Nuclear pore complexes (NPCs) are large macromolecular gateways embedded in the nuclear envelope of Eukaryotic cells that serve to regulate bi-directional trafficking of particles to and from the nucleus. NPCs have been described as creating a selectively permeable barrier mediating the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins and some viral particles. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are repelled from the NPC, unable to translocate. The NPC is made up of over 30 unique proteins, each present in multiples of eight copies. The two primary protein components of the NPC can be simplified as scaffold nucleoporins which form the main structure of the NPC and the phenylalanine-glycine (FG) motif containing nucleoporins (FG-Nups) which anchor to the scaffold and together create the permeability barrier within the pore. Advances in fluorescence microscopy techniques including single-molecule and super-resolution microscopy have made it possible to label and visualize the dynamic components of the NPC as well as track the rapid nucleocytoplasmic transport process of importing and exporting cargoes. The focus of this dissertation will be on live cell fluorescence microscopy application in probing the dynamic components of the NPC as well as tracking the processes of nucleocytoplasmic transport. / Biology

Chez les eucaryotes, les pores nucléaires (NPCs), ancrés dans l’enveloppe nucléaire (EN), régulent les échanges nucléocytoplasmiques. Ces complexes, très conservés, sont composés d’une trentaine de protéines appelées nucléoporines (Nups) présentes en multiples copies au sein de chaque NPC. Chez la levure S. cerevisiae, seules quatre Nups, dont la protéine Pom33, possèdent des domaines transmembranaires. Une étude réalisée en amont de ce projet a permis de caractériser Pom33 et de montrer que le mutant pom33∆ est viable et ne présente pas de défaut apparent de transport nucléocytoplasmique mais se caractérise par un défaut de distribution des NPCs. Pom33 joue également un rôle dans l’assemblage des pores nucléaires au sein de l’EN (biogenèse de novo des NPCs). POM33 appartient à une famille de gènes très conservés. Il possède un paralogue chez S. cerevisiae, PER33, qui code pour une protéine localisée majoritairement au réticulum endoplasmique et minoritairement aux NPCs et qui n’est pas impliquée dans la biogenèse des NPCs. Chez les mammifères, il n’existe qu’un homologue de Pom33/Per33, TMEM33. Dans le cadre de ce doctorat, nous nous sommes demandés quels étaient les déterminants contribuant à l’adressage spécifique de Pom33 au niveau des NPCs et à sa fonction dans la biogenèse de ces structures. La purification de Pom33-ProtA, suivie de spectrométrie de masse, nous a permis d’identifier un nouveau partenaire de Pom33, le facteur d’import Kap123. Des approches in vitro ont montré une interaction directe entre Kap123 et le domaine C-terminal (CTD) de Pom33, qui est perturbée en présence de RanGTP. Par ailleurs, des prédictions in silico ont révélé la présence dans ce domaine CTD de deux hélices amphipathiques, conservées chez l’humain. Des analyses par dichroïsme circulaire et flottaisons ont confirmé la capacité du CTD à s’organiser en hélice en présence de membranes lipidiques et à interagir préférentiellement avec les membranes très courbées. L’expression d’une version mutée de Pom33-CTD, incapable de se lier aux membranes et couplée à la GFP, a révélé la capacité de ce domaine à agir comme un NLS, importé spécifiquement dans le noyau par Kap123. Alors que la délétion du domaine CTD affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs, la mutation des résidus basiques impliqués dans l’interaction avec Kap123 ou des résidus permettant sa liaison aux membranes lipidiques ne récapitule pas ce phénotype. En revanche, la perte combinée de ces deux déterminants affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs ainsi qu'une interaction génétique avec le mutant nup133∆, impliqué dans la biogenèse de novo des NPCs. Les résultats obtenus lors de cette étude indiquent donc que l’adressage de Pom33 est un mécanisme actif et multifactoriel, qui met en jeu au moins deux déterminants dans son domaine CTD. Ces données indiquent également un rôle de ce domaine dans la biogenèse de novo des NPCs, qui pourrait néanmoins n’être qu’un effet indirect de son rôle dans l’adressage de Pom33 aux NPCs. Au cours de cette étude, nous avons également mis en évidence d’autres partenaires potentiels de Pom33, en particulier Myo2, une localisation de Pom33 au niveau du bourgeon lors de la division et une interaction génétique entre POM33 et KAP123. Ces observations préliminaires ouvrent de nouvelles pistes de réflexion quant au rôle de Pom33 lors de la division cellulaire. / In eukaryotic cells, nucleocytoplasmic exchanges take place through the nuclear pores complexes (NPCs). These conserved macromolecular assemblies are embedded in the nuclear envelope (NE) and composed of ~30 distinct proteins called nucleoporins (Nups), each presents in multiple copies. In the budding yeast Sacharomyces cerevisiae, there are only four transmembrane Nups, including Pom33. A previous study leds to the characterization of Pom33 and revealed that pom33∆ mutant cells, although viable and without apparent alteration in nucleocytoplasmic transport, display NPCs distribution defect. Pom33 also contributes to the biogenesis of NPCs into the intact NE (de novo biogenesis). Pom33 is highly conserved among species and has a paralogue in S. cerevisiae, Per33, which can associate with NPCs but is mainly localized at the endoplasmic reticulum (ER) and NE. Unlike Pom33, Per33 is not involved in NPCs distribution and biogenesis. In mammalian cells, there is a unique homologue of Pom33/Per33, named TMEM33. In the context of this thesis, we aimed to identify the determinants involved in the specific targeting of Pom33 to NPCs and in its function in pore biogenesis. To characterize these determinants, we first performed affinity-purification experiments followed by mass spectrometry analyses. This identified a novel Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between Pom33 C-terminal domain (CTD) and Kap123 that involves positively-charged residues within Pom33-CTD and is altered in the presence of Ran-GTP. Moreover, in silico analyses predicted the presence of two evolutionarily-conserved amphipathic ~-helices within Pom33-CTD. Circular dichroism studies and liposome co-floatation assays confirmed that this CTD domain is able to fold into ~-helices in the presence of liposomes and revealed its preferential binding to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, Pom33-CTD behaves as a Kap123-dependent nuclear localization domain. While deletion of Pom33 C-terminal domain (Pom33-∆CTD-GFP) impairs Pom33 NPC targeting and stability and leads to a NPC distribution phenotype, mutants affecting either Kap123 binding or the amphipathic properties of the ~-helices do not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects Pom33 targeting to NPCs and leads to an altered NPC distribution and a genetic interaction with the deletion of NUP133, a gene coding for a nucleoporin involved in NPCs biogenesis. Together, these results indicate that Pom33 targeting to NPCs is an active and multifactorial process that requires at least two determinants within its CTD. They also suggest a role of Pom33-CTD in the de novo NPCs biogenesis process, which could however only be an indirect consequence of its requirement for Pom33 targeting to NPCs. Our mass spectrometry analysis also identified other partners of Pom33, in particular Myo2, a molecular motor required for the cell cycle-regulated transport of various organelles and proteins and for correct alignment of the spindle during mitosis. Our studies also revealed a specific localization of Pom33 at the bud tip during mitosis and a genetic interaction between POM33 and KAP123. Taken together, these preliminary observations open new perspectives regarding additional functions of Pom33 during cell division.

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Made available in DSpace on 2016-12-12T20:27:38Z (GMT). No. of bitstreams: 1 Marcos Vinicius Bressan.pdf: 3794882 bytes, checksum: c55b9af15d7d913dc3d962a990a792c4 (MD5) Previous issue date: 2014-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work present a new three-phase hybrid multilevel inverter, that is composed of two topologies in cascaded association. The three-phase NPC and the half-bridge modules are connected in series. This con¬nection increases the number of the output voltage levels, improves the output voltage quality and rises the power process by inverter. The theoretical analysis is presented, the switching states are investigated and the average and the RMS currents values in the semiconductors are verified. Based on the current values are projected the dc capacitors, and analyzed the semicondutors loss energy and the input currents of the inverter. Two modulation scheme are presented at proposed inver¬ter, the hybrid PWM and SVM modulation scheme. The first part of the experimental results are verified behavior of the multilevel power structure developed in laboratory. The second part of the experimen¬tal results evaluates quality of output voltage with different modulation scheme using of the mathematical tools THD and WTHD. / Este trabalho apresenta um novo inversor multinível híbrido trifásico, que é formado pela associação em cascata de duas topologias. No caso, um conversor NPC trifásico conectado em série a módulos de converso¬res meia ponte. A ligação em série desses conversores permite ampliar o número de níveis de tensão sintetizados pelo inversor, melhorar a qualidade da forma de onda da tensão de saída e o aumento da energia processada pelo inversor. Uma análise teórica do inversor é apresen¬tada, averiguando as etapas de operação e determinando os valores médio e eficaz da corrente nos semicondutores de potência. Com base nas análises dos valores médio e eficaz da corrente nos semicondutores dimensiona-se os capacitores de barramento, assim com a energia dissi¬pada pelos semicondutores e a análise teórica das correntes de entradas do inversor empregando de retificadores multipulsos. Também são apre¬sentadas duas técnicas de modulação ao inversor proposto: a técnica de modulação PWM híbrida e a SVM. Os resultados experimentais, em sua primeira parte, avaliam o comportamento da estrutura multinível desenvolvida em laboratório. Na segunda parte dos resultados experi¬mentais, avalia-se qualidade da forma de onda de tensão com diferentes técnicas de modulação utilizando das ferramentas matemáticas THD e WTHD.

This study is an examination of how a music education that is run by a NGO, Non Governmental Organisation, in South Africa works. The organisation Arkwork for Art NPC, and their artistic programme Access Music Project, AMP, based in Grahamstown in Eastern Cape province, works as an case study. This study shows what different opportunities and challenges are of the work with the project and describes who the young participants enrolled in the project are and how they experience the music education. Further more this work explore the relationship between music education and social justice. Information was gathered through qualitative semi-structured interviews with the participants and project founder and leadership of the project. Partly information also was gathered through unstructured observations. The result shows that Arkwork for Art is filling an important gap where the government lack resources to offer music education for youth in marginalised communities. The organisation struggle with many things, most difficult for them is to get stable funding. AMP have many big future plans and want for instance to change the curriculum of music education in the future. My examination shows that the participants are very satisfied to be a part of AMP and that the project has an important role in their lifes and the community where they are based.

Le travail de cette thèse apporte une contribution aux méthodes de réglage de la tension dans les réseaux électriques. Il s’agit de fournir au réseau la puissance active et surtout la puissance réactive nécessaire pour réguler la tension et aboutir à un système équilibré vue du côté source. Ces puissances sont extraites d’une source d’energie renouvelable : une attention particulière a été portée à l’énergie éolienne raccordée au réseau à travers la Machine Asynchrone à Double Alimentation (MADA) pilotée par des convertisseurs statiques. Le système de contrôle le plus répandu des éoliennes est basé principalement sur la technique d’extraction du maximum de puissance. Cependant, cette technique limite la mise en oeuvre deservices auxiliaires, telle que la participation des éoliennes au réglage de la tension dans le réseau électrique. Pour cela, une nouvelle méthode d’extraction du coefficient de puissance optimal, permettant d’améliorer la participation de la MADA à la régulation de la tension dans le réseau (compensation de la puissance réactive et du déséquilibre), a été développée. Le convertisseur multiniveaux à structure NPC (Neutral Point Clamped) raccordant l’énergie renouvelable au réseau a été étudié. La commande prédictive assurant simulatnément l’amélioration du facteur de puissance, l’équilibrage du réseau électrique et du bus continu du convertisseur NPC a été proposée. Ensuite, l’application de cette commande prédictive a été elargie en lui attribuant plusieurs objectifs : amélioration du facteur de puissance avec équilibrage du réseau, équilibrage du bus continu, minimisation des pertes par commutation et réduction de la tension de mode commun. La minimisation des pertes a été obtenue en proposant une nouvelle stratégie qui consiste à exploiter les datasheets constructeurs donnant l’évolution de l’énergie dissipée durant la commutation en fonction du courant. Ces courbes expérimentales ont été transformées en modèlesmathématiques implémentés dans la commande prédictive. Les résultats de simulation et expérimentaux sont présentés pour évaluer les performances de la méthode proposée. / The work in this research thesis presents a contribution to voltage regulation in electrical networks. By considering adequate active and reactive powers injection into the grid, voltage control and load balancing are provided. These powers are generated from a grid connected renewable energy conversion system : a special attention was paid to the Wind Energy ConversionSystem (WECS) based on Doubly-Fed Induction Generator (DFIG).The typical control strategy for WECS is the maximum power coefficient tracking method. However, this method limits desirable ancillary power services, such as the participation of wind turbines in voltage regulation in the power grid. Therefore, a new method that derives the optimal power coefficient enhancing the participation of WTS in voltage regulation in the network (reactive and unbalanced power compensation), has been developed. The multilevel NPC (Neutral Point Clamped) converter, used for grid interface connection of renewable energy sources systems, has been studied. A predictive control method for the three-level NPC converter, capable of simultaneously compensating the problems of : DC link capacitors voltage balancing, load balancing and power factor correction in the power system, has been proposed. Then, the application of this predictive control was extended to simultaneously achieve multiple objectives: load balancing with power factor correction in the network, DC link capacitors voltage balancing, switching losses minimization and common mode voltage reduction. The switching losses minimization was obtained by proposing a new strategy which consists on exploiting the manufacturer datasheets that gives the evolution of the switching loss energy in function of the circulating current. The experimental curves of the datasheet are expressed in a mathematical model implemented in the predictive control. Simulation and experimental results are presented to evaluate the performance of the proposed method.

Um fator relevante para a aceitação de metáforas de mundos virtuais 3D como interfaces homem máquina é a supressão do fator estranheza. Tal fator decorre da percepção de comportamentos não naturais entre as representações de usuários (avatares) e robôs (NPCs Non Playable Characters) nestes ambientes virtuais e acontece, principalmente, devido a diferenças sutís entre o comportamento esperado de um ser humano e o apresentado pelas representações virtuais (avatares). Ao conjunto de comportamentos exibidos denominamos persona. Assim, uma maneira de reduzir a barreira de estranheza entre usuários interagindo em mundos virtuais 3D é fazendo com que suas representações (avatares) tenham personas convincentes. Os seres humanos tem suas personas regidas por seus tratos emocionais. As interações com o ambiente e com outros seres humanos fornecem estímulos que modificam o estado emocional com relação ao estado de equilíbrio e, consequentemente, alteram o comportamento exterior observado. O estado emocional de equilíbrio pode ser obtido através da análise de corpo de comunicação em linguagem natural do usuário. Neste trabalho é defendida a tese de que através da análise do corpo das interações em linguagem natural de usuários nas mídias sociais é possível estabelecer um modelo de personalidade e persona que pode ser transposto para a representação dos usuários nem mundos virtuais 3D, de forma que as representações tenham comportamentos tais que reduzem o fator de estranheza. O modelo faz uso das interações recentes nas mídias sociais e das interações nos mundos virtuais 3D para determinar o estado emocional imediato e o comportamento instantâneo e é coerente com o paradigma do modelo OCC. Também é demonstrado que este modelo é evolutivo: pela análise contínua do corpo da comunicação é acompanhada a evolução da personalidade do usuário, bem como se adequa continuamente sua persona. Os traços de personalidade são analizados no modelo Big Five (Neuroticism, Extraversion, Agreeableness, Conscientiousness, Openness) e operacionalizados no modelo PAD (Personality, arousal, dominance). O modelo constrói um campo de personalidade, no espaço PAD, de tal forma que na ausência de excitação o estado emocional do avatar fica em repouso no centro deste campo. As excitações movimentam o estado ao longo dos eixos PAD e provocam mudanças no comportamento observado. A correspondência entre as mudanças de comportamento do avatar e o movimento do estado do usuário no campo emocional aumenta a concordância entre comportamento esperado e observado, reduzindo o fator de estranheza. / A key factor for the acceptance of 3D virtual world mataphor as human machine interface is the suppression of the strangeness factor, which appears due the perception of non natural behaviour in representations of users (avatars) and bots (NPCs). Such perception raises mostly due to subtle differences in expected behavior for a human being and observed behavior in virtual representations (avatars and NPCs). In this work we will call persona the set of exhibited behaviors. Thus, a way of diminishing the strangeness valley is by supplying avatars with convincing personas. Human beings rule their personas with intermediation of their emotional traits. Interactions with environment and other beings supply stimuli that change the emotional state from the equilibrium situation and, consequently, change observed behavior. Idle emotional state can be determined through natural language communication corpora analysis. In this thesis we state that through the analysis of user interactions in social media it is possible to establish a model of personality and persona that can be transposed to their representation in 3D virtual worlds, so such representation exhibits behavior that minimize strangeness. The model uses recent interations of user in social media and his interactions inside the 3D virtual world to establish current behavior and is coherent with OCC paradigm. It is also demonstrated that such model is evolutive: by the continuous analysis of communication corpora the user personality behavior is updated and its persona is continually adjusted. Personality traits are analysed in the Big Five model (neuroticism, agreeableness, conscientiousness, openness) and operationalized in the PAD model (personality, arousal, dominance). A personality field is built such as in the absence of inputs, emotional state lies in the center of this field. Inputs move state along PAD axes and cause changes in the observed behavior. Correspondence between avatar behavior changes and state changes of user regarding its emotional field enhance the compliance between expected and observed behavior, reducing strangeness.

Um fator relevante para a aceitação de metáforas de mundos virtuais 3D como interfaces homem máquina é a supressão do fator estranheza. Tal fator decorre da percepção de comportamentos não naturais entre as representações de usuários (avatares) e robôs (NPCs Non Playable Characters) nestes ambientes virtuais e acontece, principalmente, devido a diferenças sutís entre o comportamento esperado de um ser humano e o apresentado pelas representações virtuais (avatares). Ao conjunto de comportamentos exibidos denominamos persona. Assim, uma maneira de reduzir a barreira de estranheza entre usuários interagindo em mundos virtuais 3D é fazendo com que suas representações (avatares) tenham personas convincentes. Os seres humanos tem suas personas regidas por seus tratos emocionais. As interações com o ambiente e com outros seres humanos fornecem estímulos que modificam o estado emocional com relação ao estado de equilíbrio e, consequentemente, alteram o comportamento exterior observado. O estado emocional de equilíbrio pode ser obtido através da análise de corpo de comunicação em linguagem natural do usuário. Neste trabalho é defendida a tese de que através da análise do corpo das interações em linguagem natural de usuários nas mídias sociais é possível estabelecer um modelo de personalidade e persona que pode ser transposto para a representação dos usuários nem mundos virtuais 3D, de forma que as representações tenham comportamentos tais que reduzem o fator de estranheza. O modelo faz uso das interações recentes nas mídias sociais e das interações nos mundos virtuais 3D para determinar o estado emocional imediato e o comportamento instantâneo e é coerente com o paradigma do modelo OCC. Também é demonstrado que este modelo é evolutivo: pela análise contínua do corpo da comunicação é acompanhada a evolução da personalidade do usuário, bem como se adequa continuamente sua persona. Os traços de personalidade são analizados no modelo Big Five (Neuroticism, Extraversion, Agreeableness, Conscientiousness, Openness) e operacionalizados no modelo PAD (Personality, arousal, dominance). O modelo constrói um campo de personalidade, no espaço PAD, de tal forma que na ausência de excitação o estado emocional do avatar fica em repouso no centro deste campo. As excitações movimentam o estado ao longo dos eixos PAD e provocam mudanças no comportamento observado. A correspondência entre as mudanças de comportamento do avatar e o movimento do estado do usuário no campo emocional aumenta a concordância entre comportamento esperado e observado, reduzindo o fator de estranheza. / A key factor for the acceptance of 3D virtual world mataphor as human machine interface is the suppression of the strangeness factor, which appears due the perception of non natural behaviour in representations of users (avatars) and bots (NPCs). Such perception raises mostly due to subtle differences in expected behavior for a human being and observed behavior in virtual representations (avatars and NPCs). In this work we will call persona the set of exhibited behaviors. Thus, a way of diminishing the strangeness valley is by supplying avatars with convincing personas. Human beings rule their personas with intermediation of their emotional traits. Interactions with environment and other beings supply stimuli that change the emotional state from the equilibrium situation and, consequently, change observed behavior. Idle emotional state can be determined through natural language communication corpora analysis. In this thesis we state that through the analysis of user interactions in social media it is possible to establish a model of personality and persona that can be transposed to their representation in 3D virtual worlds, so such representation exhibits behavior that minimize strangeness. The model uses recent interations of user in social media and his interactions inside the 3D virtual world to establish current behavior and is coherent with OCC paradigm. It is also demonstrated that such model is evolutive: by the continuous analysis of communication corpora the user personality behavior is updated and its persona is continually adjusted. Personality traits are analysed in the Big Five model (neuroticism, agreeableness, conscientiousness, openness) and operationalized in the PAD model (personality, arousal, dominance). A personality field is built such as in the absence of inputs, emotional state lies in the center of this field. Inputs move state along PAD axes and cause changes in the observed behavior. Correspondence between avatar behavior changes and state changes of user regarding its emotional field enhance the compliance between expected and observed behavior, reducing strangeness.

For parents caring for an intellectually or developmentally disabled child (I/DD), there can be many challenges throughout various life stages. While research has been shown to emphasize an abundance of services for parents of young children and adolescents with I/DD, there has been a lack of services for later life transitions within adulthood. Within this research, a brief literature review of what preparation and services have been provided to parents of adult children with I/DD in regard to non-parental care placement is provided. The following research discusses the various perspectives of the future planning process through interviews from participants who identify themselves as a primary caregiver of their adult child with I/DD and who are 45 years of age or older. This research also discusses the results, limitations, recommendation for social work practice, and research conclusion.

The Niemann-Pick type C (NPC) disease is a lysosomal lipid storage disorder caused by mutations in one of the two genes NPC1 or NPC2 encoding proteins of the late endosome/lysosome compartment (LE/LY). Defect in these proteins alters vesicular transport and leads to abnormal accumulation of cholesterol (Chol) in LE/LY. There are some lines of evidence suggesting that annexin A6 (AnxA6) participates in vesicular transport of Chol and may interact with membrane domains enriched in Chol and bind Chol. In this work we characterized the membrane microdomains resistant to Triton X-100, i.e., detergent-resistant membranes (DRMs) isolated from NPC patient-derived fibroblasts and from control cells. NPC cells contain a significantly higher amount of DRMs than the control cells that is consistent with the defect in Chol turnover in NPC cells. We also studied the mechanism of AnxA6 involvement in the NPC-induced changes in the membrane organization and showed that in the presence of calcium some AnxA6 molecules associate with the DRMs. This suggests that AnxA6 may play a role in the membrane lateral organization, contributing thus to the etiology of NPC disease. We then focused on the interaction of AnxA6-1 with Chol-rich membranes and on the involvement of its flexible region and VAAEIL sequence in these interactions. For this purpose, kinetics of the interfacial adsorption of human recombinant AnxA6 to Langmuir monolayers containing phosphatidylcholine, Chol and/or cholesteryl acetate were measured. Our data suggest that AnxA6 exhibits the highest affinity to Chol-containing monolayers and that the hydroxyl group of Chol plays a pivotal role in the AnxA6-lipid interactions in vitro.