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1
CUNATI, DIANA. « Ruolo dei lipid raft nel metabolismo della proteina prionica ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/27001.
Texte intégralRésumé :
The prion protein (PrP) is a GPI-anchored protein primarily concentrated in neuronal cells. Under certain conditions, the innocuous cellular form of this protein, PrPC, can convert into the lethal scrapie isoform, PrPSc, which can aggregate with other PrP molecules and exert its neurotoxic activity.
The structure of PrPC consists of two domains: an N-terminal, glycosylated, flexible disordered domain which is capable of binding copper and a C-terminal α-helical domain.
In contrast, PrPSc is enriched in β-sheet structures and characterized by its poor solubility in non-denaturing detergents, propensity for aggregation, partial resistance to proteinase K digestion.
The conversion of PrPC into PrPSc occurs in particular regions of the cell membrane, enriched with cholesterol and glycosphingolipid, called lipid rafts; these microdomains are thought to play a crucial role both in physiological functions and in the alternative folding of the prion protein.
In addition, it’s known that:
-PrPC can be cleaved at the 110/111 peptidyl bond to produce a C-terminal fragment, C1, which remains membrane bound and a N-terminal fragment, N1, released in the extracellular space. C1 fragments can’t be converted to the scrapie isoform;
- in cell cultures, ADAM10 and ADAM17 were shown to be responsible for this processing and their activation seems PKC-dependent.
The aim of our project is to establish if the alteration of cell lipid composition can modify the membrane distribution of the prion protein within rafts or non-raft regions and promote the activity of disintegrins such as ADAM10/17 upon the prion protein.
For this reason, granule cells, from the cerebella of 8-day-old rats, were incubated after 8 days in culture with GM1 or GD1a or GT1b for 4 hours at 37°C or with GM1 for 4 hours at 4°C. Detergent resistant fractions, containing lipid rafts, were isolated and proteins in all gradient fractions were separated and analyzed by EF/WB with specific antibodies.
After cell treatments with exogenous gangliosides, a good percentage of them was found in lipid rafts; immunoblotting analysis with specific antibodies showed a significant reduction in the amount of proteins, normally localized in lipid rafts, after incubation with GT1b.
The incorporation of this ganglioside, characterized by a remarkable steric encumbrance, might be responsible for lipid rafts destabilization and proteins redistribution toward non-raft regions.
Another possibility is that GT1b incorporation reduces the number of lipid rafts on the cell membrane.
Immunoblotting analysis with three different anti-PrP antibodies showed that this protein is not selectively located in lipid rafts but it is also distributed in several intracellular compartments.
Cell treatments with GM1 or GD1a at 37°C for 4 hours were not able to promote PrPC cleavage at the 110/111 peptidyl bond; cell incubation with GM1 seemed able to induce a conformational change of the prion protein toward a “simil-scrapie” isoform, partially resistant to classical denaturation protocols. Further studies are in progress to fully demonstrate that GM1-PrP interaction results in this conformational change.
2
Sang, Chieh. « Single molecule fluorescence studies of prions and prion-like proteins ». Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/287929.
Texte intégralRésumé :
Prions are infectious agents that cause fatal neurodegenerative diseases in the brain. The wide-accepted protein-only hypothesis states that the misfolded form of prion protein (PrP) is the sole constituent of prions, and the self-propagating process of PrP is considered to play a central role in prion pathogenesis. Prions are believed to propagate when a PrP assembly enters a cell and replicates to produce two or more fibrils, leading to an exponential increase in PrP aggregate number with time. However, the molecular basis of this process has not yet been established in detail. This prion-like replication is also suggested to be the mechanism in the development of other notorious neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. In this thesis, I use single-aggregate imaging to study fibril fragmentation and elongation of individual murine PrP aggregates from seeded aggregation in vitro. From fluorescence imaging of individual PrP aggregates on the coverslip surface, elongation and fragmentation of the PrP assemblies have been directly observed. PrP elongation occurs via a structural conversion from a proteinase K (PK)-sensitive to PK-resistant conformer. Fibril fragmentation was found to be length-dependent and resulted in the formation of PK-sensitive fragments. To gain more insights into the mechanism of the spread of PrP, the quantified kinetic profiles allows the determination of the rate constants for these processes through the use of kinetic modelling. This enables the estimation of a simple framework for aggregate propagation through the brain, assuming that doubling of the aggregate number is rate-limiting. In contrast, the same method was applied to measurement for α-Synuclein (αS) aggregation, which has been suggested to be prion-like and is associated with Parkinson's disease. While αS aggregated by the same mechanism, it showed significantly slower elongation and fragmentation rate constants than PrP, leading to much slower replication rate. Furthermore, the measurements in αS aggregation has been extended to the cellular environment, I use super-resolution imaging to study the amplification of endogenous αS aggregation in cells and the transcellular spread of αS. Endogenous αS showed a clear amplification in number of aggregates with time after seed transduction, and the newly-formed αS aggregates are likely to spread through cell-to-cell transmission. The proteasome was demonstrated to possess a novel disaggregase function for αS fibrils and thus produce more seeds for further replication. It partially explains that αS aggregation in cells was found to replicate at a substantially faster rate than that in vitro. Determining the nature of the oligomers formed during aggregation has been experimentally difficult due to the lack of suitable methods capable of detecting and characterising the low level of oligomers. To address this problem, I have studied the early formation of PrP oligomers formed during aggregation in vitro using various single-molecule methods. The early aggregation of PrP is observed to form a thioflavin T (ThT)-inactive and two ThT-active species of oligomers, which differ in size and temporal evolution. The ThT-active oligomers undergo a structural conversion from a PK-sensitive to PK-resistant conformer, while a fraction of which grow into mature fibrils. These results also enable the establishment of a kinetic framework for elucidating temporal evolution of PrP aggregation and the relationship between oligomers and fibrils. Overall, my research identifies fibril elongation with fragmentation are the key molecular processes leading to PrP and αS aggregate replication, an important concept in prion biology, and provides a simple framework to estimate the rate of prion and prion-like spreading in animals. The results also show that a diverse range of oligomers is formed and co-exist during PrP aggregation which differ both in their structure and properties and provides mechanistic insights into a prion aggregation. The work provides a new quantitative approach to describe the prion-like property in neurodegenerative diseases from a kinetic perspective that can be verified in extending studies in other proteins or in cells.
3
Heiseke, Andreas. « Prions and autophagy : Effect of lithium on prion infection and role of basal autophagy in primary prion infection ». kostenfrei, 2010. https://mediatum2.ub.tum.de/node?id=818228.
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Urrea, Zazurca Laura. « Funciones de la proteína priónica celular, alfa-sinucleína y reelina en enfermedades neurodegenerativas ». Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/482168.
Texte intégralRésumé :
Las enfermedades neurodegenerativas son una serie de trastornos del sistema nervioso caracterizadas por la pérdida de grupos neuronales específicos y por la presencia de cuerpos de inclusión proteicos, entre ellas las más frecuentes son la enfermedad de Alzheimer y la enfermedad de Parkinson, ambas asociadas a la edad. Su etiología, en la mayoría de los casos, aún se desconoce y su manifestación clínica es progresiva y crónica.
La enfermedad de Parkinson se caracteriza por la pérdida de neuronas dopaminérgicas de la sustancia nigra pars compacta y por la presencia de agregados intracitoplasmáticos, denominados cuerpos de Lewy (LB). Se ha identificado la α-sinucleína como el principal componente de los LB, su forma desplegada está involucrada en el proceso patológico de la EP. La α-sinucleína desplegada, se agrega para formar protofibrillas que finalmente darán lugar a los LB. La acumulación intracelular de proteínas anormales da lugar al concepto de proteinopatías. Se cree que estas proteínas anómalas son capaces de propagarse entre células. Varios mecanismos moleculares se han propuesto para la transmisión de α-sinucleína, en este caso estudiamos la proteína priónica celular (PrPc) como posible receptor de α-sinucleína.
PrPc es conocida por su participación en las enfermedades priónicas en su forma patológica, llamada PrPsc. Esta forma PrPsc se agrega y forma placas en el cerebro. Se ha demostrado que la PrPc es capaz de unirse a péptidos amiloides como los oligómeros β-amiloides que se encuentran en la enfermedad de Alzheimer. En esta tesis estudiamos el transporte de α-sinucleína según la dosis genética de PrPc. Después de realizar inoculaciones intracraneales con protofibrillas de α-sinucleína en animales con distintas dosis génica de PrPc, se observa que los animales sobreexpresantes de la PrPc presentan más agregados de α-sinucleína fosforilada que los animales deficientes de PrPc. Además, también identificamos la región de unión entre PrPc y α-sinucleína. Gracias a las construcciones delecionadas de PrPc detectamos que la región del dominio central cargada es esencial para la unión con α-sinucleína.
Además, en esta tesis hemos analizado los niveles de Reelina en distintas enfermedades neurodegenerativas. Reelina es una proteína secretable implicada en el neurodesarrollo. En el adulto, Reelina está involucrada en la plasticidad sináptica, aprendizaje y memoria. Se ha detectado que niveles bajos de Reelina da lugar a fallos en la sinapsis y neurodegeneración. Anteriormente, los niveles de Reelina se han analizado en muestras humanas, sobretodo, en la enfermedad de Alzheimer dando lugar a resultados contradictorios. En el presente estudio, determinamos los cambios del mRNA y proteicos de Reelina en la enfermedad de Alzheimer, la demencia por cuerpos de Lewy, la enfermedad de Parkinson y en la enfermedad Creutzfeldt-Jakob (CJD) esporádica. Mientras los niveles proteicos de Reelina descienden en la enfermedad de Alzheimer y en la demencia por cuerpos de Lewy, en la enfermedad de Parkinson se mantienen. Por otro lado, detectamos que los niveles de Reelina en CJD aumentan, sobretodo en los casos tipo 1. Animales sobreexpresantes de PrPc humana inoculados con extracto cerebral de CJD también presentan un aumento de sus niveles de Reelina. In vitro, se observa que la expresión de Reelina aumenta en presencia del prion sintético que imita la secuencia central de la PrPc humana. Además, el aumento de Reelina es dependiente de las especies reactivas de oxígeno (ROS), mediante el uso de inhibidores de ROS detectamos como los niveles de Reelina se mantienen.Many neurodegenerative diseases are characterized by the loss of neurons and intracellular accumulation of abnormal proteins, with the formation of inclusion bodies. Parkinson’s disease (PD) is the second most common form of neurodegenerative diseases. PD shows an abnormal accumulation of α-synuclein aggregates in neurons, called Lewy bodies (LB). Several groups have reported that abnormal form of α-synuclein can propagate through the cells and, consequently, form inclusions. Thus, it has been suggested different molecular mechanisms involved in α-synuclein propagation. It has been reported that cellular prion protein (PrPc) is a receptor of β-amyloid. In this study, we analyse whether the PrPc is a receptor for α-synuclein. Animals with different PrPc expression were intracranially injected with α-synuclein protofibrils. We observe that PrPc expression is not mandatory for α-synuclein propagation, but PrPc-overexpressing mice show more aggregates than in PrPc absence. Moreover, charge cluster domain of PrPc is essential for α-synuclein binding. In addition, we study Reelin levels in different neurodegenerative diseases. Reelin is a glycoprotein that is crucial for the correct cytoarchitectonic organization of the developing Central Nervous System. Decreased levels of Reelin lead to synaptic dysfunction or neurodegeneration. In the present study, we analyse the changes in Reelin and Reln mRNA in Alzheimer’s disease, Dementia with Lewy Bodies (DLB), Parkinson´s disease (PD) and sporadic Creutzfeldt-Jakob disease (sCJD). Meanwhile, inmunoblot results indicate decreased levels of Reelin in AD and DLB, PD do not show changes. In contrast, it has been detected an increase in sCJD(I). Reelin increased levels depends on reactive oxygen species (ROS). Using inhibitors of ROS production, as DPI and NAC, Reelin levels are maintained.
5
Bhamra, S. K. « Systematic mutagenesis of the mouse prion protein to identify critical regions for the efficient propagation of prions ». Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1443249/.
Texte intégralRésumé :
The aim of this study was to systematically investigate the contributions of various amino acids within the prion protein, on prion propagation. To test this in a cellular system, we used a sub-cloned population of N2a cells (PK1) that are highly susceptible to RML mouse prions. A library of stable PK1 cells was generated, which expressed the full length mouse prion protein (moPrP) bearing either point, double, or triple alanine replacements. The effects these changes in the prion protein sequence had on the ability of PK1 cells to propagate RML was tested using a previously established cell based assay. We found that: (i) in the unstructured region of the protein, alanine replacements in CC2 region 90-111 of the prion protein severely diminish, but do not abrogate the ability of cells to propagate prions whilst substitutions K23A.K24A.R25A and Q41A exerted a moderate inhibitory effect on propagation; (ii) alanine replacements in CC2 displayed a dominant negative effect by imposing their propagation inhibition phenotype in the presence of the wild-type protein; (iii) the diminished propagation abilities of cells expressing CC2 alanine mutants were a result of these cells being less susceptible to infection than their wild-type counterparts (iv) all alanine replacements tested in the structured region of the protein appeared to hamper prion propagation, regardless of their positioning within this globular domain. Taken together, these results suggest that integrity of the structured region is vital for successful prion propagation, and that although the flexible region of the prion protein alone (residues 23-111), does not exclusively confer infectivity and/or propagative capacity, charge interactions in this region govern the efficacy with which propagation ensues.
6
Wang, Weiqiang. « Prion inspired nanomaterials and their biomedical applications ». Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670982.
Texte intégralRésumé :
Els amiloides presenten una estructura fibril·lar molt ordenada. Molts d’aquests conjunts de proteïnes apareixen associats a malalties humanes. No obstant això, es pot aprofitar la naturalesa controlable, estable, ajustable i robusta de les fibres amiloides per crear nanomaterials amb una àmplia gamma d’aplicacions.
Els prions funcionals constitueixen una classe particular d’amiloides. Aquestes proteïnes transmissibles presenten una arquitectura modular, amb un domini prió desordenat responsable del assemblatge i d’un o més dominis globulars que proporcionen l’activitat. És important destacar que la proteïna globular original es pot substituir per qualsevol proteïna d’interès, sense comprometre el potencial de fibril·lació. Aquestes fusions genètiques formen fibres en les quals el domini global roman plegat, formant nanoestructures funcionals. Tot i això, en molts casos, els impediments estèrics poden restringir l’activitat d’aquestes fibres. Aquesta limitació es pot solucionar disseccionant els dominis priònics en seqüències més curtes que mantenen les seves propietats d’auto-assemblatge alhora que permeten un millor accés a la proteïna en estat fibril·lar.
En aquesta tesi doctoral, vam aprofitar el “soft amyloid core” (SAC) del prió de llevat Sup35p com una unitat de muntatge modular, que recapitula la propensió a l’agregació del domini priònic complet. Vam fusionar el SAC amb diferents proteïnes globulars d’interès que difereixen en la conformació i la mida, creant un mètode genètic general i senzill per generar nanofibres dotades de les funcionalitats desitjades. El modelatge computacional ens va permetre conèixer la relació entre la mida dels dominis globulars i la longitud del enllaç que els connecta al SAC, proporcionant les bases per al disseny de nanomaterials amb diferents propietats mesoscòpiques, ja siguin nanofibres o nanopartícules. Sobre aquesta base, hem dissenyat i produït, per primera vegada, nanopartícules amiloides esfèriques altament actives, no tòxiques, de mida definida, i s’han produït nanoestructures bifuncionals amb aplicació en el subministrament específic de fàrmacs. Les lliçons apreses en aquests exercicis van donar lloc a la construcció d’una nanofibrilla similar a un anticòs biespecífic amb potencial per la immunoteràpia.
En resum, els nanomaterials funcionals de tipus priònic descrits aquí aprofiten l’enfocament de la fusió genètica per crear un nou conjunt d’estructures amb aplicacions en biomedicina i biotecnologia.Los amiloides muestran una estructura fibrilar altamente ordenada. Muchos de estos ensamblajes aparecen asociados a enfermedades humanas. No obstante, la naturaleza controlable, estable, modulable y robusta de las fibras amiloides se puede emplear para construir nanomateriales notables con una amplia gama de aplicaciones. Los priones funcionales constituyen una clase particular de amiloides. Estas proteínas transmisibles exhiben una arquitectura modular, con un dominio priónico desordenado responsable del ensamblaje y uno o más dominios globulares que dan cuenta de la actividad. Cabe destacar que la proteína globular original se puede reemplazar con cualquier proteína de interés sin comprometer el potencial de fibrilación. Estas fusiones genéticas forman fibrillas en las que el dominio globular permanece plegado, lo que genera nanoestructuras funcionales. Sin embargo, en muchos casos, el impedimento estérico restringe la actividad de estas fibrillas. Esta limitación puede resolverse diseccionando los dominios de priones en secuencias más cortas que mantengan sus propiedades de autoensamblado mientras permiten un mejor acceso a la proteína en el estado fibrilar. En esta tesis doctoral, exploramos el "soft amyloid core" (SAC) del prion de levadura Sup35p como una unidad modular de autoensamblaje, que recapitula la propensión a la agregación del dominio priónico completo. Fusionamos el SAC con diferentes proteínas globulares de interés que difieren en conformación y tamaños, creando un enfoque genético general y directo para generar nanofibrillas dotadas de las funcionalidades deseadas. El modelado computacional nos permitió obtener información sobre la relación entre el tamaño de los dominios globulares y la longitud del conector que los une con el SAC, proporcionando la base para el diseño de nanomateriales con diferentes propiedades mesoscópicas, ya sean nanofibrillas o nanopartículas. Sobre esta base, diseñamos y producimos, por primera vez, nanopartículas amiloides esféricas, altamente activas, no tóxicas, de tamaño definido, y diseñamos nanoestructuras bifuncionales con aplicación en la administración dirigida de fármacos. Las lecciones aprendidas en estos ejercicios permitieron la construcción de una nanofibrilla similar a un anticuerpo biespecífico con potencial para su uso en inmunoterapia. En resumen, los nanomateriales funcionales similares a los priones descritos aquí aprovechan la metodología de fusión genética para generar un nuevo conjunto de estructuras con aplicación en biomedicina y biotecnología.
Amyloids display a highly ordered fibrillar structure. Many of these assemblies appear associated with human disease. However, the controllable, stable, tunable, and robust nature of amyloid fibrils can be exploited to build up remarkable nanomaterials with a wide range of applications. Functional prions constitute a particular class of amyloids. These transmissible proteins exhibit a modular architecture, with a disordered prion domain responsible for the assembly and one or more globular domains that account for the activity. Importantly, the original globular protein can be replaced with any protein of interest, without compromising the fibrillation potential. These genetic fusions form fibrils in which the globular domain remains folded, rendering functional nanostructures. However, in many cases, steric hindrance restricts the activity of these fibrils. This limitation can be solved by dissecting prion domains into shorter sequences that keep their self-assembling properties while allowing better access to the protein in the fibrillar state. In this PhD thesis, we exploited the "soft amyloid core (SAC)" of the Sup35p yeast prion as a modular self-assembling unit, which recapitulates the aggregation propensity of the complete prion domain. We fused the SAC to different globular proteins of interest differing in conformation and sizes, building up a general and straightforward genetic approach to generate nanofibrils endowed with desired functionalities. Computational modeling allowed us to gain insights into the relationship between the size of the globular domains and the length of the linker that connects them to the SAC, providing the basis for the design of nanomaterials with different mesoscopic properties, either nanofibrils or nanoparticles. On this basis, we designed and produced, for the first time, highly active, non-toxic, spherical amyloid nanoparticles of defined size and engineered bifunctional nanostructures with application in targeted drug delivery. The lessons learned in these exercises resulted in the construction of a bispecific antibody-like nanofibril, showing potential in immunotherapy. In summary, the prion-like functional nanomaterials described here take profit of the genetic fusion approach to render a novel set of structures with application in biomedicine and biotechnology.
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Apodaca, Jennifer J. « Regulation of prion protein in yeast and mammalian cells via ubiquitin mediated degradation a dissertation / ». San Antonio : UTHSC, 2008. http://proquest.umi.com.libproxy.uthscsa.edu/pqdweb?did=1594496391&sid=6&Fmt=2&clientId=70986&RQT=309&VName=PQD.
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Howlin, Robert. « Decontamination of prions, prion-associated amyloid and infectivity from surgical stainless steel : implications for the risk of iatrogenic transmission of CJD ». Thesis, University of Southampton, 2009. https://eprints.soton.ac.uk/150533/.
Texte intégralRésumé :
The physicochemical nature of the infectious agent in prion diseases creates a significant challenge for decontamination services. It has been shown to be both resistant to standard methods of decontamination, used to inactivate viruses and bacteria, and to associate avidly with surgical stainless steel. Moreover, the pathophysiology of the variant, iatrogenic and sporadic forms of Creutzfeldt-Jakob Disease (CJD) suggests deposition of the infectious agent across a wide range of extraneural, lymphoid tissues, as well as in the skeletal muscle and blood. Coupled with the potential for asymptomatic carriers, there is a significant risk of iatrogenic transmission of CJD through both neurosurgical procedures and standard surgery. This PhD study was undertaken in order to improve methods of instrument decontamination and to evaluate prion detection techniques and their applicability for the assessment of prion inactivation and removal. The project has provided relevant, critical assessment of hospital decontamination procedures, in addition to guidance on how working protocols should be improved to provide a cleaner and safer end product for the patient. Moreover, laboratory studies have been performed to evaluate current methods of prion decontamination in the context of hospital procedures for instrument reprocessing. Challenges faced by sterile service departments, such as soil drying and surface degradation, have been addressed and their impact on the risk of iatrogenic transmission of prions has been investigated. Critically, the use of a fluorescent amyloid fluorophore for the detection of prionassociated amyloid as a marker for disease permitted the investigation of the role of amyloid in infectious disease under denaturing conditions. Correlation of this detection technique with the identification of PrPres by Western blot and infectious disease suggested that, whilst fluorescent detection of prion-associated amyloid was more sensitive than Western blot, PrPres detection was more specific relative to infectivity. Improved fluorophores, with greater sensitivity, have been evaluated which will enhance in situ detection of prions in the future.
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Sun, Meng. « Development of the new yeast-based assays for prion properties ». Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45831.
Texte intégralRésumé :
Prion is an infectious isoform of a normal cellular protein which is capable of converting the non-prion form of the same protein into the alternative prion form. Mammalian prion protein PrP is responsible for prion formation in mammals, causing a series of fatal and incurable prion diseases. (1) We constructed, for the first time, a two-component system to phenotypically monitor the conformational status of PrP in the yeast cells. In this system, the prion domain of Sup35 (Sup35N) was fused to PrP90-230, and the initial formation of the PrPSc-like conformation stimulated prion formation of Sup35N, which in turn converted soluble Sup35 into the prion isoform, leading to a detectable phenotype. Prion-like properties of PrP were studied in this novel yeast model system. Additionally, we employed this system to study amyloidogenic protein Aβ42 aggregation in the yeast model.
It has been suggested that the ability to form transmissible amyloids (prions) is widespread among yeast proteins and is likely intrinsic to proteins from other organisms. However, the distribution of yeast prions in natural conditions is not yet clear, which prevents us from understanding the relationship between prions and their adaptive roles in various environmental conditions. (2) We modified and developed sequence and phenotype-independent approaches for prion detection and monitoring. We employed these approaches for prion-profiling among yeast strains of various origins.
(3) Lastly, we found a prion-like state [MCS+] causing nonsense suppression in the absence of the Sup35 prion domain. Our results suggested that [MCS+] is determined by both a prion factor and a nuclear factor. The prion-related properties of [MCS+] were studied by genetic and biochemical approaches.
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Dakowski, Caroline. « Rôle de la protéine prion cellulaire (PRPC) dans la différenciation neuronale : Infection par les prions (PRPSC) et bases moléculaires de la neurodégénérescence ». Thesis, Paris 5, 2012. http://www.theses.fr/2012PA05T032.
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Premzl, Marko, et Premzl@anu edu au premzl@excite com Marko. « Prion Protein Gene and Its Shadow ». The Australian National University. The John Curtin School of Medical Research, 2004. http://thesis.anu.edu.au./public/adt-ANU20050328.164529.
Texte intégralRésumé :
Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes.¶The normal function of PRNP is elusive. The Prnp knock-out mice with disrupted ORF show only very subtle phenotype. A number of hypotheses were proposed on the function of mammalian PRNP. The extracellular, GPI-anchored, glycosylated mammalian PrP^C expressed in a heterogenous set of cells could: transport copper from extracellular to intracellular milieu, buffer copper from synapse, contribute to redox signalling, act neuroprotectively, mediate cell-cell contacts, affect lymphocyte activation, participate in nucleic acid metabolism, be a memory molecule, and be a signal-transduction protein.¶ Experimental evidence demonstrated a redundancy between the PRNP and another, unknown gene. The critical issue therefore is to discover new genes homologous with PRNP, candidates for this redundancy. Using unpublished data, a sequence of zebrafish cDNA sequenced by Prof. Tatjana Simonics group (University of Milan, Italy), I discovered a new paralogue of PRNP. By searching manually, and in a targeted fashion, data deposited in public biological databases, I compiled support for the new human gene Shadow of prion protein (SPRN) including the direct evidence, homology-based evidence and ab initio gene prediction. The protein product called Shadoo (shadow in Japanese) is an extracellular, potentially glycosylated and GPI-anchored protein of a mature size of 100-odd amino acids. It is conserved from fish (zebrafish, Fugu, Tetraodon) to mammals (human, mouse, rat), and exhibits similarity of overall protein features with PrP. Most remarkably, the Sho is the first human/mammalian protein apart from PrP that contains the middle hydrophobic region that is essential for both normal and pathogenic properties of PrP. As this region is critical for heterodimerization of PrP, Sho may have potential to interact with PrP and is a likely candidate for the Protein X. Mammalian SPRN could be predominantly expressed in brain (Tatjana Simonic Lab, University of Milan, Italy).¶ Using the same approach to search public databases, I found, in addition, a fish duplicate of SPRN called SPRNB, and defined a new vertebrate SPRN gene family. Further, I also expanded a number of known fish genes from the PRNP gene family. The total number of the new genes that I discovered is 11. With the representatives of two vertebrate gene family datasets in hand, I conducted comparative genomic analysis in order to determine evolutionary trajectories of the SPRN and PRNP genes. This analysis, complemented with phylogenetic studies (Dr. Lars Jermiin, University of Sydney, Australia), demonstrated conservative evolution of the mammalian SPRN gene, and more relaxed evolutionary constraints acting on the mammalian PRNP gene. This evolutionary dialectic challenges widely adopted view on the highly conserved vertebrate PRNP and indicates that the SPRN gene may have more prominent function. More conserved Sprn could therefore substitute for the loss of less conserved, dispensable Prnp in the Prnp knock-out mice. Furthermore, the pathogenic potential of PRNP may be a consequence of relaxed evolutionary constraints.¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells.
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Fröhlich, Gallardo Estefanía Paz. « Aplicación de la inmunohistoquímica en óbex de caprinos para la detección de proteínas priónicas ». Tesis, Universidad de Chile, 2017. http://repositorio.uchile.cl/handle/2250/151607.
Texte intégralRésumé :
Memoria para optar al Título Profesional de Médico Veterinario.El Scrapie es una enfermedad neurodegenerativa y fatal que afecta a pequeños rumiantes como ovinos, caprinos y muflones. El causante de esta enfermedad es una partícula proteinácea infecciosa llamada prión, que se origina por un cambio conformacional de una proteína priónica celular del hospedero (PrPC). Los programas de vigilancia consideran a la Histopatología como método diagnóstico y a la Inmunohistoquímica (IHQ) como método confirmatorio para el Scrapie. Esta memoria fue desarrollada en el laboratorio de la Unidad de Patología del Servicio Agrícola y Ganadero de Lo Aguirre. Se utilizaron 50 muestras de óbex de caprinos mayores de 2 años, sin importar raza ni sexo, provenientes de mataderos de la IV Región de Chile. De cada muestra de óbex se obtuvieron cortes seriados con el propósito de destinar uno a la tinción de Hematoxilina y Eosina (H/E), y un corte homólogo a la técnica de Inmunohistoquímica. Así en la tinción H/E se comprobó la aptitud de la muestra evaluada a partir de la observación de la integridad de núcleos nerviosos de presentación bilateral. En total se trabajó con 50 muestras que fueron sometidas a los métodos tradicionales de histopatología para la tinción de H/E. De éstas, 50 cortes resultaron aptos para aplicar la técnica de Inmunohistoquímica. De los cortes sometidos a IHQ ninguno presentó el precipitado granular rojo característico de la inmunorreacción por la presencia de priones, por lo cual se determinó que todas las muestras de los caprinos estudiados fueron negativas, al igual que los controles negativos de óbex. Por su parte, los controles positivos de óbex siempre presentaron el precipitado granular rojo indicativo de una inmunorreacción positiva, tal como lo había indicado el Centro de Referencia para ese tipo de controles. De esta manera, este trabajo colaboró con la vigilancia pasiva anual que realizó el Servicio Agrícola y Ganadero durante el año 2015, con respecto a la detección de priones en la especie caprina en un determinado grupo de animales de nuestro país
Scrapie is a neurodegenerative and fatal disease, affecting small ruminants such as sheep, goats and mouflons. The cause of these diseases is an infectious proteinaceous particle called prion, which originates from a conformational change of a prion cellular protein of the host (PrPC). Surveillance programs consider histopathology as the diagnostic method and Immunohistochemistry (IHC) as a confirmatory method for Scrapie. This study was developed in the Laboratory of Pathology Unit of the Agricultural and Livestock Service of Lo Aguirre. Fifty obex samples of goats, older than 2 years, regardless of race or sex, were used from slaughterhouses in IV Region of Chile. Serial sections were obtained from each obex sample in order to assign one slice to the Hematoxylin and Eosin staining (H/E) and its homologous slice to the Immunohistochemistry technique. Thus, in the H/E staining the suitability of the slices were evaluated, from the observation of the integrity of the following nerve nuclei, with bilateral presentation: of the solitary tract, parasympathetic of the vagus nerve, of the spinal tract of the trigeminal nerve and motor of the hypoglossus. In total, 50 slices were performed, which were subjected to the traditional methods of histopathology and H/E staining. Of these, 50 were able to apply the technique of Immunohistochemistry (IHC). Of the samples submitted to IHC, none of them presented the red granular precipitate characteristic of the immunoreaction due to the presence of prions, whereby it was determined that all samples of the goats studied were to be classified as negative, as were the negative controls of Obex. On the other hand, positive obex controls always presented the red granular precipitate indicative of a positive immunoreaction, as indicated by the Reference Center for such controls. In this way, this work collaborated with the annual passive surveillance carried out by the Agricultural and Livestock Service during the year 2015, regarding the detection of prions in the goat species in a determined group of animals of our country
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Chen, Buxin. « Prion species barrier at the short phylogenetic distances in the yeast model ». Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/29762.
Texte intégralRésumé :
Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009.Committee Chair: Chernoff, Yury; Committee Member: Bommarius, Andreas; Committee Member: Doyle, Donald; Committee Member: Lobachev, Kirill; Committee Member: Yi, Soojin. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Chan, Hok-mo. « Medium security prison ». Hong Kong : University of Hong Kong, 1996. http://sunzi.lib.hku.hk/hkuto/record.jsp?B2595149x.
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Crozet, Carole. « Souris transgéniques pour la protéine prion ovine : transmission d'encéphalopathies subaiguës spongiformes transmissibles naturelles et expérimentales : contribution à la caractérisation des maladies à prions ». Lyon 1, 2001. http://www.theses.fr/2001LYO1T036.
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Boudet-Devaud, François. « La protéine prion cellulaire : un relai de neurotoxicité commun aux protéines amyloïdes et aux nanoparticules Protective role of cellular prion protein against TNFα-mediated inlammation through TACE α-secretase PrPSc-induced PDK1 overactivation promotes the production of seedable Amyloid-β peptides in prion diseases Corruption of cellular prion protein signaling by titanium dioxide or carbon black nanoparticles promotes the accumulation of amyloid-β peptides ». Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB127.
Texte intégralRésumé :
La protéine prion cellulaire (PrPC) est une protéine majoritairement exprimée à la surface des neurones, dont la conversion transconformationnelle en prion pathogène PrPSc, est à l'origine des maladies à prions. Il est clairement établi que la neurodégénérescence induite par la PrPSc dépend de l'expression de la PrPC dans les neurones et résulte d'une déviation de la/des fonction(s) de la PrPC par la PrPSc. Identifier le rôle de la PrPC est donc un pré-requis pour aborder les mécanismes de neurodégénérescence dans les maladies à prions. Une partie de mes travaux de thèse a permis de montrer que la PrPC exerce un rôle cytoprotecteur vis-à-vis de la cytokine inflammatoire TNFalpha. L'extinction de la PrPC dans les neurones (neurones PrPnull) rend ces cellules hypersensibles au TNFalpha en raison de l'accumulation membranaire des récepteurs au TNFalpha (TNFR). Mes travaux démontrent que la perte de la fonction régulatrice de la PrPC sur l'agrégation et la signalisation des intégrines bêta 1 dans les neurones PrPnull provoque la suractivation de la kinase PDK1, l'internalisation subséquente de l'alpha-sécrétase TACE, et un découplage de TACE vis-à-vis de l'un de ses substrats, TNFR. Étant donné la proximité phénotypique entre les neurones PrPnull (Ezpeleta et al. 2017) et les neurones infectés par la PrPSc (Pietri et al. 2013 ; Alleaume-Butaux et al. 2015), mes travaux plaident en faveur d'une perte de fonction cytoprotectrice de la PrPC dans les maladies à prions. Concernant l'infection à prions, mes travaux montrent que TACE internalisée en réponse à la suractivation de PDK1 est découplée d'un autre substrat, la protéine précurseur des peptides amyloïdes (APP), ce qui mène à l'accumulation des peptides neurotoxiques Abêta 40 et Abêta 42 caractéristiques de la maladie d'Alzheimer. Dans un contexte « infection à prions », les peptides Abêta 40/42 sont présents majoritairement sous une forme monomérique, et de façon plus discrète sous forme trimérique et tétramérique. Par des approches in vitro et in vivo, nous montrons que les peptides Abêta générés par les cellules infectées par les prions ne modifient ni la réplication ni l'infectiosité des prions. Néanmoins, nous démontrons que les formes oligomérisées d'Abêta sont capables de se déposer sous forme de plaques amyloïdes dans le cerveau des souris transgéniques APP23 infectées par les prions. Dans ces souris, les dépôts d'Abêta accélèrent la pathogenèse des prions. Le dernier axe de mon travail de thèse concerne les nanoparticules, des matériaux de taille nanométrique couramment utilisés dans de nombreux produits et procédés industriels. Mes travaux mettent en évidence que, à l'instar de la PrPSc et d'Abêta, des assemblages de nanoparticules de dioxyde de titane ou de noir de carbone se lient à la PrPC exprimée à la surface des neurones et dévient sa fonction de signalisation. Cette interaction PrPC/nanoparticules provoque, entre autres, la suractivation de PDK1, l'internalisation de TACE, et l'accumulation membranaire de TNFR. Les cellules neuronales exposées aux nanoparticules deviennent alors hypersensibles au stress inflammatoire TNFalpha. Le découplage de TACE à APP induit par les nanoparticules augmente aussi la production de peptides Abêta par les neurones. Même si aucune donnée épidémiologique n'associe une exposition aux nanoparticules à la maladie d'Alzheimer, mes travaux suggèrent une implication causale des nanoparticules dans l'initiation voire l'amplification de cette maladieThe cellular prion protein (PrPC) is a protein mostly expressed at the plasma membrane of neurons. Its transconformation into the pathogenic prion PrPSc is at the root of prion diseases. It is clearly established that the PrPSc-induced neurodegeneration depends on the expression of PrPC in neurons and results from the corruption of PrPC function(s) by PrPSc. Unravelling the role of PrPC is thus a prerequisite to grasp neurodegeneration mechanisms in prion diseases. Part of my work shows that PrPC exerts a cytoprotective function against TNFalpha inflammatory cytokine. PrPC silencing in neurons (PrPnull-neurons) renders these cells highly sensitive to TNFalpha due to surface accumulation of TNFalpha receptor (TNFR). My work demonstrates that the loss of PrPC regulatory function on the clustering and signaling downstream of bêta 1 integrins in PrPnull neurons provokes the overactivation of the kinase PDK1, subsequent internalization of TACE alpha-secretase, and uncoupling of TACE from TNFR substrate. Because of the phenotypic proximity between PrPnull neurons (Ezpeleta et al. 2017) and PrPSc-infected neurons (Pietri et al. 2013; Alleaume-Butaux et al. 2015), my work supports the view of a loss of PrPC protective function in prion diseases. As concerns prion infection, my work shows that after PDK1 overactivation, internalized TACE is uncoupled from another substrate, the amyloid peptides precursor protein (APP), leading to the accumulation of neurotoxic peptides Abêta 40 and Abêta 42, hallmarks of Alzheimer's disease. Within a prion infectious context, Abêta 40/42 peptides are predominantly present as monomers, and to a lesser extent, as trimers and tetramers. By combining in vitro and in vivo approaches, we show that Abêta peptides produced by infected neurons do not alter replication nor the infectivity of prions. Nevertheless, we demonstrate that oligomerized Abêta is able to form amyloid plaques in the brain of transgenic APP23 mice infected by prions. In these mice, Abêta deposits accelerate prion pathogenesis. The last axis of my work deals with nanoparticles, that is, nanometric materials commonly found in manufactured products and industrial processes. My work shows that, as PrPSc and Abêta, titanium dioxide or carbon black assemblies interact with PrPC at the surface of neurons and deviate its signaling function, which leads, inter alia, to PDK1 overactivation, TACE internalization, TNFR accumulation at the plasma membrane, and neuronal cells hypersensitivity to TNFalpha inflammatory stress. We also found that nanoparticle-induced TACE uncoupling from APP increases Abêta peptide production by neurons. Even if no epidemiological study has demonstrated to date a link between nanoparticle exposure and Alzheimer's disease, my work suggests an causal implication of nanoparticles in the initiation or amplification of this disease
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Coyle, Andrew G. « The organisational development of the Scottish Prison Service, with particular reference to the role and influence of the prison officer ». Thesis, University of Edinburgh, 1986. http://hdl.handle.net/1842/7557.
Texte intégralRésumé :
This thesis argues that the Prison Service, while it has several unique features, is a bureaucratic structure with a typical mix of organisational strengths and weaknesses. The study of the development of the organisation of the Scottish Prison Service is, therefore, as possible and as proper as is the study of any large organisation. The first substantive chapter of the thesis analyses the historical development of the Scottish Prison Service within an organisational context. This has taken place in 3 main phases, the first two of which were sequential, the third less obviously so and more the result of the increasing involvement of central bureaucratic processes. Historically the Scottish prison system has been properly located within the criminal justice process and throughout the first 100 years of its modem existence the judiciary and the legal establishment played a central role in its development. The first phase or its history covers the years between 1835 and 1877 when it was taken progressively under central control. Particular attention is paid to William Brebner, the founding father of the Scottish prison system, and to the place of the General Prison at Perth. The second historical phase covers the tenure of office of the Scottish Prison Commission between 1877 and 1929. The significance of the Elgin Report of 1900, which has not previously been the subject of research, is described. The third phase of development which began in 1929 and continues today-has attempted to take the prison system out of the criminal justice process and to place it inappropriately within the mainstream of the administrative Civil Service. The thesis analyses the reasons for this and suggests that this structural change, rather than any lack of resources, is responsible for many of the present difficulties facing the Prison Service. The second substantive chapter of the thesis examines the place of the prison system within the sociology of organisations. By definition, an organisation can have only one primary goal. A feature of bureaucratic organisations is that those who work within them will not be satisfied with a single objective and are likely to develop secondary goals. One consequence of the location of the prison system within the mainstream of the civil service has been an emphasis on the secondary goals of imprisonment, principally that of rehabilitation, to the neglect of the primary goal which is the punishment involved in the deprivation of liberty for the length of time laid down by the court. A second consequence is the influence which staff are able to exert on the development of the service. The manner in which the trade unionism of prison staff has evolved in Scotland makes this area particularly worthy of study; an important and topical example is the control of difficult prisoners. The Thesis suggests that the management of the Scottish Prison Service is more participative in style than either the Official or the Staff Side recognise. Throughout the thesis many of the arguments presented are given support by responses to a questionnaire which was issued to serving members of staff and which is fully documented into appendices.
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Ezpeleta, Juliette. « Du rôle physiologique de la protéine prion cellulaire à l'infection par les prions : régulation/dérégulation du module de signalisation PDK1/TACE α-secrétase Protective role of cellular prion protein against TNFα-mediated inflammation trough TACE α-secretase Cerebellar compartmentation of prion pathogenesis Production of seedable Amyloid-β peptides in prion diseases upon PrPSc-induced PDK1 overactivation ». Thesis, Sorbonne Paris Cité, 2019. http://www.theses.fr/2019USPCB004.
Texte intégralRésumé :
Les maladies à prions sont des maladies neurodégénératives caractérisées par l'accumulation dans le système nerveux central d'une protéine anormalement conformée et neurotoxique, la protéine prion Scrapie (PrPSc). La PrPSc est l'isoforme transconformationnelle d'une protéine normale de l'hôte, la protéine prion cellulaire (PrPC). Il est établi que la toxicité de la PrPSc est restreinte aux neurones et que la neurodégénérescence résulte d'une corruption de la/des fonction(s) physiologique(s) de la PrPC par la PrPSc. Néanmoins, nul ne sait s'il s'agit d'une perte de fonction protectrice ou d'un gain de fonction toxique de la PrPC, ou d'une combinaison des 2 événements, en partie parce que les fonctions de la PrPC restent difficiles à cerner. C'est pourquoi, identifier la/les fonction(s) de la PrPC est un prérequis pour comprendre comment la PrPSc exerce sa neurotoxicité. Mes travaux de thèse montrent pour la première fois une fonction protectrice de la PrPC vis-à-vis de la cytokine pro-inflammatoire sTNF-alpha Nous démontrons que la PrPC ajuste la sensibilité des cellules au sTNF-alpha en contrôlant le clivage des récepteurs au sTNF-alpha (TNFR1) par TACE. Au niveau mécanistique, la PrPC exerce un double contrôle sur TACE en gouvernant (i) son activité enzymatique, via le couplage de la PrPC à la NADPH oxydase/production de dérivés réactifs de l'oxygène, et (ii) sa localisation, en modulant négativement la voie de signalisation intégrines bêta-1/ROCK/PDK1, ce qui assure le maintien de TACE sous forme active à la membrane plasmique. La déplétion en PrPC provoque la micro-agrégation des intégrines bêta-1, la suractivation du duo de kinases ROCK/PDK1, et l'internalisation subséquente de l'alpha-secrétase TACE dans des microvésicules enrichies en Cavéoline-1. TACE est alors découplée de son substrat TNFR1, qui s'accumule à la membrane plasmique et rend les neurones déplétés en PrPC hypersensibles au stress inflammatoire de type sTNF-alpha. Ces mêmes défauts ont été retrouvés, avec des intensités comparables, dans les neurones infectés par les prions, ce qui appuie l'idée que la perte de la fonction cytoprotectrice de la PrPC dans les neurones vis-à-vis du sTNF-alpha participe à la progression des maladies à prions. Concernant l'infection à prions, un travail en collaboration révèle que les cellules de Purkinje du cervelet qui n'expriment pas les zébrines sont plus sensibles à la toxicité de 2 souches de prions, 22L et ME7, que celles qui expriment les zébrines. Cette étude suggère un rôle protecteur des zébrines vis-à-vis des prions. Un axe majeur de ma thèse identifie une nouvelle cible déréglée en aval du module de signalisation PDK1/TACE dans les maladies à prions, la protéine précurseur des amyloïdes (APP) avant tout connue pour son rôle dans la maladie d'Alzheimer. En abrogeant le clivage non-amyloïdogène d'APP par TACE, la PrPSc est à l'origine d'une surproduction d'Abêta40/42. Les peptides Abêta40/42 sont majoritairement sous forme monomérique, mais des formes multimériques (trimères et tétramères) d'Abêta40/42 sont aussi générées. Cette production d'Abêta40/42 dépend de la suractivation de PDK1 puisque l'inhibition pharmacologique de la kinase permet de réduire la production des peptides Abêta40/42 monomériques et rend les multimères indétectables. Il est à noter que les peptides Abêta produits ne modifient ni la réplication ni l'infectiosité de la PrPSc. Toutefois, l'Abêta40/42 généré par l'infection à prions est capable de former des dépôts dans les cerveaux de souris si, et seulement si un « seed » d'Abêta exogène a été co-transmis avec la PrPSc. De manière importante, la conjonction infection à prions/dépôts d'Abêta accélère la mort des souris infectées par les prions. Ces travaux définissent les conditions qui permettent la formation de plaques Abêta et mettent en exergue l'émergence d'une pathologie mixte causée par la présence de PrPSc et des dépôts d'Abêta dans un contexte infection à prionsPrion diseases are neurodegenerative disorders characterized by the accumulation into the central nervous system of an abnormally folded protein called Scrapie prion protein (PrPSc). PrPSc is the transconformational isoform of a ubiquitous protein of the host named cellular prion protein (PrPC). It is well established that the toxicity of PrPSc is restricted to neurons and arise from a corruption of the physiological function(s) of PrPC. However, the mechanisms by which PrPSc exerts its neurotoxicity remain poorly understood, partly because the physiological function(s) of PrPC is/are still elusive. Currently, no one knows if PrPC loses a protective role or acquires a toxic function upon its conversion into PrPSc, a combination of both events is also possible. Identifying PrPC-associated function(s) is thus a prerequisite to understand how PrPSc provokes neurodegeneration. The present work reports for the first time a protective role of PrPC towards the pro-inflammatory cytokine sTNF-alpha-associated toxicity. We show that PrPC adjusts cell sensitivity to sTNF-alpha by controlling TACE-dependent TNFR1 shedding. Mecanistically, PrPC governs both (i) TACE activity, through PrPC coupling to NADPH oxidase/Reactive Oxygen Species production, and (ii) TACE localization, by downregulating the beta-1 integrins/ROCK/PDK1 signaling pathway, thus PrPC ensures the bioavailability of an active TACE at the plasma membrane. PrPC depletion provokes the micro-aggregation of beta-1 integrins, the overactivation of ROCK and PDK1 kinases, and the subsequent internalization of TACE into Caveolin-1 enriched micro-vesicles. This leads to a defect of TNFR1 shedding, which accumulates at the plasma membrane and renders PrPC-depleted neurons highly vulnerable to sTNF-alpha insult. These alterations have also been reported in prion-infected neurons with the same intensities, supporting the view that a loss-of-the protective function of PrPC towards sTNF-alpha likely occur along prion diseases. Within a prion infectious context, a collaborative work revealed that the cerebellar Purkinje cells that do not express zebrins are highly vulnerable to the toxicity of two prion strains, 22L and ME7, compared to Purkinje cells that express zebrins. This suggest a protective role of zebrins against PrPSc-associated toxicity. A major part of my thesis identifies a new target deregulated downstream from the PDK1/TACE signaling module, the amyloid precursor protein (APP), well-known for its implication in Alzheimer's disease. By abrogating the non-amyloidogenic cleavage of APP by TACE, PrPSc provokes the overproduction of Abeta40/42 peptides. Abeta40/42 predominates as monomers but are also found as multimeric assemblies, i.e. trimers and tetramers. PrPSc-induced Abeta40/42 overproduction relates to PDK1 overactivation as pharmacological inhibition of PDK1 attenuates production of Abeta monomers and renders multimers undetectable. Of note, our work reveals that Abeta peptides do not impact on PrPSc replication nor infectivity. Nevertheless, Abeta40/42 peptides generated upon prion infection can deposit in mice brains only if an exogenous Abeta seed is co-transmitted with PrPSc. Importantly, Abeta deposition leads to early death of prion-infected mice. This work delineates the conditions that allow Abeta plaques formation and highlights the onset of a mixed-pathology caused by the co-occurrence of PrPSc and Abeta deposition within a prion infectious context
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Schwengler, Franziska. « Prion Diseases ». Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-36790.
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Bamia, Aline. « Identification de nouvelles molécules anti-prions et caractérisation de leurs modes d'action ». Thesis, Brest, 2019. http://www.theses.fr/2019BRES0047.
Texte intégralRésumé :
Le prion est un agent pathogène infectieux de nature protéique responsable de maladies neurodégénératives à la fois chez l’homme et les animaux. Le prion est responsable de la tremblante chez le mouton et la chèvre et de la maladie de Creutzfeldt-Jakob chez l’homme. Les maladies à prions sont fatales et il n’existe aucun traitement efficace de nos jours. C’est la raison pour laquelle dans mon laboratoire nous nous intéressons à l’identification de nouvelles molécules antiprions.La flunarizine a été identifiée comme étant active contre les prions [PSI+] et [URE3] de levure et contre PrPSc de mammifères in vitro, ex vivo et in vivo. L’efficacité de la flunarizine contre les prions sur tous ces modèles fait d’elle une bonne molécule candidate contre les maladies à prions. Une étude des relations structure-activité (RSA) autour de la flunarizine a été effectuée sur 47, dont 31 étaient actives contre PrPSc in vitro.Une étude des relations structure-activité (RSA) autour de la flunarizine a été effectuée sur 47, dont 31 étaient actives contre PrPSc in vitro. Six des molécules les plus actives en culture organotypique ont aussi montré leur efficacité contre PrPSc. L’effet de la flunarizine et de ses analogues contre le prion PrPSc ne dépendait pas de leurs modes d’actions connus.Les molécules les plus actives contre PrPSc inhibent la PFAR (protein folding activity of ribosome) une activité chaperon de protéines qui est impliquée dans la propagation du prion de levure [PSI+]. L’efficacité de ces molécules contre les prions fait d’elles de bons candidats pour un repositionnement thérapeutique pour les maladies à prions. Par ailleurs, nos travaux suggèrent que la PFAR pourrait être utilisée comme cible thérapeutique pour les maladies à prionsPrion is infectious protein responsible of neurodegenerative diseases in human and animal. Scrapie in goat and sheep and Creutzfeldt-Jakob disease in human are prion-related diseases. Prion diseases are fatal and to date there is no efficient treatment against these troubles. This is why in our lab we focus on identification of new compounds efficient against prions. Flunarizine was identified as new anti-prion compound efficient against yeast prion [PSI+] and [URE3], and against mammalian prion PrPSc in vitro, ex vivo and in vivo. Flunarizine may be good drug candidate against prion diseases due to its anti-prion potential in different model. Structure-activity relationship (SAR) around flunarizine hightlights 31 compounds out of 47 which inhibit prion PrPSc propagation in vitro. Six of most efficient compounds cleared prion PrPSc in organotypic slice culture. There were no relationship between flunarizine and related compound activities against prion PrPSc and their known mode of action. The most potent compounds against PrPSc inhibit PFAR (protein folding activity of ribosome). PFAR is a protein chaperon activity which is involved in yeast prion [PSI+] propagation. Many tested compounds are good candidates for drugs repurposing against prion diseases because of their important activity against PrPSc prion.Inhibition of PFAR by all the hightly effective flunarizine related compounds, suggest that PFAR may be consider as cellular target for prion related-diseases treatment
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Peyrin, Jean-Michel. « Implication des cellules microgliales dans la neuropathologie des maladies à prions ». Paris 5, 1998. http://www.theses.fr/1998PA05P226.
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Bariar, Bhawana. « Effects of the components of the Get pathway on prion propagation ». Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/26659.
Texte intégralRésumé :
Thesis (M. S.)--Biology, Georgia Institute of Technology, 2008.Committee Chair: Chernoff,Yury; Committee Member: Cairney,John; Committee Member: Choi,Jung; Committee Member: Doyle,Donald; Committee Member: Lobachev,Kirill. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Saijo, Eri. « INVESTIGATING THE ROLE OF PRION PROTEIN POLYMORPHISMS ON PRION PATHOGENESIS ». UKnowledge, 2012. http://uknowledge.uky.edu/microbio_etds/4.
Texte intégralRésumé :
Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are lethal and infectious neurodegenerative diseases of humans and animals. The misfolding of the normal, or cellular isoform of the prion protein (PrPC) into the abnormal disease-associated isoform of PrP (PrPSc) could change the properties of PrP, consequently, PrPSc has lethal infectivity to transmit diseases. The proteinaceous infectious particle consisting mainly of PrPSc is called prion. Transmissibility of prions is strongly influenced by multiple factors including PrP polymorphisms, species barriers (PrP sequence specificity) and prion strains (conformational specificity) by unknown mechanisms. Even though the ability of prions to cross a species barrier has been recognized, the precise mechanisms of interspecies prion transmission remain unclear.
This dissertation research was conducted in order to learn more about the molecular mechanisms of conversion, propagation and transmission of PrPSc; about determinants of genetic susceptibility to infection in prion diseases; and about understanding those mechanisms, which might govern the zoonotic potential of prion diseases.
First, we investigated the transmissibility risk of multiple strains of Chronic Wasting Disease, which is a cervid TSE, with humanized transgenic mice and showed that the transmission barriers between cervid and the humanized mice are high. Next, the structural factors underlying the species barrier of prion diseases were studied using cell culture systems by systematically introducing amino acid substitutions in the regions of PrP, where the most divergences of different PrP species are recognized. Thirdly, we investigated the effects of the genetic susceptibility to prions as well as conversion kinetics and properties of PrPSc using Tg mice expressing ovine PrP polymorphism (OvPrP) at codon 136 either alanine (A) or valine (V). The templating characteristics of OvPrPSc-V136 were dominant over OvPrPSc-A136 under co-expressions of OvPrPC-A136 and OvPrPC-V136. Finally, the function of PrP was studied in relation to the pathogenesis of Alzheimer’s disease.
These studies demonstrated that the conformational compatibility between PrPC and PrPSc contributed to the conversion kinetics and species barrier. We concluded that the conformational compatibility of PrPC to PrPSc is controlled not only by the PrP sequence specificity but also by the tertiary structure of PrPC.
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Rumscheid, Frank. « Die figürlichen Terrakotten von Priene : Fundkontexte, Ikonographie und Funktion in Wohnhäusern und Heiligtümern im Licht antiker Parallelbefunde ». Wiesbaden Reichert, 2006. http://bvbr.bib-bvb.de:8991/F?func=service&docl̲ibrary=BVB01&docn̲umber=015027207&linen̲umber=0001&funcc̲ode=DBR̲ECORDS&servicet̲ype=MEDIA.
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Krejciova, Zuzana. « Exposure and response of human non-neuronal cells to prions in vitro ». Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/8186.
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Despite intensive research, the cellular and molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined, in part due to the continuing lack of cultured human cells that are susceptible to infection with human prions. Such culture models would present distinct advantages including speed and expense compared with animal models, and would provide systems in which to investigate the interaction between PrPC and PrPSc, the basis of cellular susceptibility, the nature of the species barrier and the mechanism of prion propagation in situ. This study sought to examine whether non-neuronal cells might provide opportunities to establish human cell lines replicating human prions. A human follicular dendritic cell-like cell line (termed HK) was obtained, further characterised and then tested for its ability to support human prion replication. The mechanisms of internalisation, intracellular trafficking and the eventual fate of exogenous PrPSc taken up by these cells were also examined. This thesis similarly examined the cellular response of human embryonic stem cells (hESC) to acute exposure to human and animal prions. PrPC was found to be abundantly expressed by HK cells and HK cell extracts were found to support conversion to PrPSc in a cell-free conversion assay. However, HK cells exposed to infectious brain homogenates failed to accumulate PrPSc or become infected in vitro. Exposed HK and hESC did display a readily detectable, time dependent uptake of PrPSc from medium spiked with prion-infected brain homogenates that was independent of the species, disease phenotype and PRNP codon 129 genotype of the human source and the recipient cells. The exposed cells showed intensely labelled intracellular accumulations of PrPSc with coarse granular morphology, largely in the juxtanuclear region of cytoplasm. However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished. Co-localisation studies implicated caveolae-mediated endocytic uptake of exogenous PrPSc, apparently preceding uptake via clathrin coated pits in HK cells. Evidence suggesting that the endosomal recycling compartment and lysosomes are involved in intracellular trafficking and degradation of exogenous PrPSc was also found. Understanding the cell biology of these processes may help to explain why the majority of cultured cells are refractory to prion infection in vitro. Internalization of misfolded PrP and its subsequent degradation in the lysosomal compartment might function as a self-protective cellular mechanism, serving to eliminate non-native, presumably dysfunctional and potentially dangerous PrP conformers, whether generated endogenously or acquired through exposure to exogenous prion infectivity.
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Mirabile, I. « Prion pathology in the brainstem : clinical target areas in prion disease ». Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1346475/.
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Prion diseases are fatal transmissible neurodegenerative disorders characterized by spongiform changes, neuronal loss, reactive astrocytosis, and deposition of disease associated prion protein (PrP). Our aim was to investigate "clinical target areas" for prion disease, responsible for disease onset, progression, and the clinical phenotype, using PrP overexpressing MloxP and PrP depleted NFH-Cre/MloxP transgenic mouse lines. Upon infection with different prion strains NFH-Cre/MloxP mice have significantly longer survival than MloxP mice (first set of experiments: Me7, ~29 weeks vs. ~17 weeks; Mouse-adapted BSE , ~33 weeks vs. ~20 weeks; second set of experiments: RML, ~35 weeks vs.12 weeks; Me7 ~29 weeks, vs. ~17 weeks; MRC2 ~31 weeks vs. ~22 week). As we found that the first pathological changes in the brains of Me7 and Mouse–adapted BSE infected mice are localized in the brainstem, and clinical signs of prion disease point to brainstem failure, we quantitatively scored spongiosis, abnormal PrP accumulation and astrogliosis at early and late stage of disease in specific brainstem nuclei of RML and Me7 infected MloxP and NFH-Cre/MloxP mice. The first target areas showing abnormal PrP accumulation and gliosis in both prion infections are the locus coeruleus (LC), the nucleus of the solitary tract (NTS) and the pre-Bötzinger complex (PBC). We then studied the pathology progression, scoring prion pathology in these and other brainstem nuclei of infected MloxP and NFH-Cre/MloxP mice in the course of the disease. We show that neural degeneration in the LC, NTS, and PBC correlate with clinical signs characteristic of terminally ill mice. We therefore propose that these areas are potential clinical target areas of prion disease. We also studied the spatial and temporal characteristics of Cre-mediated recombination. With immunohistochemistry in reporter mice, we estimated that in the LC, NTS, and PBC, Cre-mediated recombination is 60% or lower, and this can explain why mice proceed to terminal stage of the disease. In NFH-Cre/MloxP mice we found that recombination is a progressive event and in the hippocampus it is complete by 5 weeks post-natally, differently from previous data. Finally, we produced anti PrP RNAi –encoding lentivirus which could be used as focal therapy in the clinical target areas we propose.
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Carulla, Martí Patricia. « La proteína priónica celular : Análisis de su función neuroprotectora y reguladora del ciclo Celular ». Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/129157.
Texte intégralRésumé :
Las encefalopatías espongiformes transmisibles (EETs) son un conjunto de enfermedades neurodegenerativas letales que afectan tanto animales como humanos. Están causadas por unas partículas proteicas denominadas “priones”, resultantes de la transformación de la proteína priónica celular (PrP(C)) a una forma patogénica denominada PrP(SC), que se acumula en el cerebro en forma de agregados e induce la transformación de más moléculas a expensas de la reducción de los niveles de PrP(C).
Hasta el día de hoy, el estudio de las prionopatías se ha centrado en la bioquímica, toxicidad y transmisibilidad de PrP(SC). Sin embargo, la pérdida de la proteína endógena puede también contribuir al desarrollo de la enfermedad. A día de hoy, la función fisiológica de PrP(C) está aún por esclarecer, aunque los análisis realizados la involucran en procesos de: i) ciclo celular y proliferación, ii) homeostasis del cobre, iii) neuroprotección, iv) transmisión sináptica y v) señalización intracelular, entre otros.
Esta Tesis Doctoral aborda la función de PrP(C) en la regulación del ciclo celular y la neuroprotección, ampliando los conocimientos existentes mediante la descripción de las vías moleculares implicadas en ambos procesos. Nuestros datos describen a PrP(C) como una proteína capaz de integrar gran variedad de señales extracelulares y generar respuestas a través de la modulación de diferentes vías de señalización asociadas.
Hemos observado como la modulación transitoria de los niveles de PrP(C) en una línea celular de neuroblastoma produce cambios dosis-dependientes sobre los mecanismos de proliferación celular. Estas alteraciones son debidas a la interacción de la proteína con EGFR y la consiguiente activación de las vías ERK1/2 y AKT, que incluyen entre sus dianas a elementos reguladores del ciclo celular. PrP(C) también parece ejercer cierta influencia sobre la formación de filopodios en este tipo celular, regulando la dinámica del citoesqueleto de actina por su acción sobre las Rho GTPasas, concretamente, vía Cdc42-N-WASP.
Por otro lado, el uso de un modelo in vivo de excitotoxicidad por KA nos ha permitido entrar en detalle en la función neuroprotectora de PrP(C). Se conoce que tanto la susceptibilidad incrementada de los ratones Prnp-knockout a agentes convulsionantes como el daño neurotóxico derivado a nivel del hipocampo, son debidos a la activación de la vía JNK3 de muerte celular. Sin embargo, nuestros resultados obtenidos in vivo de ratones Prnp0/0 Jnk3+/+ y Prnp0/0 Jnk30/0 describen por primera vez la participación de PrP(C) en la modulación de esta vía de señalización. Hemos descrito como la proteína priónica interactúa a nivel postsináptico con la subunidad GluR6/7 de los receptores de glutamato, bloqueando por un lado la formación del complejo trimérico GluR6/7-PSD-95-MLK3 que lleva a la activación de la vía JNK3 y, por otro, modulando la actividad del receptor, que es un factor determinante de la excitabilidad neuronal incrementada propia de un episodio epiléptico.
Esta Tesis Doctoral supone, por lo tanto, un avance en el conocimiento de la función fisiológica de PrP(C) y, consecuentemente, en el estudio de la fisiopatología de la enfermedad priónica. La implicación de esta proteína en procesos proliferativos esenciales durante la neurogénesis así como en la regulación de la transmisión sináptica, deja entrever que PrP(C) es esencial para el desarrollo y mantenimiento de los circuitos neuronales. Probablemente, las alteraciones derivadas de la reducción de los niveles de PrP(C) se suman a los efectos neurotóxicos de la agregación de PrP(SC), dando lugar al desarrollo de la enfermedad. Asimismo, la descripción de los mecanismos moleculares y vías de señalización reguladas por PrP(C) también abre las puertas a la identificación de nuevas dianas terapéuticas para la prevención o tratamiento de las EETs.Transmissible spongiform encephalopathies constitute a group of fatal neurodegenerative disorders affecting both animals and humans. The causative agents of these diseases are “prions”, which are defined as proteinaceous infectious particles that result from an abnormal conformational folding of the cellular prion protein (PrPC) into a pathogenic form called PrPSC. Although the role of PrPSC has been intensively studied, the physiological function of PrPC still remains unclear. Several evidences support the notion that PrPC plays a role in different cellular processes including: i) cell cycle and proliferation, ii) copper homeostasis, iii) neuroprotection, iv) synaptic transmission and v) intracellular signaling, among others. Here we expand previous data concerning PrPC function by describing the molecular mechanisms by which PrPC regulates cell proliferation in vitro and excitotoxic cell death in vivo. First, we describe how transient overexpression of PrPC in Neuro2a cells enhances cell cycle progression after mitogenic stimulation, while the opposite effect is observed when PrPC is silenced. These effects are due in part to the interaction of PrPC with the epidermal growth factor receptor (EGFR) in the cell membrane and the consequent downstream activation of ERK1/2 and protein kinase B (AKT) pathways. We also describe PrPC-dependent filopodia formation in Neuro2a cells through the modulation of AKT-Cdc42-N-WASP pathway. In a second study, we took advantage of the Prnp0/0Jnk3+/+ and Prnp0/0Jnk30/0 mice models to analyze the neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death. Our results indicate that PrPC regulates kainate receptor-mediated neurotransmission through its interaction with the GluR6/7-PSD-95-MLK3 complex and the downstream modulation of JNK3 neurotoxic signaling. These new insights into the molecular functions of PrPC help us to understand the physiopathological events underlying prion disease and other related neurodegenerative pathologies.
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Smith, Juliana Jamel. « The cultural dynamic of the prison industrial complex a critique of political rhetoric and popular film during the 1980's / ». Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1450190.
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Thesis (M.A.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed April 7, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 121-129).
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Kiachopoulos, Sophia. « Biogenese des Prion-Proteins ». Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-44118.
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Sarafoff, Nikolaus. « Amplifikation von Prionen in vitro ». Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-57072.
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Radreau, Félicie. « Cellules souches embryonnaires et neurales humaines : quand la PrP et l'APP "s'en mêlent" ou "s’emmêlent" ». Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT045/document.
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La Protéine Prion cellulaire (PrPc) est une protéine ubiquitaire mais majoritairement présente dans le système nerveux central. Elle est plus particulièrement connue pour sa conversion conformationnelle en PrPSc dans les maladies à Prions qui sont des Protéinopathies comme la maladie d’Alzheimer (MA). La MA est en partie associée à des dépôts de peptides beta-amyloïdes (Aβ) agrégés de façon extracellulaire et issus des clivages successifs par la β- puis la γ-sécrétase de la protéine précurseur amyloïde (APP) exprimée dans les neurones. La PrPc et l’APP partagent des fonctions et des voies protéolytiques communes (α- ou β-sécrétase) les impliquant dans la prolifération, la différenciation, la synaptogenèse et la survie cellulaire. La PrPc est impliquée dans la régulation de la prolifération et la différenciation de différentes cellules souches : neurales adultes (NSC), hématopoïétiques (HSC), embryonnaires humaines (hESC). Si la PrP et l’APP partagent des fonctions communes, plusieurs publications montrent que la PrPc régule négativement le clivage de l'APP en Aβ et positivement le clivage de l’APP en sAPPα suggérant ainsi un rôle anti-amyloïdogénique de la PrPc. La PrP agirait également comme récepteur des Aβ à la surface neuronale induisant notamment l’inhibition des LTP et l’altération synaptique.Dans ce contexte, les objectifs spécifiques de la thèse sont :- L’étude de l’expression de la PrP, de l’APP et ses résidus de clivage au cours de l’induction neurale des hESC en NSC et de la différenciation neuronale- L’impact de la modulation de l’expression de la PrP sur le clivage de l’APP ainsi que sur les propriétés des cellules souches (survie, prolifération, différenciation).1. Induction neurale des hESC en NSC Pour ce projet nous avons utilisé des Cellules Souches Embryonnaires Humaines (hESC) pour lesquelles le laboratoire dispose d’une autorisation de l’Agence de la Biomédecine.Pour l’induction neurale, nous avons testé deux protocoles : l’un permet d’obtenir des neurosphères en suspension puis des «rosettes» constituées de NSC, l’autre protocole en monocouche mime quant à lui la corticogenèse. Une optimisation de ces protocoles a été nécessaire (densité de départ, méthodes de fixation des cellules pour améliorer la détection de la PrP) ainsi que la détermination des conditions d’analyse de l’expression de PrP, d’APP et ses résidus clivés (Aβ, sAPPα/β). 2. Différenciations à partir des NSC Les NSC obtenues ont ensuite été amplifiées puis différenciées en neurones et/ou astrocytes. Les cellules ont été caractérisées notamment par immunofluorescence et RT-qPCR pour l’expression des principaux marqueurs astrocytaires (GFAP) et neuronaux (BIII-tubuline, Doublecortine, Synaptophysine) et la disparition progressive des marqueurs de NSC. Là encore nous avons établi des conditions précises de densité cellulaire ainsi que les points des analyses cinétiques de nos différents paramètres.3. Modulation de l’expression de la PrPc Nous avons utilisés des vecteurs lentiviraux permettant l’expression ou l’inhibition de la PrPc humaine pour transduire des hESC au moment d’initier l’induction neurale et des NSC. Pour cela nous avons également dû réaliser des optimisations de différents paramètres : densité cellulaire, taille des supports d’ensemencement ou MOI de lentivirus afin d’avoir une transduction efficace tout en limitant la cytotoxicité. De même, les échantillons récoltés nous ont permis d’évaluer l’impact de la modulation de la PrPc sur le clivage de l’APP ainsi que sur la biologie des cellules souches (survie, prolifération, différenciation)The cellular Prion Protein (PrPc) is a ubiquitary protein mainly expressed in the central nervous system. It is particularly known for its conformational conversion in PrPSc in Prion diseases, which are proteinopathies such as Alzheimer’s disease (AD). AD is associated with extracellular deposits of aggregated beta-amyloid peptides (Aβ) derived from successive β- and the γ-secretase cleavages of the amyloid precursor protein (APP) expressed by neurons. PrPc and APP share some common functions and proteolytic pathways (α- or β-secretase), involving them in proliferation, differentiation, synaptogenesis and cellular survival. PrPc is involved in the regulation of proliferation and differentiation of many stem cells: adult neural (NSC), hematopoietic (HSC) and human embryonic (hESC). Several publications also show that PrP downregulates the cleavage of APP in Aβ and positively regulates the cleavage of APP in sAPPα suggesting an anti-amyloïdogenic role of PrPc. PrP could also act as a receptor of Aβ at the neuronal surface inducing LTP inhibition and synaptic alteration. In this context, the specific objectives of my thesis were:- Study of the expression of PrP, APP and its cleavage residues during neural induction of hESC in NSC and neuronal differentiation.- Impact of the modulation of PrP expression on APP cleavages as well as on stem cells properties (survival, proliferation, differentiation). 1. Neural induction of hESC in NSCFor this project, we have used Human Embryonic Stem Cells (hESC) for which the laboratory has an authorization from the “Agence de la Biomédecine”.For the neural induction, we have tested two protocols, the first one allows the obtention of neurospheres in suspension and then figures of “rosettes” composed of NSC, and a “monolayer” protocol that mimics the beginning of corticogenesis. An optimization of these protocols has been necessary (starting cell density, cell fixation methods to improve PrP detection). We have also determined the best conditions to analyze the expression of PrP, APP and its derived peptides (Aß, sAPPα/β). 2. Differentiation of NSCNSC derived from hESC were amplified and differentiated into neurons and/or astrocytes. Cells were characterized in particular by immunofluorescence and RT-qPCR for the expression of the major astrocytic (GFAP) and neuronal markers (BIII-tubulin, doublecortin, synaptophysin) and the progressive decrease of NSC markers. Again we have determined the best conditions for cell density and kinetic time points for our analysis.3. Modulation of PrPC expression We have used lentiviral vectors allowing the expression of an anti-PrP shRNA, human PrP and respective controls. To achieve this task, lentiviral transductions of hESC and NSC were optimized: cell density, size of the seeding culture wells or MOI of lentivirus. Finaly, samples collected allowed us to evaluate the impact of PrPc modulation on the APP cleavages as well as on stem cells properties (survival, proliferation, differentiation)
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Mead, Simon Harvey. « Molecular genetic analysis of the prion protein gene locus in human prion disease ». Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417947.
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Shi, Song. « Screening anti-prion compounds and diagnosing prion diseases by amplifying PrPSc in vitro ». Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-179963.
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Mahmoud, Mohamed Karmi Hussein. « Studies on pathogenic mechanisms of prion diseases and evaluation of prion strains properties ». Diss., Munich Verl. Dr. Hut, 2009. http://d-nb.info/992892376/04.
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Klingeborn, Mikael. « The prion protein in normal cells and disease : studies on the cellular processing of bovine PrPC and molecular characterization of the Nor98 prion / ». Uppsala : Department of Molecular Biosciences, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/2006105.pdf.
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Sanghera, Narinder. « The interaction of the prion protein with lipid membranes and implications for prion conversion ». Thesis, University of Warwick, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247140.
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Mashima, Tsukasa. « The structural analysis of RNA aptamer against prion protein and its anti-prion activity ». Kyoto University, 2013. http://hdl.handle.net/2433/170071.
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Lenuzza, Natacha. « Modélisation de la réplications des Prions : Implication de la dépendance en taille des agrégats de PrP et de l'hétérogénéité des populations cellulaires ». Phd thesis, Ecole Centrale Paris, 2009. http://tel.archives-ouvertes.fr/tel-00453321.
Texte intégralRésumé :
Les maladies à Prions sont des maladies neurodégénératives fatales, touchant l'homme et l'animal. Même si le risque de transmission de la maladie de la vache folle à l'homme semble maîtrisé, il persiste actuellement un risque de santé publique lié à la transmission iatrogène de cette forme, notamment par transfusion sanguine. Pour contrôler cette transmission, il est donc essentiel de mieux comprendre les mécanismes moléculaires et cellulaires de réplication et de dissémination des Prions. Ces mécanismes de réplication se produisent à des échelles de temps et de taille difficilement accessibles expérimentalement, et ont ainsi fait l'objet de nombreuses modélisations théoriques utiles pour aider à la compréhension des mécanismes. L'objectif de cette thèse est de compléter ces modèles mathématiques, afin d'étudier plus spécifiquement les conséquences dynamiques sur la réplication des Prions, des propriétés de réplication taille-dépendante d'une part, et de l'hétérogénéité des cellules impliquées dans la réplication d'autre part. Dans un premier temps, nous avons généralisé un modèle de polymérisation nucléée pour prendre en compte un taux d'élongation des fibrilles dépendant de leur taille. Nous avons principalement déduit de cette étude que la distribution en taille des agrégats semble une donnée expérimentale très informative sur les mécanismes élémentaires de réplication, au contraire du profil cinétique d'accumulation de la PrPres peu sensibles aux propriétés de réplication taille-dépendantes. Dans un second temps, après une caractérisation expérimentale de l'hétérogénéité cellulaire de réplication, nous avons intégré le mécanisme de réplication intracellulaire à un modèle multicellulaire par automate cellulaire continu stochastique. De manière appliquée, cette étude nous a permis d'identifier des étapes du processus de culture cellulaire critiques pour l'établissement d'une infection chronique, et nous a permis de proposer plusieurs protocoles pour augmenter la sensibilité des cultures cellulaires aux infections à Prions.
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Chu, Clement SM. « Towards the structure of yeast prions ». Diss., Search in ProQuest Dissertations & ; Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3390039.
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Ragagnin, Audrey. « Mort neuronale et maladies à prions ». Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ094/document.
Texte intégralRésumé :
La conversion conformationnelle de la protéine prion cellulaire PrPC neuroprotectrice en protéine prion PrPSc infectieuse et pathogène caractérise les maladies à prions. Dans le cerveau infecté par les prions, la perte de PrPC, le gain de PrPSc neurotoxique et l’inflammation concourent à la mort neuronale par des mécanismes encore mal connus.Ces travaux valident les cultures organotypiques de cervelet de souris comme système expérimental ex vivo favorable à l’étude de ces mécanismes et montrent que l’absence de PrPC aussi bien que PrPSc activent des mécanismes apoptotiques et autophagiques qui conduisent à la mort des cellules de Purkinje du cervelet. Une deuxième étude in situ chez la souris montre que la compartimentation anatomo-fonctionnelle du cervelet est un paramètre endogène de la pathogenèse des prions de tremblante 22L. Une troisième série d’expériences in situ montre que les prions provoquent l’augmentation du récepteur TNFR1 de la cytokine pro-inflammatoire TNF-α à la membrane des astrocytes enveloppant les synapses excitatrices des cellules de Purkinje dans le cortex cérébelleux de souris infectées. Ceci implique une composante astrocytaire dans la réaction des complexes synaptiques aux prionsThe conversion of the protective cellular prion protein PrPC into an infectious, neurotoxic conformer PrPSc is a feature of prion diseases. In the prion-diseased brain, the loss of PrPC, the production of pathogenic PrPSc and inflammation contribute to neuronal death by still unknown mechanisms.The present results validate cerebellar organotypic cultures as a valuable experimental system to study ex vivo these mechanisms and provide insight into the apoptotic and autophagic processes activated by the absence of PrPC in Prnp-deficient mice and by PrPSc prions and lead to the death of the cerebellar Purkinje cells. A second line of research in situ showed that the anatomo-functional compartmentation of the mouse cerebellum is an endogenous parameter of the pathogenesis of the 22L scrapie prions. Finally, another in situ approach revealed that prions increase the levels of TNFR1, a receptor for the pro-inflammatory cytokine TNF-α at the membrane of the astrocytes enveloping Purkinje cell excitatory synapses in the cerebellar cortex of infected mice. This implies that the response of synaptic complexes to prions involves a glial component
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Gierusz, Leszek A. « Folding and fibril formation of prions ». Thesis, University of Warwick, 2012. http://wrap.warwick.ac.uk/56927/.
Texte intégralRésumé :
Prions diseases are a group of fatal neurodegenerative disorders called the transmissible spongiform encephalopathies (TSEs), which include bovine spongiform encephalopathy in cattle, scrapie in sheep and Creutzfeldt-Jakob disease (CJD) in humans. TSEs are associated with the conversion of normal cellular form of the prion protein (PrPC) to an altered pathological form (PrPSc). An important phenomenon known as the species barrier affects prion transmission, resulting in longer incubation time and lower incidence of disease upon transfer between species. Another feature of prion diseases is diseasemodulating polymorphisms in PrP sequence which can alter individual‟s susceptibility to infection. This thesis investigates two properties of PrP that may elucidate the mechanisms underlying both species barrier and disease resistance; (i) effect of diseasemodulating mutations on folding kinetics of PrP and (ii) impact of diseasemodulating mutations on formation of PrP fibrils. Equilibrium and kinetic folding studies demonstrate that the folding pathway of PrP is affected by mutation Q167R which confers disease resistance, and mutations S170N, N174T and S170N/N174T characteristic for Chronic Wasting Disease in cervids, which are known to increase disease susceptibility. The destabilising effect of Q167R mutation previously observed via equilibrium folding studies was confirmed through direct kinetic observations. Subsequent fibrilisation experiments suggested a possible link between the stability of mouse prion protein and its propensity to form fibrils, elucidating a potential mechanism of increased disease resistance conferred by Q167R mutation. Equilibrium folding studies of S170N, N174T and S170N/N174T revealed a surprising correlation between the structural effects of these mutations and fibrilisation propensity. Based on these findings, a disease resistance mechanism centred on decreased formation of neurotoxic particles in the organism as well as diminished ability of infectious oligomers from both inside and outside to propagate oligomerisation of PrP has been proposed.
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Toupet, Karine. « Stratégies thérapeutiques des maladies à prions ». Montpellier 2, 2009. http://www.theses.fr/2009MON20128.
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Les maladies à prions sont des maladies neurodégénératives qui touchent l'homme et l'animal, et dont l'issue est fatale. Ces maladies sont provoquées par l'accumulation dans le cerveau de la PrPSc, l'isoforme mal repliée de la protéine prion cellulaire. L'apparition de nouveaux risques de transmission de ces maladies et l'absence de traitement efficace, nous ont incité à explorer de nouvelles stratégies et cibles thérapeutiques. Nous avons développé deux approches thérapeutiques innovantes. La première à consister à rechercher des molécules capables de piéger les formes préamyloïdes de la PrPSc (dimères et trimères), décrites comme éléments essentiels du cycle de réplication des prions. Une technique de criblage de drogues in silico et in cellulo nous a permis de mettre en évidence des composés thiényl pyrimidiques et thiényl azines capables d'oligomériser spécifiquement la PrPSc. Ces oligomères de PrPSc réduisent l'infectiosité des prions in vivo, soulignant le potentiel thérapeutique de ces composés. Notre deuxième stratégie est une stratégie de thérapie génique utilisant les propriétés dominantes négatives de certains polymorphismes de la protéine prion, comme les mutants Q218K et Q167R. Notre objectif a été d'évaluer le potentiel thérapeutique de vecteurs lentiviraux portant les mutants PrPQ218K et PrPQ167R, chez des souris au stade tardif de la maladie. Nous avons réussi à prolonger significativement la durée de vie des souris de 20% grâce à 2 injections chroniques de vecteurs lentiviraux portant le mutant PrPQ167R. Nos résultats ouvre la voie sur de nouvelles perspespectives thérapeutiques pour les maladies à prions et autres maladies neurodégénérativesPrion diseases are fatal neurodegenerative disorders that affect both humans and animals. These diseases are induced by the accumulation in the brain of the misfolded isoform of the normal cellular prion protein: PrPSc. The emergence of new risks of transmission for these diseases and the lack of efficient treatments, prompt us to search for new therapeutic strategies and targets. We developed two innovative therapeutic approaches. The first one consisted in searching for molecules able to trap preamyloid forms of PrPSc (dimers and trimers), known as key elements in the replication cycle of prions. A drugs screening approach, in silico and in cellulo, allowed us to discover thienyl pyrimidine and thienyl azine compounds able to specifically oligomerize PrPSc molecules. These PrPSc oligomers decrease prions infectivity in vivo, highlighting the therapeutic potential of these compounds. Our second strategie is a gene therapy approach using the dominant negative properties of certain polymorphisms of the prion protein, such as the Q218K and Q167R mutants. Our objective was to evaluate the therapeutic potential of lentiviral vectors carrying the PrPQ218K and PrPQ167R mutants, in mice, at the terminal stage of the disease. We succeeded in significantly prolonging the survival time of mice of 20%, with two intracerebrally chronic injections of lentiviral vectors carrying the PrPQ167R mutant. All our results not only open the way for new therapeutic strategies against prion diseases but also will benefit for therapies of other neurodegenerative disorders
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Ekwa, Robert. « Les maladies à prions : problèmes épistémologiques ». Paris 1, 2012. http://docelec.u-bordeaux.fr/login?url=http://www.harmatheque.com/ebook/les-maladies-a-prions--problemes-epistemologiques--volume-2--vache-folle-et-raisonnements-causals-41085.
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Entrée en Europe au XVIIIe siècle, portant des noms différents attachés au contexte de lecture de ses symptômes, la tremblante resta longtemps un mystère. Dès le milieu des années 1960 sous l'impulsion de Carleton Gadjusek, l'analogie étiologique et pathologique entre cette neuropathologie animale et la maladie du kuru aujourd'hui disparue, permit de tester la transmissibilité de plusieurs maladies neuropsychiatriques et d'établir un groupe de maladies nommées Encéphalopathies spongiformes subaiguës transmissibles. Dès 1982, grâce à Stanley Prusiner, les outils conceptuels et techniques de la biologie moléculaire ont élucidé l'énigme de la tremblante, établi la protéine prion dans un statut d'agent causal de toutes les maladies neurodégénératives transmissibles humaines et animales. Puis, apparue au Royaume-Uni en 1985, l'ESB a étendu cet ensemble. L'analogie entre cette maladie bovine et la tremblante a conduit à inférer de l'innocuité de la tremblante à l'innocuité de l’ESB pour la santé humaine. L'étude menée introduit dans une trame philosophique la réflexion scientifique sur la difficulté de la biologie et de la médecine des maladies à prions, qui s'est constituée avec les inférences sur l'ESB, difficulté dont le résultat fut l'émergence d'un nouveau variant de la MC1. Cette étude prolonge les activités scientifiques de la biologie moléculaire. Des neurosciences fondamentale et clinique des troubles neurodégénératifs, sur la signification des inférences, de la causalité biologique, du temps réel de l'action causale, de l'analogie dans le raisonnement causal. Elle jette la lumière sur la nature des causes biologiques, ouvre la voie à une biologie moléculaire du futur.
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Zahn, Ralph. « Prion propagation and molecular chaperones ». Zürich : Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, 2002. http://e-collection.ethbib.ethz.ch/show?type=habil&nr=4.
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Uelhoff, Armgard. « Polarisierter Transport des Prion-Proteins ». Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-40163.
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Rambold, Angelika. « Funktionelle Charakterisierung des Prion-Proteins ». Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-91541.
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Norton, Jennifer Diane, et Jennifer Diane Norton. « Mechanisms of Prion Variant Competition ». Diss., The University of Arizona, 2017. http://hdl.handle.net/10150/625845.
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The assembly of some misfolded proteins into aggregates can cause dramatic changes in cellular phenotypes. In prion diseases, these phenotypes are self-perpetuating because the highly ordered aggregates are capable of templating the conversion of soluble form of the protein into the aggregated form. Interestingly, the aggregated form of the protein can exist as a range of unique self-replicating conformations, referred to as prion "strains" or "variants," conferring distinct phenotypic characteristics to their hosts. The presence of more than one prion variant has been implicated in the alteration of prion phenotypes, interspecies transmission and anti-prion drug-resistance. When more than one variant arises or is introduced into the same host, usually the faster replicating variant phenotypically dominates over slower replicating variants; however, this dominance is not absolute. Studies in mammals suggest that these outcomes are determined by competition between variants for the conversion of soluble protein. However, other steps in prion replication have not been thoroughly examined. In this study we show that prion variant dominance is indeed determined by competing for the conversion of soluble protein, but this advantage arises from other cell-based factors that influence prion biogenesis, such as fragmentation rates and the number of templates present within the cell.
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Uppington, Kay Marie. « Cell death in prion disease ». Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488879.
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Prion diseases are a group of fatal neurodegenerative diseases, including CJD and scrapie, which are thought to be caused by a protein termed a prion (PrP). As manganese has previously been suggested to be involved in prion disease we have investigated manganese binding to PrP and its role in the toxicity of the protein. We have shown that manganese bound PrP (MnPrP) has several of the characteristics of the disease form of PrP, including protease resistance and toxicity that is dependent on cellular PrP expression. Further investigation into the mechanism of toxicity revealed that MnPrP is significantly more toxic to neuronal cells than nonmanganese bound PrP and that toxicity requires the presence of known metal binding residues within the protein. We have demonstrated that treatment of neuronal cells with MnPrP causes caspase 3 activation and apoptosis, as demonstrated by DNA laddering, and we hypothesise that caspase 3 is activated by a p38 pathway. Treatment of neurones with MnPrP also caused a significant increase in cellular ROS production, although this did not appear to be a major cause of cell death as antioxidants were unable to save cells from cell death. We also investigated mechanisms by which cells can survive scrapie infection and MnPrP toxicity. We have shown that cells infected with scrapie have increased ERK activation which was important for their survival. Cells that survived MnPrP treatment were also found to have increased ERK activation. This suggests that ERK may have a protective role in prion diseases and may be a potential therapeutic target.
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Lee, Kil Sun. « Biologia da proteína prion celular ». Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22082018-104148/.
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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.
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Apetri, Constantin Adrian. « Folding of the Prion Protein ». Case Western Reserve University School of Graduate Studies / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=case1080747299.
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