Made available in DSpace on 2017-07-10T17:30:33Z (GMT). No. of bitstreams: 1 Andressa_Fachin.pdf: 7147263 bytes, checksum: c3f02dba66f39392f3087baf7f763360 (MD5) Previous issue date: 2013-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This thesis presents the paleovalley head evolution in the Chopinzinho drainage basin inside Araucária Plateau (Planation Surface 2), aiming to understand the landscape evolution in the South Brazil during the Upper Quaternary. The stratigraphic record was characterized using the combined criteria of pedo-, litho-, allo-, and cronostratigraphic, for which chronology was establish by 14C (AMS) and thermoluminescence. As base these criteria was utilized following technical: field survey, laboratory analyses (textural, ten major oxides more zirconium oxide, clay mineralogy, and dating) and office work (cluster statistical analysis and organization of date). It has been done the reconstitution spatial distribution of the paleosol and paleogullies by drilling. The field survey showed stratigraphic section in the cutting rural road. Therein was identified paleosol with Ab horizon (27.36 + 0.14 Kyr BP 44.13 Kyr cal. BP), paleogullies field of colluvium (15 beds), colluvium-alluvial (4 beds) and alluvial (1 bed). The colluvium beds was divided into clogging faces and ramp faces. The paleogullies were generated in Last Glacial Maximum (< 24 Kyr to > 17 Kyr BP.) and in Medium Holocene (< 7 Kyr to > 4 Kyr BP). This last moment were generated two paleogullies generations promoting the larger erosion phenomenon in the paleovalley head. The paleovalley head happens to be clogging in the Upper Holocene (< 4 Kyr BP) and change of the morphology becoming colluvium ramp. The palleovalley head was eroded by spot drainage and becoming one convex mound in the end of period. This moment there was one inversion of the relief. / Esta dissertação apresenta a evolução de uma paleocabeceira de drenagem no Planalto das Araucárias (Superfície 2),buscando contribuir para o entendimento do Quaternário Tardio do Sul do Brasil. O registro estratigráfico foi caracterizado utilizando-se dos critérios conjugados da pedo-, lito-, alo- e cronoestratigrafia, cuja cronologia foi estabelecida pelo 14C (AMS) e termoluminescência (TL). Com base nesses critérios foram utilizadas as seguintes técnicas: levantamento de campo, análises laboratoriais (granulométrica, análise química dos 10 principais óxidos + Zircônio,mineralogia da fração argila e ainda datações por 14C e luminescência) e trabalho de gabinete (análise estatística por similaridade Cluster e organização dos dados). Foi feita a reconstituição da distribuição espacial do paleossolo e das paleovoçorocas por meio de sondagens. O levantamento de campo revelou seção estratigráfica exposta em corte de estrada. Nela identificou-se: paleossolo com horizonte A húmico (27.360 + 140 anos AP. 44.130 cal. AP.), paleovoçorocas colmatadas por colúvios (15 camadas), colúvio-alúvio (4 camadas) e alúvios (1 camada). As camadas colúviais foram subdivididas em fácies de colmatação e fácies de rampa.As paleovoçorocas foram geradas no Último Máximo Glacial (< 24.000 a> 17.000 anos AP) e no Holocêno Médio (< 7.000 anos a > 4.000 anos AP). Neste último período foram geradas duas gerações de paleovoçorocas, promovendo o maior fenômeno erosivo na cabeceira de drenagem. A paleocabeceira de drenagem passa a ser colmatada no Holoceno Superior (< 4.000 anos AP) e muda de morfologia para rampa de colúvio. No final desse período, a rampa de colúvio é dissecada pela drenagem local e torna-se uma colina convexa. Nessafasehouveumainversão de relevo.

A espécie em estudo (Brycon orbignyanus) vem apresentando um declínio populacional importante nos últimos anos em função de interferências antrópicas. Por este motivo, populações naturais na bacia do rio Uruguai são praticamente inexistentes, e os estoques mantidos em cativeiro apresentam razões sexuais desproporcionais e perdas importantes de variabilidade genética, o que prejudica o uso desta espécie em programas de conservação. Esta tese tem como objetivo principal avaliar parâmetros populacionais e reprodutivos de Brycon orbignyanus sob condições de cativeiro visando a aplicabilidade deste conhecimento em programas de conservação. Com isso, objetivou-se no primeiro experimento avaliar as flutuações na razão sexual ao longo do desenvolvimento ontogenético e os diferentes padrões morfológicos de crescimento entre os sexos. Quinhentos exemplares foram amostrados de uma piscicultura comercial e os dados biométricos foram registrados para a realização de uma regressão linear entre peso e comprimento. A partir do teste Qui-Quadrado (χ²) foi possível observar que a razão sexual apresentou desproporções nas classes de comprimento superiores. Os machos e fêmeas apresentaram crescimento alométrico positivo (b=3,35 e b=3,44), enquanto indivíduos sexualmente indiferenciados apresentaram alometria negativa (b=2,96) O objetivo do segundo estudo foi descrever os processos de diferenciação sexual, afim de definir o período termossensível. Após eutanásia dos animais, as gônadas foram coletadas e fixadas em solução formaldeído tamponado 4%. Os cortes seriados (3μm) foram corados com Azul de Toluidina e analisados por microscopia de luz. Os primeiros sinais de diferenciação sexual foram observados apenas aos 323 dias após a fertilização (DAF) e, aos 730 DAF todos os indivíduos já estavam diferenciados sexualmente. O objetivo do terceiro estudo foi descrever o ciclo reprodutivo de machos de B. orbignyanus, a fim de se estabelecer uma escala de maturação para machos desta espécie. Para isto, exemplares da espécie foram coletados periodicamente em uma estação de piscicultura comercial. Assim como no experimento anterior, as gônadas foram coletadas e fixadas para análise histológica. As análises das imagens foram obtidas através de microscópio óptico e a descrição das fases reprodutivas seguiu recomendações de Brown-Peterson et al. (2011). Foram estabelecidas 5 fases reprodutivas de acordo com as características macroscópicas dos testículos, presença e distribuição das células espermatogênicas e variações no índice gonadossomático. Os primeiros indivíduos aptos à reprodução foram observados aos 435 DAF. / The species under study (Brycon orbignyanus) has undergone a important population decline in the last years due to anthropic interferences. For this reason, natural populations in the Uruguay River basin are practically non-existent, and stocks kept in captivity suffer from disproportionate sex ratios, and significant losses of genetic variability, impairing the use of this species in conservation programs. The main objective of this thesis is to evaluate the population and reproductive parameters of Brycon orbignyanus under captive conditions aiming the applicability of this knowledge in conservation programs. Thus, the objective of the first experiment was to evaluate the fluctuations in sexual ratio along the ontogenetic development and the different morphological patterns of growth between the sexes. Five hundred specimens were sampled from commercial fish farming and biometric data were recorded for a linear regression between weight and length. From the chi-square test (χ²) it was possible to observe that the sex ratio showed disproportions in the upper length classes. Males and females showed positive allometric growth (b = 3.35 and b = 3.44), while sexually undifferentiated individuals showed negative allometry (b = 2.96). The objective of the second experiment was describe the processes of sexual differentiation, in order to define the thermosensitive period After euthanasia of the animals, the gonads were collected and fixed in 4% buffered formaldehyde solution. Serial sections (3 μm) were stained with Toluidine Blue and analyzed by light microscopy. The first signs of sexual differentiation were observed only at 323 days after fertilization (DAF), and at 730 DAF all individuals were already sexually differentiated. The objective of the third experiment was to describe the reproductive cycle of B. orbignyanus males, in order to establish a maturation scale for males of this species. For this, specimens were collected periodically in a commercial fish farm. As in the previous experiment, the gonads were collected and fixed for histological analysis. The images were obtained through an optical microscope and analyzed according to the description of the reproductive phases suggested by Brown-Peterson et al. (2011). Five reproductive phases were established according to macroscopic characteristics of the testes, presence and distribution of spermatogenic cells and variations in the gonadosomatic index. The first individuals able to reproduce were observed at 435 DAF.

Made available in DSpace on 2017-05-12T14:42:07Z (GMT). No. of bitstreams: 1 Andressa_Fachin.pdf: 7147263 bytes, checksum: c3f02dba66f39392f3087baf7f763360 (MD5) Previous issue date: 2013-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This thesis presents the paleovalley head evolution in the Chopinzinho drainage basin inside Araucária Plateau (Planation Surface 2), aiming to understand the landscape evolution in the South Brazil during the Upper Quaternary. The stratigraphic record was characterized using the combined criteria of pedo-, litho-, allo-, and cronostratigraphic, for which chronology was establish by 14C (AMS) and thermoluminescence. As base these criteria was utilized following technical: field survey, laboratory analyses (textural, ten major oxides more zirconium oxide, clay mineralogy, and dating) and office work (cluster statistical analysis and organization of date). It has been done the reconstitution spatial distribution of the paleosol and paleogullies by drilling. The field survey showed stratigraphic section in the cutting rural road. Therein was identified paleosol with Ab horizon (27.36 + 0.14 Kyr BP 44.13 Kyr cal. BP), paleogullies field of colluvium (15 beds), colluvium-alluvial (4 beds) and alluvial (1 bed). The colluvium beds was divided into clogging faces and ramp faces. The paleogullies were generated in Last Glacial Maximum (< 24 Kyr to > 17 Kyr BP.) and in Medium Holocene (< 7 Kyr to > 4 Kyr BP). This last moment were generated two paleogullies generations promoting the larger erosion phenomenon in the paleovalley head. The paleovalley head happens to be clogging in the Upper Holocene (< 4 Kyr BP) and change of the morphology becoming colluvium ramp. The palleovalley head was eroded by spot drainage and becoming one convex mound in the end of period. This moment there was one inversion of the relief. / Esta dissertação apresenta a evolução de uma paleocabeceira de drenagem no Planalto das Araucárias (Superfície 2),buscando contribuir para o entendimento do Quaternário Tardio do Sul do Brasil. O registro estratigráfico foi caracterizado utilizando-se dos critérios conjugados da pedo-, lito-, alo- e cronoestratigrafia, cuja cronologia foi estabelecida pelo 14C (AMS) e termoluminescência (TL). Com base nesses critérios foram utilizadas as seguintes técnicas: levantamento de campo, análises laboratoriais (granulométrica, análise química dos 10 principais óxidos + Zircônio,mineralogia da fração argila e ainda datações por 14C e luminescência) e trabalho de gabinete (análise estatística por similaridade Cluster e organização dos dados). Foi feita a reconstituição da distribuição espacial do paleossolo e das paleovoçorocas por meio de sondagens. O levantamento de campo revelou seção estratigráfica exposta em corte de estrada. Nela identificou-se: paleossolo com horizonte A húmico (27.360 + 140 anos AP. 44.130 cal. AP.), paleovoçorocas colmatadas por colúvios (15 camadas), colúvio-alúvio (4 camadas) e alúvios (1 camada). As camadas colúviais foram subdivididas em fácies de colmatação e fácies de rampa.As paleovoçorocas foram geradas no Último Máximo Glacial (< 24.000 a> 17.000 anos AP) e no Holocêno Médio (< 7.000 anos a > 4.000 anos AP). Neste último período foram geradas duas gerações de paleovoçorocas, promovendo o maior fenômeno erosivo na cabeceira de drenagem. A paleocabeceira de drenagem passa a ser colmatada no Holoceno Superior (< 4.000 anos AP) e muda de morfologia para rampa de colúvio. No final desse período, a rampa de colúvio é dissecada pela drenagem local e torna-se uma colina convexa. Nessafasehouveumainversão de relevo.

O trabalho foi realizado com o objetivo de avaliar algumas variáveis estruturais e bromatológicas de Panicum maximum Jacq cv. Massai submetido a quatro doses de nitrogênio (0, 40, 80 e120 mg/dm3) e três momentos de aplicação após o corte (um, três, e sete dias), divididos em três cortes, em um esquema fatorial 4 x 3, em um delineamento inteiramente casualizado, com quatro repetições. O incremento nas taxas de aparecimento foliar (TApF), alongamento foliar (TAIF) e aparecimento de perfilhos (TApP), no número de folhas vivas (NFV), no comprimento final da folha (CFF), na densidade populacional de perfilhos (DPP), na produção de matéria seca de folha total (MSFT) e nos teores de proteína bruta (PB), foi de 79%, 115%, 42%, 23%, 48%, 65%, 770% e 35%, respectivamente para a dose de 120 mg/dm3 em relação à ausência de adubação nitrogenada. O filocrono (fil), a duração de vida da folha (DVF), a fibra em detergente neutro (FDN) e a fibra em detergente acido (FDA), apresentaram decréscimo de 58%, 47%, 6% e 9%, respectivamente, com a adição de N. A taxa de alongamento de colmo (TAlC) não respondeu a adubação nitrogenada. Apenas o CFF, a TAlC e a MSFT responderam às épocas de aplicação de N após o corte, com valores pouco expressivos. A adubação nitrogenada exerce efeito positivo sobre as variáveis estudadas, porém novos estudos devem ser conduzidos para a definição da época de adubação de N após o corte, em que o nutriente seja melhor aproveitado pelas plantas. / This work was carried out to evaluate same morphogenic, structural, bromatologic and productive variables of the Panicum maximum Jacq cv. Massai in response to four nitrogen doses (0, 40, 80 and 120 mg/dm3) and with three application times after cutting (one, three and seven days) in a 4 x 3 factorial treatment combination, in a completely randomized design, with four replications. The increase in therates of leaf appearance (LAR), leaf elongation (LER) and tillering (TR), number of live leaves (NLL), final leaf length (FLL), tiller population density (TPD), dry matter total leaf production (DMTLP) and crude protein (CP) levels was 79%, 115%, 42%, 23%, 48%, 65%, 770% and 35%, respectively for the dose of 120 mg/dm3 regarding the absence of fertilization. The phyllochron (phy) showed a decrease of 58%, the leaf life span (LLS) of 47%, the neutral detergent fiber (NDF) of 6% and acid detergent fiber (ADF) of 9%, with N addition. The stem elongation rate (SER) didnot respond to nitrogen fertilization. Only the FLL, the SER and DMTLP responded times of N application after cutting, with little significant values. Nitrogen fertilization has a positive effect on the variables studied, but news studies should be conducted to better define the time of N application after cutting, for better utilization of this nutrient by plants.

A dissertação aborda os aspectos formadores da habitação nas áreas de várzea da região Amazônica, especificamente nas margens do Rio Solimões, hoje alvo de intensos investimentos governamentais, principalmente os da indústria nacional petrolífera em função da construção do Gasoduto Coari - Manaus. Com forte incidência de impactos ambientais e culturais, tais intervenções apontam para um quadro amplo e complexo, servindo também de base experimental para outros tipos de interferência em menor escala. A Amazônia corresponde a aproximadamente 5% da superfície terrestre, equivalente a 2/5 da América do Sul. A Bacia Amazônica contém por volta de 1/5 de toda a água doce mundial, colaborando para que o Brasil tenha a maior biodiversidade do mundo, com mais de 20% das espécies. Integrante desta região, a ampla bacia do Rio Solimões é a terceira área sedimentar com produção petrolífera no Brasil. O que é produzido nesta reserva, que é estimada em 132 milhões de barris de petróleo, percorre aproximadamente 600 km até chegar ao mercado consumidor. Para tanto, está sendo construído um poliduto capaz de atender a essa demanda; sua construção atinge cerca de 135 comunidades ribeirinhas, que ainda conservam valores, hábitos e costumes originais devido ao isolamento territorial e informacional. Assim, este trabalho lança mão de três momentos específicos da ocupação da floresta amazônica. No primeiro momento, busca-se uma análise da gênese da forma da habitação ribeirinha. A forma definida enquanto comunicação, informação, mediação, signo, fundamentado na Semiótica de Charles S. Peirce, buscando referências não só em um contexto imediato, mas também em um contexto estendido, histórico, onde os significados da relação do homem com o meio ambiente deixam marcas em sua produção. No segundo momento, a pesquisa volta-se à conjuntura atual das populações ribeirinhas e, a partir dos dados colhidos pelo projeto PIATAM Inteligência Socioambiental Estratégica da Indústria de Petróleo na Amazônia\", estabelece um plano de desenvolvimento socioambiental regional integrado, envolvendo as áreas de habitação, saneamento, desenvolvimento humano, atividades singulares nas áreas de floresta e de pesca, educação e comunicação, entre outras. Isso permitiria a criação de uma matriz de sustentabilidade, procurando determinar com clareza o grau de seus impactos ambientais. Neste contexto, o presente trabalho volta-se finalmente à atuação do arquiteto como um gestor de informações no processo de formação das frentes de desenvolvimento humano, enquanto propositor de organização de espaços e ambientes sustentáveis, a partir da ação de um sistema cultural, visando à troca dialética de saberes entre ciência e moradores, na qual o ribeirinho amazônico torna-se um agente ativo na construção repertorial da região, integrado aos conhecimentos científicos de sistemas indiciais característicos da floresta amazônica. Disto resulta a transformação de hábitos e costumes dos próprios moradores ribeirinhos frente às novas demandas socioambientais, bem como em novas posturas das próprias áreas científicas de conhecimento envolvidas nesse processo. É nessa mudança de estado, nesse processo, que poderão ser construídas as diretrizes gerais para um desenvolvimento regional sustentável, matrizes em constante processo de formação, constituintes e constituidoras de caminhos mais justos, fundindo homem e natureza como agentes de uma única realidade. / This dissertation addresses the training aspects of housing in lowlands areas of the Amazon region, specifically on the banks of the Solimoes River, nowadays the target of intense government investments, particularly by the national oil industry in terms of building the gas pipe Coari-Manaus. With strong cultural and environmental impacts, such interventions reveal a broad and complex panorama, which also serves as an experimental basis for other types of interventions on a smaller scale. The Amazon accounts for approximately 5% of the Earths surface, and is equivalent to 2/5 of South America. The Amazon Basin contains around 1/5 of all fresh water worldwide, which makes Brazil the country with the greatest biodiversity in the world, with more that 20% of all known species. As part of this region, the large basin of the Solimoes River is the third largest oil-producing sediment area in Brazil. With a reserve estimated as 132 million oil barrels, between extraction sites and the marketplace the products cover approximately 600 km. In this scenario, a duct able to meet this demand is being constructed , and its construction will affect approximately 135 riparian communities that retain values, habits and customs due to the original territorial and information isolation. This work resorts to three specific moments of the occupation of the Amazon forest. The first moment analyzes how the riparian families build their houses. The focus lies on form defined as communication, information, mediation, on the sign based on the Semiotic of Charles S. Peirce, seeking references not only in an immediate context, but also in an extended historical context, where the meanings of the relationship between man and the environment takes their toll on his production. Next, the search returns to the current situation of the riparian families. Based on data collected from PIATAM- Intelligence Social Environment Strategic Petroleum Industry in the Amazon region, an regional integrated social environment plan is developed to address housing, sanitation, human development, activities in the areas of the natural forest and fishing education and communication, among others, allowing the generation of a sustainability matrix, seeking to clearly determine the extent of their environmental impacts. In this context, our work finally returns to the role of the architect as a manager of information in the process of formation of the lines of human development, as an agent of organization of spaces and sustainable environments, from the action of a cultural system, aiming at the dialectical exchange of knowledge between science and residents, where the Amazon natives become active agents in the reportorial construction of the region, integrated to scientific knowledge system that is characteristic of the Amazon forest. This leads to the transformation of habits and customs of the natives in the light of new social environmental demands, and new attitudes in their own areas of scientific knowledge involved in this process. It is in this change of state, in this process, that general guidelines for sustainable regional development could be built, in a constant process of training, elements and at the same time promoters of more equitable ways, casting man and nature as agents of a sole reality.

L’émergence récente de nouvelles thérapies antivirales efficaces est une avancée considérable pour lutter contre l'infection chronique par le virus de l'hépatite C (VHC). Cependant, un pic de carcinomes hépatocellulaires, représentant l'atteinte hépatique ultime liée à l'infection, est attendu dans la prochaine décennie. Approfondir les connaissances des différentes étapes du cycle viral et de l’interférence du VHC avec l'hépatocyte hôte permet de mieux comprendre la pathogénèse associée à ce virus. Les travaux présentés dans cette thèse ont eu pour objectif d'identifier le réseau de partenaires cellulaires et viraux de la protéine non-structurale NS2 du VHC et de mieux comprendre les mécanismes d'action et de régulation de cette protéine transmembranaire multi-fonctionnelle, qui est un acteur clé du clivage protéolytique de la polyprotéine virale et de la morphogénèse des virions. Dans une première partie, nous avons analysé comparativement les mécanismes moléculaires de l’activité enzymatique des protéines NS2 du VHC et de plusieurs hepacivirus non-humains, qui infectent des primates du Nouveau Monde (GBV-B) ou qui ont été récemment identifiés chez plusieurs autres espèces animales (NPHV, RHV, BHV et GHV). Des analyses phylogénétiques, des modèles structuraux tridimensionnels et des Études dans un contexte d'expression transitoire de précurseurs polypeptidiques viraux ou dans des modèles d'infection ont montré que l’activité des protéases NS2 de divers hepacivirus (1) s'exerce à la jonction NS2/NS3 sous la forme d'homodimères formant deux triades catalytiques composites ; (2) est régulée dans le contexte de la polyprotéine virale par quelques résidus de surface du domaine N-terminal de NS3 (NS3N) nécessaires à son activation ; (3) est efficace en l'absence complète de NS3N, suggérant un rôle négatif ou régulateur, plutôt qu'activateur de NS3N, contrairement au dogme en vigueur actuellement. Ces travaux soulignent l'importance fonctionnelle des mécanismes protéolytiques de NS2 conservés parmi les différents hepacivirus. Dans une deuxième partie, nous avons identifié un réseau de facteurs cellulaires et viraux interagissant avec NS2 au cours du cycle infectieux par un crible interactomique reposant sur la purification par affinité et l'analyse par spectrométrie de masse des complexes protéiques isolés de cellules hépatocytaires infectées, ainsi que par un test de complémentation enzymatique fonctionnelle. Par une approche d'ARN interférence, nous avons ensuite montré qu'un nombre limité de facteurs cellulaires interagissant avec NS2 sont impliqués dans la production et la sécrétion de particules virales infectieuses, incluant des protéines du complexe de la peptidase signal (SPCS) au sein du réticulum endoplasmique, des protéines chaperonnes (DNAJB11, HSPA5) et une protéine impliquée dans le transport intracellulaire (SURF4). Notamment, nos Études suggèrent que plusieurs membres du SPCS forment un complexe multi-protéique avec NS2, impliquant Également la glycoprotéine virale E2, qui jouerait un rôle dans une Étape précoce de l'assemblage ou lors de l’enveloppement de la particule virale. En conclusion, mes travaux de thèse ont permis d'identifier pour la première fois une série limitée de facteurs hépatocytaires interagissant spécifiquement avec la protéine NS2 du VHC au cours de l'infection et de déterminer parmi ceux-ci les facteurs essentiels la morphogenèse virale. Par ailleurs, nos résultats ont permis d’enrichir les connaissances naissantes des hepacivirus non-humains récemment identifiés et de montrer que ceux-ci partageaient avec le VHC des mécanismes clés mis en jeu au cours du cycle viral, ce qui contribue consolider leur intérêt comme modèles animaux de substitution. / The recent emergence of a panel of direct acting antivirals will certainly help combat chronic hepatitis C in the future. However, in the current context worldwide, a peak of hepatitis C virus (HCV)-induced hepatocellular carcinoma is expected in the next decade. Deepening our understanding of HCV life cycle and HCV interference with host cells may help monitor HCV-associated pathogenesis. The aim of my PhD work was to identify the network of host and viral interactors of HCV nonstructural protein 2 and to unravel the mechanisms of action and regulation of this multifunctional, transmembrane protein, which is key both for the viral polyprotein cleavage and virion morphogenesis.In the first part of the work, we comparatively characterized molecular mechanisms underlying the enzymatic activity of NS2 proteins from HCV and from various non-human hepaciviruses that infect small New World primates (GBV-B) or that were recently identified in the wild in several mammalian species (NPHV, RHV, BHV, GHV). A combination of phylogenetic analyses, tridimensional structural models, and studies relying on the transient expression of viral polypeptide precursors or on infection models showed that NS2 proteases of the various hepaciviruses (1) act as dimers with two composite active sites to ensure NS2/NS3 junction cleavage, (2) are regulated in the polyprotein backbone via a hydrophobic patch at the surface of NS3 N-terminal domain (NS3N) that is essential to activate NS2 protease, and (3) are efficient in the complete absence of NS3N, which is unprecedented and suggests that NS3N has rather a negative or regulating role on NS2 activity. These data underline the functional importance of NS2 proteolytic mechanisms that are conserved across hepaciviruses.In the second part, we identified a network of cellular factors and viral proteins that interact with NS2 in the course of HCV infection using an interactomic screen based on affinity purification and mass spectrometry analysis of protein complexes retrieved form HCV infected hepatoma cells, as well as a split-luciferase complementation assay. Next, using a gene silencing approach, we found that a limited set of NS2 interactors among these host factors were involved in HCV particle assembly and/or secretion. This includes members of the endoplasmic reticulum signal peptidase complex (SPCS), chaperone proteins (DNAJB11, HSPA5) and a factor involved in intracellular transport (SURF4). Notably, our data are in favor of the existence of a multiprotein complex involving NS2, several members of the SPCS, and the viral E2 glycoprotein, which likely plays a role in an early step of HCV particle assembly or during particle envelopment. Altogether, my PhD work allowed us to identify a limited set of hepatocyte factors interacting with HCV NS2 during infection and to pinpoint those that are essential for HCV morphogenesis. Additionally, our results contributed to the molecular characterization of the recently identified non-human hepaciviruses and revealed that these hepaciviruses share with HCV key mechanisms in the course of their infectious life cycles. This highlights the value of non-human hepaciviruses as surrogate animal models of HCV infection.

[Copie de la conclusion de la thèse, en l'absence de résumé disponible] La morphogénèse bronchique est-elle l’œuvre de programmes et sous-programmes ou bien est-elle un phénomène partiellement auto-organisé à la faveur d’une régulation moléculaire parcimonieuse ? L’étude tridimensionnelle in-vivo que nous avons menée au stade pseudo-glandulaire précoce révèle que la structure de l’arbre est moins stéréotypée que ce qui est classiquement décrit et qu’elle ne peut être interprétée indépendamment de la croissance du mésenchyme et des tissus environnants. Ensemble, les variations observées dans le temps, l’espace ou la morphologie des branchements rendent peu probable l’existence d’un programme qui prédéfinirait où, quand, comment chaque branchement doit s’effectuer. Sans compter la complexité d’un tel programme, la manière dont il pourrait être écrit en langage moléculaire demeure obscure. En contexte sauvage, les variations – y compris celles qui ont été considérées comme d’authentiques erreurs – n’entravent jamais le processus de branchement. Celui-ci continue à se dérouler de telle sorte que l’espace est rempli sans conflits ni lacunes. Cela suggère fortement que les tubes épithéliaux sont capables de s’adapter en temps réel à la configuration des bourgeons et du mésenchyme environnant. Intuitivement, il est sans doute beaucoup plus simple de coordonner la croissance épithélio-mésenchymateuse et d’autoriser les bourgeons à remplir l’espace en s’auto-évitant que d’écrire un programme contenant le plan complet de l’organe.Nous montrons qu’un mécanisme simple fondé sur la diffusion d’un facteur de croissance clef (FGF10) permet d’organiser une croissance branchée. La dynamique laplacienne qui se met en place dans une géométrie mouvante provoque spontanément l’apparition de doigts dont l’extrémité se déstabilise de manière itérative pour produire une arborescence. Chaque branche de l’arbre remplit au fur et à mesure l’espace qui se développe dans le mésenchyme en s’auto-évitant. Les deux articles présentés introduisent donc une rupture conceptuelle en proposant que le plan de l’arbre bronchique ne soit pas « préprogrammé ». La morphogénèse de l’arbre bronchique est vraisemblablement régulée en temps réel par instruction réciproque de l’épithélium et du mésenchyme, au moyen d’un nombre réduit de boucle de régulation. / [This abstract is the conclusion of the dissertation] Is bronchial morphogenesis the result of programs and sub-programs or is it a partially self-organized phenomenon thanks to parsimonious molecular regulation? The three-dimensional in vivo study that we conducted at the early pseudo-glandular stage reveals that the structure of the tree is less stereotyped than is conventionally described and that it can not be interpreted independently of the growth of mesenchyme and surrounding tissues. Together, the variations observed in the time, the space or the morphology of the connections make it unlikely the existence of a program that predefines where, when, how each connection must be made. Not to mention the complexity of such a program, the way it could be written in molecular language remains unclear.In the wild context, variations - including those that have been considered genuine mistakes - never hinder the connection process. It continues to unfold so that the space is filled without conflicts or gaps. This strongly suggests that the epithelial tubes are able to adapt in real time to the configuration of buds and surrounding mesenchyme. Intuitively, it is probably much simpler to coordinate the epithelio-mesenchymal growth and to allow the buds to fill the space by self-avoidance than to write a program containing the complete organ plan.We show that a simple mechanism based on the diffusion of a key growth factor (FGF10) allows to organize a trendy growth. The Laplacian dynamics that is set up in a moving geometry spontaneously causes the appearance of fingers whose end is destabilized iteratively to produce a tree. Each branch of the tree gradually fills the space that develops in the mesenchyme by self-avoiding.The two articles presented thus introduce a conceptual break by proposing that the plane of the bronchial tree is not "preprogrammed". Morphogenesis of the bronchial tree is likely to be regulated in real time by reciprocal instruction of the epithelium and mesenchyme, by means of a reduced number of regulation loop.

La morphogenèse épithéliale est essentielle à la formation des organes. J'utilise le follicule ovarien de Drosophila comme modèle d'étude de l’aplatissement des cellules. Un follicule est composé de cellules germinales en croissance entourées d'une monocouche de cellules épithéliales cuboïdes. À un stade de développement spécifique, une part de ces cellules s'aplatit en suivant une vague régulée. Cet aplatissement est en partie contrôlé par un gradient de pression provenant d’un groupe de cellules germinales (les cellules nourricières). Toutes les cellules germinales sont connectées via des ponts cytoplasmiques. Cette thèse étudie les mécanismes conduisant à la génération du gradient de pression, et à la modulation moléculaire induite par cette force mécanique pour permettre l'aplatissement. J'ai montré que le nombre et le diamètre des ponts cytoplasmiques influaient sur la pression. En utilisant des reconstructions tridimensionnelles de follicules, j’ai étudié le rôle de la croissance différentielle des cellules nourricières en mesurant le changement de volume des cellules germinales lors de l'aplatissement des cellules. Enfin, j’ai cherché le mécanisme moléculaire conduisant à l’aplatissement des cellules et influencé par un stimulus mécanique à partir de la pression germinale, en proposant un rôle de la voie Hippo dans ce processus. En conclusion, nous proposons que la croissance des cellules germinales influe de manière mécanique et génétique sur les cellules épithéliales pour permettre l’élongation, et donc l'acquisition de la forme finale du follicule. / Epithelial morphogenesis is essential for organ formation. I use the Drosophila ovarian follicle as a model for studying cell flattening. A follicle is composed of growing germ cells surrounded by a monolayer of cuboidal epithelial cells. At a specific stage of development, some of these cells flatten out following a regulated wave. This flattening is partly controlled by a pressure gradient from part of the germ cells (the nurse cells). All germ cells are connected via cytoplasmic bridges. This thesis studies the mechanisms leading to the generation of the gradient of pressure, and to the molecular modulation induced by this mechanical force to allow flattening. I have shown that the number and diameter of cytoplasmic bridges affect the pressure. Using three-dimensional reconstructions of follicles, I studied the role of differential growth of nurse cells by measuring the change in germ cell volume during epithelial cell flattening. Finally, I looked for the molecular mechanism leading to the flattening and influenced by a mechanical stimulus from the germinal pressure, supporting a role of the Hippo pathway in this process. In conclusion, we propose that germ cell growth mechanically and genetically influences epithelial cells to allow elongation, and thus the acquisition of the final form of the follicle.

Congenital heart disease (CHD) is the major cause of death in the first year of life, the estimated incidence being 0.5-5% of live births; therefore it is important to understand the genetic causes underlying the complex process of heart formation to help prophylaxis, diagnosis and treatment of affected patients. CHD is the commonest phenotype associated with left-right (LR) disorders. LR asymmetry is determined during embryonic development. The three major body axes ? antero-posterior, dorso-ventral and left-right ? are patterned at gastrulation. LR asymmetry is established shortly after the two other major axes are patterned. The process of LR determination can be sub-divided into four integrated steps: 1. breaking of molecular symmetry in the gastrulation organizer; 2. transfer or relay of this asymmetric information to the lateral plate mesoderm (LPM), from which most internal organs will be formed; 3. reinforcement and propagation of asymmetric cues throughout the LPM and 4. conversion of asymmetric molecular information into proper organ morphogenesis. The goal of this work is to investigate mechanisms involved at two specific points in the laterality pathway: the initial generation/maintenance of asymmetric gene expression in the LPM and the morphogenetic translation of these early events into correct heart formation in the mouse. My emphasis has been on the characterization of laterality targeted cells via careful analysis of Pitx2c expression using a Pitx2c-lacZ reporter transgene, the role of BMP signalling, via Smad1, in generation/maintenance of early asymmetric signalling in the LPM, and the later involvement of both Smad1 and Pitx2 in cardiac morphogenesis through analyses of knockout mice.

Doctor of Philosophy / This thesis looks at social interaction from the point of view of social-institutional process. In doing so, it aims to account for i) how broader institutional processes are instantiated in local interaction, and ii) how western technologisation (in the Foucaultian sense) relates to or is instantiated in local interaction.