held by prisoners and religious experience in everyday life imprisonment.
O presente estudo se refere a uma pesquisa teórica e de campo no sentido de entender as estratégias políticas e simbólicas de conversão religiosa para as igrejas evangélicas de sujeitos sociais marcados em suas histórias de vida pelas práticas, carreiras e perspectivas criminosas. Os atores sociais aqui investigados são prisioneiros da Penitenciária de Segurança Máxima Criminalista Geraldo Beltrão, localizada no bairro de Mangabeira VI, em João Pessoa, estado da Paraíba. Nosso objetivo é demonstrar o impacto da conversão religiosa no processo de regeneração, recuperação e re-inclusão social de prisioneiros na sociedade de origem, frente ao sistema penitenciário vigente. Procuramos identificar o universo prisional como campo fértil para o proselitismo religioso neopentecostal. Apresentamos também o relato de um culto na prisão, onde analisamos as celebrações e a vivência religiosa pelos presidiários no cotidiano da prisão.

In this dissertation, I consider the relationship between the rhetoric of conversion that informs the American prison system and the pervasive use of the conversion narrative in the life writing of American prisoners. I argue that ever since the first penitentiary opened its gates at the beginning of the nineteenth century, prison reformers have relied on the conversion narrative to redefine the rehabilitative goals of the modern prison. Prison reformers, moreover, have historically deployed a variety of strategies—indeterminate sentencing, the “mark system,” the parole board, and the prison file—to ensure that prisoners articulate their experiences behind bars according to a conversion paradigm. Reflecting the master discourse of the American prison system, the prison life writing archive contains myriad versions of the conversion story, particularly in the post-war period when conversion was reconfigured as “rehabilitation” and prisoners had to define themselves as rehabilitated before they could be released from prison. Hence I explore how the ideology of the prison is implicated in the life writing of prisoners and ex-prisoners who have achieved a notable, or notorious, visibility in American culture: Jimmy Santiago Baca (A Place to Stand), Jack Henry Abbott (In the Belly of the Beast), and James Carr (Bad). More importantly, I show how these writers complicate any notion that prison life is inherently emancipatory. Some prisoners and ex-prisoners reinscribe the ideology of the American prison system by shaping their narratives according to the conversion paradigm. But others use the conversion narrative (consciously or unconsciously) in ways radically different from those intended by prison reformers. Their creative, frequently subversive deployments of the conversion narrative end up complicating the attempt to define the emancipatory role of prison writing or the teleology of post-prison citizenship.

This thesis provides a sociological analysis of religious conversion in prison. In depth-interviews were conducted in order to obtain accounts of prisoners' experiences and subjective perceptions of conversion. Four main research objectives are addressed. Firstly, questions about prisoners' identity are considered. Secondly, the social processes involved in religious conversion are analysed, paying particular attention to the meaning of religious conversion for prisoners. Thirdly, an analysis of the role of religion in the everyday lives of converts is provided. Lastly, the process of personal change that accompanies conversion is discussed, to enhance the analysis of self-identity and personal change more generally. The way in which prisoners' self-identity was questioned prior to conversion is discussed. The importance of experiences other than imprisonment in this process is particularly highlighted. This thesis emphasises the role of both reflective and interactive processes in religious conversion. The ways in which self-reflection led to a contemplation of religious ideas is highlighted, as is how participation in religious activities drew individuals into religious life and belief These considerations are placed within the context of prison life. The role of religion in the everyday lives of prisoners is explored. In particular, the role of religion in enabling inmates to transcend the restrictions of the prison environment, and reconceptualise themselves and their lives, is highlighted. Although the thesis is about religious conversion within the particular context of the prison, changes in self-identity and behaviour are viewed more widely. The analysis highlights the ways in which self-identity is negotiated in everyday life, and emphasises the dual roles of self reflection and interaction in its construction and maintenance.

Príons são proteínas que causam um grupo de doenças neurodegenerativas invariavelmente fatais, sendo uma das mais conhecidas a encefalopatia espongiforme bovina (ou doença da vaca louca). A proteína príon celular (PrPc), rica em estrutura α-helicoidal, sofre uma mudança na sua estrutura secundária produzindo a proteína patológica (PrPSc; o príon) na qual prevalecem folhas-β. Devido à falta de dados estruturais de alta resolução dos príons, simulações de dinâmica molecular (DM) podem ser particularmente úteis para estudar o redobramento de PrP. Estudos experimentais e computacionais, descritos na literatura, indicam que a utilização de pH ácido é capaz de criar certa instabilidade estrutural, produzindo um ganho de estrutura-β na região N-terminal antes desestruturada. Este trabalho se propõe a investigar computacionalmente as mudanças estruturais na proteína príon celular do hamster Sírio induzidas por alteração de pH. Para isso, foi avaliada a influência de diferentes campos de força (GROMOS96 53a6, GROMOS96 43a1, AMBER99SB, AMBER99SB-ILDN, CHARMM27 e OPLS) simulados para as condições de pH neutro e ácido. A partir das análises, observou-se uma forte dependência dos resultados com o campo de força empregado. Além disso, somente os campos de força GROMOS96 53a6 e AMBER99SB demonstraram tendência à expansão do núcleo de folhas-β na região N-terminal da proteína simulada sob pH ácido e conseguiram representar adequadamente a condição neutra. As estruturas correspondentes a esses campos de força em pH ácido, foram, então, utilizadas como ponto de partida para novas simulações de DM em pH neutro (pH 7,4). Essa situação de retorno ao pH neutro ocorre quando o príon sai do compartimento endossomal (submetido a pH ácido) e retorna à superfície externa celular (onde estaria submetida novamente a pH neutro). Os resultados desse estudo de retorno ao pH neutro apontaram para a não reversibilidade de PrPSc, com a manutenção da cauda N-terminal voltada para a extremidade N-terminal da α-hélice HB.
Prions are proteins that cause a group of invariably fatal neurodegenerative diseases, one of the most known being bovine spongiform encephalopathy (or mad cow disease). The cellular prion protein (PrPC), rich in α-helical structure, undergoes a change in its secondary structure producing the pathological protein (PrPSc; the prion) in which β-sheet prevails. Due to the lack of high resolution structural data of the prions, molecular dynamics simulations (MD) may be particularly useful to study the refolding of PrP. Experimental and computational studies, described in the literature, indicate that the use of acidic pH is capable to create some structural instability, producing a gain of β-structure in the previously unstructured N-terminal region. This work proposes to investigate computationally the structural changes in the cellular prion protein of the Syrian hamster induced by pH change. For this, the influence of different force fields (GROMOS96 53a6, GROMOS96 43a1, AMBER99SB, AMBER99SB-ILDN, CHARMM27 and OPLS) were evaluated for neutral and acid pH conditions. From the analysis, a strong dependence of the results with the force field was observed. In addition, only the GROMOS96 53a6 and AMBER99SB force fields showed a tendency to expand the β-sheet nucleus in the N-terminal region of the simulated protein under acid pH and were able to adequately represent the neutral condition. The structures corresponding to these force fields under acidic pH were then used as the starting point for new MD simulations under neutral pH. This situation of return to the neutral pH occurs when the prion leaves the endosomal compartment (submitted to acid pH) and returns to the external cellular surface (where it would be submitted again to neutral pH). The results of this neutral pH return study pointed to the non-reversibility of PrPSc, with the maintenance of the N-terminal tail facing the N-terminal end of the α-helix HB.

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A presente pesquisa aborda a discussão sobre a religiosidade cristã no cárcere, enquanto possibilidade de discurso norteador de condutas individuais e coletivas. A presença de religiosos de confissões cristãs nas instituições penais demonstra a importância desse segmento na dinâmica social da prisão e evidencia de maneira clara as diferenças entre o comportamento dos detentos não religiosos e os que professam a religiosidade cristã, sobretudo evangélica. Nesse sentido, a pesquisa, realizada na Penitenciária Professor Ariosvaldo de Campos Pires, em Juiz de Fora, trata das especificidades das condutas adotadas pelos detentos convertidos à religiosidade evangélica, sua influência na dinâmica social do cárcere e no cotidiano das relações que ali se desenvolvem. Para tanto, utilizo dois conceitos principais: o de “proceder” – que designa um complexo número de regras criadas por e seguidas ou não pelos detentos – conforme elaboração de Marques (2009) - e o de “conversão religiosa”, utilizado no sentido de mudança de visão de mundo do fiel. Constatei que, a partir da conversão, o detento passaria por uma reforma moral subjetiva que se reflete em sua interação com o coletivo e, assim, daria seguimento aos preceitos e valores cristãos em forma de uma conduta moralmente aceitável, resultando o “proceder evangélico”, ou seja, espécie de código de condutas seguido por presos religiosos. Os detentos que aderem a tal “proceder” sofrem intensa vigilância (tanto por parte dos próprios detentos, como pelos funcionários em geral) como forma de teste de sua fé, de modo que não podem cometer nenhuma contravenção, o que nos leva à compreensão de que há certa noção de moralidade religiosa que norteia tanto quem vigia quanto quem é vigiado. Assim, o “proceder” religioso consistiria numa prática ou conduta que se orienta pela moralidade – palavra – cristã. A pesquisa foi realizada na Penitenciária Professor Ariosvaldo de Campos Pires, Juiz de Fora.
The present research deals with the discussion about Christian religiosity in prison, as a possibility of guiding discourse of individual and collective conduct. The presence of religious of Christian confessions in penal institutions demonstrates the importance of this segment in the social dynamics of prison and clearly evidences the differences between the behavior of non-religious detainees and those who profess Christian religiosity, especially evangelical. In this sense, the research, carried out at Professor Ariovasdo de Campos Pires Penitentiary, in Juiz de Fora, deals with the specificities of the conduct adopted by detainees converted to evangelical religiosity, its influence on the social dynamics of the prison and the daily life of the relationships that are developed there. For this purpose, I use two main concepts: that of "proceeding" - which designates a complex number of rules created by and followed or not by detainees - as elaborated by Marques (2009) - and that of "religious conversion", used in the sense of worldview change of the faithful. I observed that, from the conversion, the detainee would go through a subjective moral reform that reflects in his interaction with the collective and, thus, would follow up Christian precepts and values in form of a morally acceptable conduct, resulting in "evangelical conduct", in other words, a kind of code of conduct followed by religious prisoners. Detainees who adhere to such "proceeding" are subjected to intense vigilance (both by detainees themselves and by officials in general) as a way of testing their faith, so that they cannot commit any contravention, which leads us to a comprehension that there is a certain notion of religious morality that guides both those who watch and those who are watched. Thus, religious "proceeding" would consist of a practice or conduct that is oriented by morality – Christian word. The research was carried out at Professor Ariosvaldo de Campos Pires Penitentiary, Juiz de Fora.

The conversion of high-occupancy vehicle (HOV) lanes to high-occupancy toll (HOT) lanes is currently being implemented in metro Atlanta on a demonstration basis and is under consideration for more widespread adoption throughout the metro region. Further conversion of HOV lanes to HOT lanes is a major policy decision that depends on knowledge of the likely impacts, including the equity of the new HOT lane. Rather than estimating these impacts using modeling or surveys, this study collects revealed preference data in the form of observed vehicle license plate data and vehicle occupancy data from users of the HOV corridor. Building on a methodology created in Spring 2011, researchers created a new methodology for matching license plate data to vehicle occupancy data that required extensive post-processing of the data. The new methodology also presented an opportunity to take an in-depth look at errors in both occupancy and license plate data (in terms of data collection efforts, processing, and the vehicle registration database). Characteristics of individual vehicles were determined from vehicle registration records associated with the license plate data collected during AM and PM peak periods immediately prior to the HOV lanes conversion to HOT lanes. More than 70,000 individual vehicle license plates were collected for analysis, and over 3,500 records are matched to occupancy values. Analysis of these data have shown that government and commercial vehicle were more prevalent in the HOV lane, while hybrid and alternative fuel vehicles were much less common in either lane than expected. Vehicle occupancy data from the first four quarters of data collection were used to create the distribution of occupancy on the HOV and general purpose lane, and then the matched occupancy and license plate data were examined. A sensitivity analysis of the occupancy data established that the current use of uncertain occupancy values is acceptable and that bus and vanpool occupancy should be considered when determining the average occupancy of all vehicles on the HOV lane. Using a bootstrap analysis, vehicle values were compared to vehicle occupancy values and the results found that there is no correlation between vehicle value and vehicle occupancy. A conclusions section suggests possible impacts of the findings on policy decisions as Georgia considers expanding the HOT network. Further research using these data, and additional data that will be collected after the HOT lane opens, will include emissions modeling and a study of changes in vehicle characteristics associated with the HOT lane conversion.

L’agregació de proteïnes ha passat de ser gairebé una curiositat biofísica sense major interès a un dels camps més actius de la recerca, especialment des que es va esbrinar que podia ser la causa de diverses malalties en humans. L’agregació en proteïnes ve determinada en un primer terme per la seva seqüència aminoacídica, que és qui delimita les possibles interaccions entre els seus aminoàcids. Diferents factors modulen aquesta propensió intrínseca a agregar. Sovint les proteïnes assoleixen un plegament natiu que és energèticament més estable i que usualment amaga regions propenses a agregar, i d’aquesta forma es prevé una oligomerització no funcional. No totes les proteïnes requereixen un plegament amb una estructura tridimensional definida; les proteïnes intrínsecament desordenades són un grup de polipèptids que manquen una arquitectura espacial definida, amb lo qual tenen una significativament major exposició al solvent; fet que incrementa el seu risc de formar contactes aberrants. Un cas especial de proteïnes desordenades o amb regions desestructurades són els prions i les proteïnes del tipus prió. Aquestes proteïnes es caracteritzen per tenir regions amb una baixa complexitat amb regions amb propensió críptica a agregar, que són capaces d’automodelar una conformació aberrant que s’acobla en forma d’agregats. La bioinformàtica ha assistit en l’estudi d’aquests diferents grups de proteïnes i dels diferents nivells estructurals que adopten, dotant-nos d’un seguit d’eines en forma d’algoritmes per modelar els seus comportaments en processos fisiopatològics. Aquests models computacionals van ser dissenyats fent servir el coneixement del qual es disposava en el seu moment. Però el ràpid increment en l’enteniment dels fenòmens que dirigeixen els processos com l’agregació proteica fan imperatiu una contínua revisió i millora en el desenvolupament d’aquests programes. La present tesi presenta una anàlisi bioinformàtica dels fenòmens darrere la compactació de proteïnes des de múltiples angles. Analitzant l’agregació de proteïnes des de l’estat natiu, proposem millores a la funcionalitat i la usabilitat d’un dels programes de predicció de referència. Tanmateix, s’analitzarà l’efecte del pH (com un primer intent d’integrar la situació on es troba la proteïna als càlculs) en els processos d’agregació i de plegament condicional en proteïnes intrínsecament desordenades. Els resultats obtinguts seran utilitzats per construir servidors web de caràcter obert, pensats com a solucions efectives a la vegada que econòmiques per a múltiples línies de recerca. El fenomen darrere la conversió priònica o de tipus prió serà analitzada per entendre els determinants que ho regulen i el rol funcional de les proteïnes que es sotmeten a aquesta transició; un aspecte sovint eclipsat per la seva associació amb malalties neurològiques. En general, el treball presentat en aquesta tesi intenta comprendre els determinants inter i intramoleculars que regeixen la compactació de les proteïnes, tant en condicions natives com canviants, i d’aquesta manera d’entendre el paper d’aquest procés tant en condicions fisiològiques com quan esdevé malaltia.
La agregación de proteínas ha pasado de ser prácticamente una curiosidad biofísica sin mayor interés a uno de los campos más activos de la investigación, especialmente desde que se dilucidó como el causante de diversas enfermedades en humanos. La agregación en las proteínas viene determinada en primer lugar por su secuencia aminoacídica, que es quién delimita las posibles interacciones entre sus aminoácidos. Diferentes factores modulan esta propensión intrínseca a agregar. A menudo las proteínas adquieren un plegamiento nativo que es energéticamente más estable y que usualmente esconde regiones propensas a agregar, y de este modo se previene una oligomerización no funcional. No todas las proteínas requieren un plegamiento con una estructura tridimensional definida; sino que las proteínas intrínsecamente desordenadas son un grupo de polipéptidos sin una arquitectura espacial definida, con lo que tienen una significativamente mayor exposición al solvente; hecho que incrementa su riesgo de formar contactos aberrantes. Un caso especial de proteínas desordenadas o con regiones desestructuradas son los priones y las proteínas de tipo prion. Estas proteínas se caracterizan por tener regiones de una baja complejidad con regiones con propensión críptica a agregar, que son capaces de automodelar una conformación aberrante que se acopla en forma de agregados. La bioinformática ha asistido en el estudio de estos diferentes grupos de proteínas y de los diferentes niveles estructurales que adoptan, dotándonos de un conjunto de herramientas en forma de algoritmos para modelar sus comportamientos en procesos fisiopatológicos. Estos modelos computacionales fueron diseñados utilizando el conocimiento del cual se contaba en su momento. Pero el rápido incremento en la comprensión de los fenómenos que dirigen los procesos como la agregación proteica hacen imperativo una continua revisión y mejora en el desarrollo de estos programas. La presente tesis presenta un análisis bioinformático de los fenómenos detrás de la compactación de proteínas desde múltiples ángulos. Analizando la agregación de proteínas desde su estado nativo, propondremos mejoras en la funcionalidad y la facilidad de uso de uno de los programas de predicción de referencia. Asimismo, se analizará el efecto del pH (como un primer intento de integrar la situación en la que se encuentra la proteína en los cálculos) en los procesos de agregación y de plegamiento condicional en proteínas intrínsecamente desordenadas. Los resultados obtenidos serán utilizados para construir servidores web de carácter abierto, pensados como soluciones efectivas a la vez que económicas para múltiples líneas de investigación. A su vez, el fenómeno detrás de la conversión priónica o de tipo prion será analizada para entender los determinantes que lo regulan y el rol funcional de las proteínas que se someten a dicha transición; un aspecto muchas veces eclipsado por su asociación con enfermedades neurológicas. En general, el trabajo presentado en esta tesis intenta comprender los determinantes inter e intramoleculares que rigen la compactación de las proteínas, tanto en condiciones nativas como cambiantes, y de esta manera entender el papel de dicho proceso tanto en condiciones fisiológicas como cuando deriva en una enfermedad.
Protein aggregation has moved from being an almost neglected biophysical curiosity to a central research field mostly due to aggregating proteins causing debilitating conditions in humans. The aggregation propensity of polypeptidic sequences is primarily dictated by their amino acid sequence, which delimits the possible interactions between amino acids. Different factors can modulate aggregation propensity. Achieving an energetic stable folded native state usually conceals aggregation prone-regions preventing aberrant self-oligomerization. Not all proteins fold into a defined three-dimensional structure; intrinsically disordered proteins are a group of polypeptides without a defined spatial architecture and therefore are significantly exposed to solvent; which increases the risk of forming aberrant contacts. A special case of disordered proteins or proteins with disordered regions are prions and prion-like proteins. These are characterized by low complexity regions with a cryptic aggregation propensity and able to self-template an aberrant conformation that self-assembles into aggregates. Bioinformatics has assisted the study of these different kinds of proteins and protein structural levels by providing a toolbox of algorithms to model their behaviour in physiology and disease. These computational models were designed using methodology approximations that exploited the available knowledge at that time. Our understanding of the phenomena that govern processes such as protein aggregation is growing rapidly; therefore, the underlying principles behind these programs should be continuously revisited. The present thesis provides a bioinformatics analysis of the phenomena behind protein compaction from multiple angles. By analysing protein aggregation in the native state, we propose improvements to both functionality and usability of a state-of-the-art globular prediction method. At the same time, the effect of pH (as a first approach integrating protein environment on calculations) on intrinsically disordered proteins aggregation and conditional folding was analysed. The obtained results will be used to build publicly accessible web servers as cost-effective tools for multiple research lines. The phenomenon behind prion and prion-like conversion will be studied to gain insight into the determinants that regulate this conversion and the functional role of proteins that undergo this transition; an aspect often overshadowed by their association with neurological diseases. Overall, the work presented in this thesis attempts to understand fundamental inter- and intra-molecular determinants governing protein compaction in near-native and in changing environmental conditions, as a proxy to understand the role of this process in physiology and disease.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Cette thèse a pour objet l'analyse du sens de l'adhésion à des croyances et à des pratiques religieuses dans l'expérience carcérale des détenus. A partir d'une approche cognitive basée sur l'individualisme méthodologique, il s'agit d'examiner trois dimensions spécifiques et transversales de cet objet d'étude. D'une part, la pratique d'une religion dans un contexte aussi spécifique que la prison interroge le degré d'autonomie des individus évoluant dans un milieu contraignant. D'autre part, il s'agit de comprendre la dynamique des croyances à travers l'interaction entre l'offre religieuse et l'expérience carcérale. Enfin, il est question d'appréhender l'organisation et le vécu de la pluralité religieuse au niveau institutionnel et au niveau individuel. En s'appuyant sur des observations et des entretiens qualitatifs menés dans trois types d'établissements pénitentiaires, l'étude a été réalisée auprès de détenus, d'aumôniers et des personnels pénitentiaires. Elle met en lumière les processus sociologiques qui sous-tendent l'adhésion et la mobilisation des ressources religieuses en prison, en proposant une modélisation des rapports sociocognitifs des détenus. Cette approche considère le vécu carcéral comme une expérience des incertitudes à la fois cognitives et en finalités. Ainsi l'adhésion à des croyances religieuses, la fréquentation des aumôneries et la pratique religieuse constituent des ressources efficaces de réduction des expériences de l'incertitude
This thesis aims at analyzing the meaning of adherence to religious beliefs and practices in prison inmates experience. Starting from a cognitive approach based on methodological individualism, we will examine three specific and transverse dimensions of this object of study. In the one hand, the practice of a religion in such a specific context as prison, questions the degree of autonomy of individuals evolving in a restricting environment. On the other hand, the aim is to understand the dynamics of beliefs through the interaction between the religious offer and the prison experience. Finally, we are to comprehend the organization and experience of religious pluralism at institutional and individual level. Based on observations and qualitative interviews conducted in three different types of prisons, the study was carried out with inmates, chaplains and prison staff. It highlights the sociological processes that underlie the adhesion to religious beliefs and the mobilization of religious resources in prison, by proposing a modelling of cognitive reports of detainees. This approach considers the prison experience as an experience of cognitive and instrumental uncertainties. Thus, adherence to religious beliefs, attendance to chaplaincies and religious practice are effective resources concerning the reduction of uncertainty experiences

Orientador: Paulo Dalgalarrondo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A religião voltou a ser considerada uma questão importante para a sociedade contemporânea. Fenômeno bastante complexo é atualmente considerado um elemento extremamente significativo para a sua vida sócio-cultural. Atribui-se à religião um papel fundamental para as pessoas encarceradas, atuando como fator protetor da Saúde Mental e incentivando a recuperação do infrator. Existem poucos estudos que relacionem saúde mental e religiosidade em presidiárias . Também não se conhecem pesquisas e estudos que enfoquem a religiosidade das mulheres presas e a forma como esta é experienciada no cotidiano de uma prisão de segurança máxima e as conseqüências nas vidas destas mulheres. Objetivos: Verificar o perfil de Saúde Mental e a relação entre religião e Saúde Mental. Analisar também como a Saúde Mental e a religião influenciam a vida das presidiárias e como lhes fornecem maior suporte psíquico e emocional. Descrever o cotidiano da penitenciária durante o período em que ocorreu a pesquisa, o perfil das presas e o ambiente em que vivem. Investigar alguns aspectos da experiência subjetiva das detentas enfocando os aspectos relacionados à espiritualidade (enfatizando-se as manifestações religiosas de origem pentecostal e neopentecostal), conversão religiosa e ao sofrimento. Método: Foi utilizado um questionário de autopreenchimento que incluiu dados sócio-demográficos, religiosidade (atual e anterior ao aprisionamento), Saúde Mental e perfil criminal. Para os aspectos psicopatológicos foi utilizado o General Health Questionnaire (GHQ-12). Foram entrevistadas 358 mulheres, detentas da Penitenciária Feminina da Capital - São Paulo - SP de Março de 2003 a Dezembro de 2004. Foram feitas análises estatísticas descritivas e bivariadas, comparando-se todas as variáveis com o GHQ-12. Foi utilizada também a análise de regressão logística multivariada para as respostas dicotômicas para as variáveis que possivelmente interagiram com a Saúde Mental.
Abstract: Nowadays religion is an important issue for the modern society, a complex and fundamental socio-cultural phenomenon. There is evidence that religious commitment tends to correlete positively with the mental health. The religion seems to have an important role or the mental health of jailed women. It provides psychological and emotional support, bringing them comfort, meaning and hope. This study intents to verify the mental health profile and the relation between religion and mental health, to analyse how mental health and the religion influence the life of jailed women as well, and to provide more psychological and emotional support. It describes too, the quotidian of female detainees, their profiles and the prisional enviroment where they are living and investigates some aspects of their subjective experience. A self-filling questionaire was used wich includes social, demographic, religious (present and before jailing), mental health and crime profile data. Concerning the psychopathologic matters General Health Questionnaire (GHQ-12) was used. 358 women were interviewed from "Penitenciária Feminina da Capital" (São Paulo, SP - Brazil ) from March, 2003 to December, 2004. Descriptive and bivaried statistcs analysis was accomplished comparing all the variables with GHQ-12. Multivaried regression logistic analysis was used to get the dichotomic answers to the variables that would possibly interact with mental health. A bigger persona' religiosity was associated to better mental health. Associations between crime type and mental health were not found.
Mestrado
Saude Mental
Mestre em Ciências Médicas

La recherche porte sur la formation universitaire à distance adaptée à des personnes détenues, dispositif qui se situe à l'intersection de plusieurs domaines d'activités, universitaire et carcérale. Cette étude vise à analyser plusieurs étapes d'un processus d'innovation pédagogique. Une première consiste à appréhender le terrain de la prison dans une démarche ethnographique constituée de pratiques d'accès aux activités de cours. Une seconde est conduite de façon à épouser les spécificités sociales et pédagogiques d'un tel terrain. Une troisième analyse les méthodes et les procédures de distribution de la parole dans des interactions enseignant-étudiants. Le corpus a été constitué lors d'activités de cours en prison.Celles-ci ont été recueillies sous forme d'enregistrements sonores et visuels, eux-mêmes explorés à l'aide de l'analyse de conversation. Une quatrième porte sur une collection de propositions techno-pédagogiques développées tout au long de la démarche de terrain. Cette recherche propose une façon d'accéder aux activités sociales en prison. Elle ouvre un champ exploratoire quant à l'étude de l'interaction pédagogique en prison, sa portée sociale et pédagogique, qui va bien au-delà des murs de la prison.

Abstract The study examines ‘recovery through repetition’, investigating how speakers repeat their own utterances in order to return to prior talk. The phenomenon comprises instances of everyday, casual conversation in which speakers indicate that their utterance was either not taken up at all or not taken up to an adequate degree. By repeating the utterance more or less word-for-word, speakers suggest to their recipients that a (different type of) response is relevant and offer the utterance for re-consideration. The data consist of American-English and Finnish conversations. The segments come from the Santa Barbara Corpus of Spoken American English and from the Corpus of Conversational Finnish that is maintained by the Department of Finnish Language and Literature at the University of Helsinki (Keskusteluntutkimuksen arkisto). The theoretical and methodological framework of the study is based on interactional linguistics and conversation analysis. First, the study details the typical composition and position of recovery through repetition and discusses the interactional implications that the repeated utterances may have. The study focuses on the functions of recovery through repetition and their implications for stance taking. Two overall interactional environments are identified: speakers employ recovery through repetition either to seek the attention of recipients and to take a stance towards an activity in progress, or to redirect the attention of recipients and to take a stance towards a recipient response. The different functions of recovery through repetition in the two environments are further examined. Moreover, the study contrasts repetition with other means of recovery and suggests that the different means have divergent implications for stance taking. Finally, the study concludes that recovery through repetition provides speakers with a means of negotiating the input of their utterances and simultaneously taking a stance towards an aspect of the ongoing interaction
Tiivistelmä Tutkimus tarkastelee toistoa elvytyskeinona keskustelussa eli sitä, kuinka puhuja toistaa oman lausumansa palatakseen aiempaan puheeseen. Ilmiö muodostuu arkisista, epämuodollisista keskustelutilanteista, joissa puhuja osoittaa, että jotakin hänen lausumaansa ei ole joko otettu lainkaan huomioon tai sitä ei ole käsitelty asianmukaisesti. Toistamalla lausuman lähes sanatarkasti puhuja ilmaisee keskustelukumppaneilleen, että jonkinlainen (tai mahdollisesti tietyntyyppinen) vastaanotto olisi odotuksenmukainen, ja tarjoaa lausumaansa käsiteltäväksi uudelleen. Tutkimuksen aineisto koostuu amerikanenglannin- ja suomenkielisistä keskusteluista, jotka ovat peräisin Santa Barbaran puhutun amerikanenglannin kokoelmasta (Santa Barbara Corpus of Spoken American English) ja Helsingin yliopiston suomen kielen ja kotimaisen kirjallisuuden laitoksen Keskusteluntutkimuksen arkistosta. Tutkimuksen teoreettisen ja menetelmällisen viitekehyksen muodostavat vuorovaikutuslingvistiikka ja keskustelunanalyysi. Aluksi tutkimuksessa kartoitetaan yksityiskohtaisesti elvyttävän toiston tyypillistä rakennetta ja paikkaa sekä pohditaan toistettujen lausumien mahdollisia vuorovaikutuksellisia seuraamuksia. Tutkimus keskittyy elvyttävän toiston tehtäviin ja niiden merkitykseen asennoitumiselle. Tutkimuksessa tunnistetaan kaksi yleistä esiintymisympäristöä: puhujat käyttävät elvyttävää toistoa joko hakeakseen vastaanottajien huomiota ja ottaakseen kantaa meneillään olevaan toimintaan tai ohjatakseen vastaanottajien huomiota ja ottaakseen kantaa edeltävään vastaanottajan vuoroon. Elvyttävän toiston tehtäviä näissä kahdessa ympäristössä eritellään tutkimuksessa tarkemmin. Lisäksi tutkimuksessa verrataan toistoa muihin elvytyskeinoihin keskustelussa ja esitetään, että eri elvytyskeinoilla rakennetaan asennoitumista eri tavoin. Tutkimus osoittaa, että elvyttävä toisto tarjoaa keskustelijoille keinon neuvotella sanomansa merkityksestä ja samalla rakentaa asennoitumistaan meneillään olevaan vuorovaikutustilanteeseen

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
The theme of the research is inserted at a field of intersection between the Sociology of Religion and Sociology of Violence, having as the general objective study the sociological meaning of the conversion of prisoners that lives at the biggest prison (Prison of Alca?uz) of Rio Grande do Norte to the evangelical churches. The research is justified, because Brazil shelter the fourth greater arrested population arrested of the world, with projections indicating that it can turn the greatest in 2034. Besides, this study about religious conversion of prisoners to the Social Sciences is too important, because is a theme little developed in Brazil and deserves attention, one time that as the arrested people as the evangelicals are in expansion in our country. Starting from the precedent observations, we guide ourselves by the following problematic of research: the religious practice in Alca?uz presents a mere instrumental perspective, where the actions of prisoners converted was on purpose oriented to conquest material or symbolic privileges; or purely religious, where seek a moral renovation? To develop the work, the scientific methodology adopted was exploratory and explanatory, using the Goffman?s theory about total institutions and presentation of self, and Blumer?s doctrine relating to Symbolic Interacionism and the Story life method, besides considerations about evangelical religion. Having this theoretical basis, was accomplished the Field research, when were made interviews and applied questionnaires to 11 Jailer Agents, 31 prisoners, Director and Vice-Dictor (in November, 2011), the coordinator of social projects of the prison and the coordinator of evangelization at the prisons in Rio Grande do Norte. As results, it was seeing in Alca?uz that the prisoners can be separated in two groups: the one of Pavilions and other one of the Medical Section. The Pavilions are branded for managerial and structural problems, where are found idle prisoners in collective cells and with a historical of escaping attempts, mutinies and murders. The Medical Section has some individual cells or destined for two people, besides few collective also, and the prisoners work and have a more disciplined behavior, there isn t escapes or rebellions and that, for these reasons end for have more confidence from the Administration. About the presence of evangelical prisoners, most are at Medical Section, where exist a specific place to the cults (what doesn t at Pavilions). At the end, the conclusion is that the prisoner that says himself evangelical in Alca?uz, although can be seeing with distrust about your real conversion, he gets win a trust vote and until the opposite being demonstrated in other words, that he is not hiding himself behind the bible to divert the vigilance of Direction and practice disciplinary faults without make any suspicions, is treated with more respect and has more opportunities live at Medical Section; have work, that most of times is paid and guarantee the homologation of your payment of penalty with work, besides other benefits, diminishing his time in jail
O tema da pesquisa encontra-se inserido em um campo de interse??o entre a Sociologia da Religi?o e a Sociologia da Viol?ncia, tendo como objetivo geral estudar o significado sociol?gico da convers?o de presos que vivem no maior estabelecimento prisional (Penitenci?ria de Alca?uz) do Rio Grande do Norte ?s igrejas evang?licas. A pesquisa se justifica, pois o Brasil abriga a quarta maior popula??o carcer?ria do mundo, com proje??es indicando que ela poder? se tornar? a maior do mundo em 2034. Al?m disso, esse estudo sobre convers?o religiosa de presos para as Ci?ncias Sociais ? muito relevante, pois ? um tema pouco desenvolvido no Brasil e merece aten??o, uma vez que tanto a massa carcer?ria quanto os evang?licos est?o em expans?o em nosso pa?s. A partir das observa??es precedentes, orientamo-nos pela seguinte problem?tica de pesquisa: a pr?tica religiosa em Alca?uz apresenta uma perspectiva meramente instrumental, na qual as a??es dos presos convertidos estariam propositalmente orientadas para conquistar privil?gios materiais ou simb?licos; ou puramente religiosa, onde se busca uma renova??o moral? Para desenvolver o trabalho, a metodologia empregada foi explorat?ria-explicativa, utilizando-se a teoria de Goffman sobre institui??es totais e a cerca da representa??o do eu, e a doutrina de Blumer relativa ao Interacionismo Simb?lico e o m?todo da Hist?ria de Vida, al?m de considera??es sobre a religi?o evang?lica. De posse dessa base te?rica, foi realizada a pesquisa de campo, na qual foram feitas entrevistas e aplicados question?rios a 11 Agentes Penitenci?rios, 31 presos, Diretor e Vice Diretor (em novembro de 2011), o coordenador dos projetos sociais do Pres?dio e o coordenador de evangeliza??o nos pres?dios do Rio Grande do Norte. Como resultado, verifica-se que em Alca?uz os presos podem ser separados em dois grupos: o dos Pavilh?es e os do Setor M?dico. Os Pavilh?es s?o marcados por problemas estruturais e gerenciais, onde se encontram presos ociosos em celas coletivas e com um hist?rico de tentativas de fugas, motins e assassinatos. O Setor M?dico tem algumas celas individuas ou para duas pessoas, al?m de coletivas tamb?m, e os presos trabalham e s?o mais comportados, n?o h? fugas ou rebeli?es e que, por esses motivos acabam por ter maior confian?a por parte da Administra??o. Quanto ? presen?a de presos evang?licos, a maioria est? no Setor M?dico, onde h? um local espec?fico para os cultos (o que n?o existe nos Pavilh?es). Por fim, concluise que o preso que se diz evang?lico em Alca?uz, embora seja visto com desconfian?a quanto a sua real convers?o, acaba por ganhar um voto de confian?a e at? que se prove o contr?rio ou seja, que n?o est? se escondendo atr?s da b?blia para desviar a vigil?ncia da Dire??o e praticar faltas disciplinares sem levantar maiores suspeitas, ? tratado com mais respeito e tem mais oportunidades morar no Setor M?dico; ter trabalho, que na maioria das vezes ? remunerado e garantir o reconhecimento de sua remi??o de pena, al?m de outros benef?cios, encurtando assim o seu tempo de pris?o

The master degree project presents design of conversion and extension of an old former prison situated between the streets Cejl, Soundni and Bratislavska for cultural, commercial and manufacturing purposes and forming multifunctional cultural center. The proposal consists of the conversion of two floors of existing building on the street Soudni, demolition of additional building on the street Bratislavska, while the additional building on street Cejl is belonging to other owner and will remain in existing status. Before the new building it is created a small square, that connects new and old building and the surroundings. Main goal is to create center for “creative” specialization, in which will be placed as working (studio, offices, conference hall) and performing spaces, but there also will be residential part (hostel and renting flats). Main purpose – create interesting space for collaboration of creative professions and to create the new cultural center.

碩士
國立中正大學
分子生物研究所
103
Prion diseases are neurodegenerative disorders carried out in mammalian. These diseases are due to structural conversion of prion protein (PrP) from normal cellular isoform (PrPC) to pathogenic isoform (PrPSc). PrPC is most of α-helical with soluble and protease sensitive. After conversion to PrPSc, protein is β-sheet rich and become insoluble and protease-resistant. According to previous studies, the C-terminal region of prion protein, PrP121-230, is the toxic component to from PrPSc and can be against protease K digestion. Therefore, the flexible N-terminal domain has not been considered to be disease-related. However, recent studies have demonstrated that the onset of familial prion diseases is associated with both point mutation and repetition of octapeptide repeats (OR) insertion in N-terminal OR region, PrP51-91 with 5 OR. OR region of PrPC has Cu2+-binding sites. However, copper binding on PrPC structure and the role in disease have not been fully clarified. Base on these studies, we speculate that the repetition of OR insertion can influence the structure, the fibril formation and protease K-resistant strength of C-terminal region. In this study, we prepared four protein variants (MoPrP ∆octa, MoPrP 1-OR, MoPrP 5-OR (WT), MoPrP 8-OR with and without Cu2+-treatment) for the structure comparison, kinetic study of fibril formation and further characterization of the fibrils. We found that increasing number of the repeats decrease protein α-helical content and the stability of C-terminus, and also affects the kinetics of fibril formation. On the other hand, PrP-8OR proteins and fibrils are more resistant to protease K digestion than other variants. Further, we tested the cell toxicity of the fibrils converted from these protein variants. Cell viability decreased with increasing numbers of OR. This study conclusively indicates that increasing octapeptide repeat affects structure of prion and the conversion to more toxic fibrils. This result provides evidence to elucidate the molecular mechanism of prion disease.

碩士
國立中正大學
分子生物研究所
103
Transmissible spongiform encephalopathy (TSE) belongs to fetal neurodegenerative diseases. The disease is infected by the pathogen called PrPSc, a robustly beta-sheet-riched isoform of cellular prion protein (PrPC). The accumulation of PrPSc forms amyloid plaque resulting in the loss of neuron. Different from other neurodegenerative diseases, prion disease can spread between species by PrPSc. In the past years, the amyloidogenic region of C-terminal prion protein is believed to be the major domain carring of proteinase-resistance resulting in the occurrence of disease. However, the new evidence indicated that the N-terminal prion protein can participate in the mediation of prion disease. The prion protein is involved in metal ion regulation in human physiology. In our study, we used mouse PrP23~230, PrP23~89, PrP23~140, PrP23~170 to identify the role of N-terminal fragment in fibril formation of PrP23~230. According to CD spectra, the N-terminal fragments of prion protein are random coil. The addition of N-terminal fragments in fibril conversion affects the efficiency of fibril formation. To reveal the effect of divalent ion, we conducted copper ion titration experiment to identify the structural instability of prion protein. The copper binding of N-terminal octapeptide repeat domain affects C-terminal globular domain structure. The N-terminal prion protein function in fibril formation can provide the treatment of prion disease.

博士
國防醫學院
生命科學研究所
100
Immense diversity of prion strains is observed, but its underlying mechanism is less clear. Three [PSI] prion strains—named VH, VK, and VL—were previously isolated in the wild-type yeast genetic background. Here we report the generation and characterization of eight new [PSI] isolates, obtained by propagating the wild-type strains with Sup35 proteins containing single amino-acid alterations. The VH strain splits into two distinct strains when propagated in each of the three genetic backgrounds, harboring respectively single mutations of N21L, R28P, and Gi47 (i.e. insertion of a glycine residue at position 47) on the Sup35 N-terminal prion-forming segment. The six new strains exhibit complex inter-conversion patterns, and one of them continuously mutates into another. However, when they are introduced back into the wild-type background, all 6 strains revert to the VH strain. We obtain two more [PSI] isolates by propagating VK and VL with the Gi47 and N21L backgrounds, respectively. The two isolates do not transmit to other mutant backgrounds but revert to their parental strains in the wild-type background. Our data indicate that a large number of [PSI] strains can be built on three basic Sup35 amyloid structures. It is proposed that the three basic structures differ by chain folding topologies, and sub-strains with the same topology differ in distinct ways by local structural adjustments. This ‘‘large number of variations on a small number of basic themes’’ may also be operative in generating strain diversities in other prion elements. It thus suggests a possible general scheme to classify a multitude of prion strains.

碩士
國立臺灣大學
生化科學研究所
101
Prion diseases are not only fetal but also infectious neurodegenerative disorders. The critical molecular event of prion diseases is the structural conversion of a normal cellular prion protein, PrPC, into a misfolded, infectious form, PrPSc. The overall structure of the prion protein transits from α- to β-dominant state, giving rise to formation of toxic amyloid fibrils. Up to now, the structural transition mechanism is still elusive. Recently, our lab found that disulfide-bond reduced mouse prion protein could be fixed in α-helical or β-rich structure under neutral condition. This finding provides us an opportunity to dissect the conversion process in details. To examine the role of three α-helices in mPrP during this structural conversion process, site-directed spin-labeling technique (SDSL), electron spin resonance spectroscopy (ESR), analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), circular dichroism spectroscopy (CD), and single molecule fluorescence resonance energy transfer (smFRET) were employed. In this study, we suggest that helix 1 and helix 3 are intact no matter in α- or β-state; however, helix 2 is unfolded after structural converted to β-oligomers. Only the residues in helix 2 are involved in intermolecular association in β-state, suggesting helix 2 is crucial for oligomerization process. In addition, the tertiary structural contact between helix 3 and loop is dragged open after structural transition. In fibril state, helix 2 and helix 3 cooperatively participate in association of amyloid core and helix 1 or loop supplies peripheral interaction to stabilize the fibril structure as well.

碩士
國立臺灣大學
生化科學研究所
100
Transmissible Spongiform Encephalopathies (TSE), also called prion diseases are infectious neurodegenerative disorders. The key molecular event in the pathogenesis of prion diseases is the conformational conversion of a cellular prion protein, PrPC, into a misfolded form, PrPSc. The α to β conformational transition leads to protein aggregation and the formation of toxic amyloid fibrils. However, the mechanism of protein misfolding and the pathogenic pathway are still unclear. In our lab’s previous study, we found that the disulfide-deleted mouse PrP could undergo a spontaneous structural conversion under native condition from the native α-helical structure to β-oligomers, amorphous aggregate, even amyloid fibrils. Here, we made spin-labeling on each of three helices of mouse prion protein individually and combined circular dichroism spectroscopy and electron spin resonance (ESR) spectroscopy to investigate the structural conversion process. Our study illustrated that helix 1 and helix 2 were partially unfolded when converted into soluble β-structures. On the other hand, spin labeled on helix 3 showed slow mobility, suggesting that the local environment of that spin is in an ordered state. Moreover, when the helix3-labeled protein was transformed into amyloid fibrils, the spin-labeled fibrils showed intermolecular spin interaction with a distance of 10 A.

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.

Avec ce mémoire, j’ai souhaité cerner ce qui serait le propre d’une conversion, soit ce que j’ai appelé un processus de trans-formation. Avec ce concept original, j’ai voulu orienter le regard de l’observateur vers les points de basculement de l’intimité à la publicité qui caractérisent les conversions. Pour ce faire, il m’est apparu fertile de mobiliser et de réhabiliter l’étude des valeurs, un thème classique en sociologie. Des valeurs portées par des individus aux valeurs publiques, la notion de « valeur » recèle le potentiel heuristique nécessaire pour étudier les conversions à différentes échelles d’analyse et par-delà des qualifications a priori religieuses, politiques, sexuelles, etc. Avec cette perspective théorique pragmatique inspirée par Dewey et articulée à la sensibilité aux positions sociales des cultural studies, je me suis donné les moyens d’analyser la façon dont change au cours d’une vie ce à quoi les gens tiennent. Cette représentation dynamique de la conversion vient ajouter des éléments de compréhension à un phénomène trop souvent appréhendé à la lumière de « lectures préférées » modernes et coloniales qui demandaient à être subverties pour redonner place à l’exercice de l’imagination sociologique. Les apports du concept de trans-formation sont illustrés à partir de la comparaison de quatre études de cas individuels : Paul Claudel, un écrivain converti au catholicisme ; Michelle Blanc, une transsexuelle québécoise ; Joe Loya, un Mexican American qui modifie ses conceptions du bien et du mal en isolement carcéral ; et Mlle Pigut qui est devenue « vegan ».
In this master thesis, I had the objective to pinpoint what is peculiar to conversions, that being what I have called a process of trans-formation. With this original concept, I wanted to guide the observer’s eyes toward the shifting points from intimacy to publicity, which are central in conversions process. To do so, I considered fruitful to acknowledge and reinstate the study of values, a classical theme in sociology. From values held by individuals to public values, the notion of “value” comprises the necessary heuristic potential to study conversions at different analytical scales and across assumed qualifications such as religious, political, sexual, etc. My pragmatic theoretical approach informed by Dewey and the cultural studies’ sensibility to social positions allowed me to analyse how what people value changes in the course of their life. This dynamic representation of conversions adds some insights to grasp a phenomenon often tackle from the perspective of modern and colonial “preferred readings”; readings that need to be challenged and revisited as to allow the exercise of sociological imagination. The contributions of the trans-formation’s concept are illustrated by the comparison of four individual case studies: Paul Claudel, a writer converted to Catholicism; Michelle Blanc, a québécoise transsexual; Joe Loya, a Mexican American who modifies his conceptions of good and bad in solitary confinement; and Mlle Pigut who becomes vegan.