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 prions. / The 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.

<p>Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques.</p><p>The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles.</p><p>Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins.</p><p>We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB).</p><p>The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism.</p><p>The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.</p>

Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques. The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles. Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins. We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB). The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism. The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.

Die Graphenwissennschaft vervendet man bei praktischem Bedarf in mehreren Gebieten der Wissenschaft (zum Beispiel: in der Chemie, in der Biologie, in der in der Psychologie, in der Informatik). Die Graphen vereinfachen viele kompliezierte Schemen, Probleme. Die Graphentheorie wird in der Schulmathematik bei der Kombinationslehre (die Baumgraphe, doppel Graph) benutzt. In der Diplomarbeit werden wichtige Arte und Eigenschaften der Gfaph verallgemeinert. Es wurde bewiesen: 1. Jeder vollständige Graph besitzt Verbindungen und der Graph ist gleichmäβig mit dem Grad r=p-1. 2. Jedem (p,q)-Graph und beliebigem v gehört der Menge V regelgerechte Ungleichheit 0≤deg v ≤p-1. 3. Wenn p≠8, dann G – kanten Graph Kp dann: 1) G hat Scheitel; 2) G – regelgerecht Graph mit Grad 2(p-2). 4. Jeden Naturzahlen m,n und vollständigem doppel Graph Km,n ist solche Gleichheit Km,n = regelgerecht. 5. Wenn m≠4 und n≠4, dann ist vollständiger doppel Graph Km,n kanten Graph L(Km,n): 1) hat Scheitel; 2) ist regelgerecht Graph mit Grad r=m+n-2.

This dissertation serves as an analysis and performance guide for No-Re I for Flute Solo (2018) by Gyu-Bong Yi (이규봉), a work that combines Korean elements and Western compositional techniques. The modern flute, developed in the mid-nineteenth century with the Boehm's Schema, has pushed the boundaries of the instrument through experimentation with new techniques, leading to numerous contemporary works presenting unique challenges for flutists. No-Re I includes passages with various extended techniques and unique combinations, requiring specific solutions to overcome the challenges they present. For example, this dissertation offers specific fingering diagrams and descriptions for executing the double trills used in No-Re I. Additionally, certain notations used by the composer may have limitations in projection or clarity, and this dissertation elucidates the composer's intent based on an interview and provides solutions for these limitations. This research also discusses the fusion of Korean and other East-Asian aesthetics with Western musical language including the works of Isang Yun (1917–1995), a Korean-born musical pioneer, who had a successful career as a composer and educator, especially elaborating on his unique compositional technique, the Haupttöne, which is based on tonal centers influenced by East-Asian philosophy, Korean folk music, and its unique characteristic of ornamenting the main tone.

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

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

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

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