Background: The 22q11 deletion syndrome (22q11DS), also known as DiGeorge Syndrome, affects ~1/5000 live born children. Congenital heart defects (typically outflow tract and interrupted aortic arch) are present in 75% of individuals with 22q11DS and are the major cause of mortality. Other defects are cleft palate, thymus hypoplasia, inner ear defects and neuropsychiatric abnormalities. Df(16)1 mice carry a ~1 Mb hemizygous deletion on mouse chromosome 16 in a region syntenic with 22q11 and phenocopies 22q11DS. TBX1 is a DNA-binding transcription factor located in this interval and is required for neural crest cell proliferation and migration and for cardiac development. TBX1 point mutations have been identified in patients with DiGeorge syndrome. Thus TBX1 is thought to be a major gene responsible for the cardiac phenotype in 22q11DS. A key unresolved issue is the mechanism of reduced penetrance of cardiac malformations. One possibility is environmental variation during cardiogenesis. A second possibility is that variation in the TBX1 protein interaction network results in variable penetrance of the phenotype. Mutations in TBX1 or interacting partners could affect the structure of this protein interaction network. Aim: The aim of this thesis is to characterize the molecular mechanism of TBX1 function using biochemical and genetic approaches and to define the role of environmental variation on the DiGeorge phenotype. Results First part. Interaction of Df(16)1 with high-fat maternal diet. To determine if a maternal high-fat diet affects the penetrance of cardiac and thymus malformations in the Df1 deletion mouse model, wild-type and Df1 heterozygous embryos from control and high-fat diet groups were analyzed. No significant difference in the penetrance or the severity of cardiac malformations between these groups was found. These results do not support the idea that change in the fat content of maternal diet affects phenotype in this model. Thus, it is possible that high-fat diet interacts specifically with left-right patterning rather than with the genetic control of pharyngeal arch development and neural crest cell migration and survival. Second part. George, a novel ENU induced mutation in Tbx1. The George mutation, identified and mapped to Chr16 between rs4161352 and D16Mit112, results in fully penetrant cleft palate, cardiac malformations (VSD, IAA, CAT), absent cochlea and abnormal semicircular canals, and absent thymus resembling the human DiGeorge phenotype. Tbx1 lies in this interval and sequencing identified a G > A point mutation in exon 3 which is predicted to cause a Arginine to Glutamine change at amino acid position 160. George fails to genetically complement a Tbx1 null allele, confirming that it is causative and that George is functionally a null allele. RT-PCR showed that the George mutation affects splicing, resulting in a transcript lacking exon 3. This causes the loss of 34 amino acids within the TBX1 T-box domain, thus predicting that it affects DNA binding. Transactivation assays show that while the R160Q amino acid substitution significantly reduces the transactivation capacity of TBX1, surprisingly the loss of exon 3 does not affect this function. Analysis of endogenous TBX1 in developing embryos by Western blot showed that the protein expression is absent or significantly reduced. This finding suggests that the observed George phenotype is caused primarily by a loss of TBX1 protein expression. Third part. Investigation of the protein interaction network surrounding TBX1. In order to get a better insight into the protein network surrounding TBX1, a TBX1 split renilla-luciferase protein complementation assay was set up which allowed to test the physical interaction between TBX1 and several putative interactors. It was found that GATA4, SMARCAD1, RBBP5 and PTDSR interact with wild-type TBX1 in HEK293T cells. The R160Q point mutation and the loss of exon 3 affect some of these interactions supporting the idea that variation in the protein interaction network may, at least in part, be responsible for the DGS phenotype.

La Cirrhose Amérindienne Infantile (CAI, NAIC) est une forme de cholestase non-syndromique héréditaire à transmission autosomique récessive, décrite uniquement chez les enfants autochtones du Nord-Ouest québécois et issue d’un effet fondateur. La maladie se présente d’abord sous la forme d’une jaunisse néonatale chez un enfant autrement en bonne santé, qui progresse en cirrhose de type biliaire dans l’enfance et dans l’adolescence. Le taux de survie à l’âge adulte est inférieur à 50% et la seule thérapie efficace à ce jour pour les patients avancés dans la maladie demeure la transplantation hépatique. Les recherches antérieures menées par le groupe ont permis d’identifier le locus ainsi que le gène responsable de NAIC, qui encode la protéine nucléolaire Cirhin. Cirhin est exprimée uniquement dans le foie et tous les patients sont homozygotes pour la mutation R565W. La fonction de Cirhin est inconnue, mais les motifs WD40 retrouvés dans sa séquence indiquent qu’elle participerait à des interactions protéine-protéine et serait impliquée dans un mécanisme moléculaire de base. Cirhin interagit avec la protéine nucléaire Cirip, qui a un effet positif important sur la transcription de l’élément activateur HIV-1 LTR et qui a un rôle dans la prolifération cellulaire. L’interaction de Cirhin et Cirip est affectée par la mutation R565W. À l’aide de la technique du double hybride chez la levure, la protéine nucléolaire Nol11 a été identifiée comme étant un partenaire d’interaction de Cirhin. Par son interaction avec MARK3 et c-Myc, Nol11 serait impliquée dans des processus cellulaires tels que le contrôle du cycle cellulaire, la polarité, la croissance cellulaire et possiblement la biogenèse des ribosomes. La portion C-terminale de Nol11 interagirait avec Cirhin, et la mutation R565W abolit cette interaction. Le résidu R565 serait donc important pour la fonctionnalité de Cirhin. / North American Indian childhood cirrhosis (NAIC) is a nonsyndromic form of hereditary autosomal recessive cholestasis, described only in children of Algonquin origin from the Abitibi region of north-western Québec and issued from a founder effect. NAIC typically presents with transient neonatal jaundice in a child who is otherwise healthy, but progresses to biliary fibrosis in childhood or young adulthood. The survival rate at adulthood is lower than 50% and liver transplantation is currently the only effective therapy for patients with advanced disease. Previous research by the group allowed the identification of the locus and the gene responsible for NAIC, which encodes the nucleolar protein Cirhin. Cirhin is expressed only in the liver and all patients are homozygous for the R565W mutation. Cirhin’s function is unknown, but it is a WD40-repeat containing protein, which indicates that it would participate in protein-protein interactions and would be involved in a basic molecular mechanism. Cirhin interacts with the nuclear protein Cirip, which has a significant positive effect on the transcription of the HIV-1 LTR enhancer element and has a role in cellular proliferation. The interaction between Cirhin and Cirip is affected by the R565W mutation. Using the yeast two-hybrid technique, the nucleolar protein Nol11 was identified as an interacting partner of Cirhin. By its interaction with MARK3 and c-Myc, Nol11 may be involved in cellular processes such as cell cycle control, polarity, cell growth and possibly ribosome biogenesis. The C-terminal portion of Nol11 interacts with Cirhin and the R565W mutation abolishes the interaction. The residue R565 may thus be important for Cirhin’s functionality.

Neospora caninum (filo Apicomplexa) é um parasita obrigatório intracelular como todos os membros deste filo, alguns reconhecidos por causarem doenças com impacto relevante na saúde humana (Plasmodium e Toxoplasma) e veterinária (Babesia, Eimeria e Cryptosporidium). Causador da neosporose, N. caninum vem emergindo como um dos maiores causadores de abortos infecciosos em bovinos, levando a consideráveis perdas econômicas na bovinocultura mundial. Devido à sua recente descoberta, o conhecimento sobre diversos processos bioquímicos de N.caninum ainda é limitado, demandando novas pesquisas para a compreensão de seus mecanismos de sobrevivência e consequente identificação de alvos para intervenção terapêutica. O processo de invasão celular é bastante investigado em pesquisas envolvendo apicomplexas, uma vez que a sobrevivência desses parasitas depende do sucesso de sua entrada na célula hospedeira. Proteínas secretadas de organelas filo-específicas (micronemas, roptrias e grânulos densos) estão intimamente envolvidas com a invasão celular. Elas são responsáveis pela interação inicial com a célula hospedeira, participam da junção de movimento formada no momento da invasão, e contribuem para a estabilização do vacúolo parasitóforo. Neste trabalho as proteínas secretadas por taquizoítas de N. caninum foram investigadas de duas formas: (1) por caracterização molecular de proteínas com domínio Apple; e (2) por estudo do secretoma do parasita. Os domínios proteicos do tipo Apple são caracterizados pela capacidade de interação proteína-proteína e proteína-carboidrato, e estão presentes em algumas proteínas micronêmicas com propriedades adesivas. Neste trabalho três proteínas de N. caninum contendo domínios Apple foram caracterizadas: MIC17A, MIC17B e MIC17C. A análise das sequências proteicas e das estruturas dos domínios Apple, obtidas por modelagem molecular, mostraram alta identidade sequencial e estrutural entre MIC17A e MIC17C. Apesar de ser paráloga às outras duas, MIC17B apresenta diferenças importantes em sua sequência e estrutura. Para MIC17B e MIC17C foram realizados experimentos de detecção das proteínas nativas nos extratos total e secretado do taquizoíta que sugerem diferentes formas de processamento entre essas proteínas no parasita. Para MIC17B foi confirmada a localização em micronemas, num padrão diferente do observado para MIC17C. Os ensaios de invasão combinados aos de localização indicam que estas proteínas estejam relacionadas ao processo de invasão celular, porém, suas funções permanecem desconhecidas. O secretoma é o conjunto de proteínas secretadas pelo parasita e, para explorar a composição deste extrato (ESA) no taquizoíta de N. caninum, duas abordagens complementares foram utilizadas. Na primeira abordagem foram identificadas as proteínas presentes no ESA por espectrometria de massas. Na segunda abordagem realizou-se uma ii quantificação relativa das proteínas, marcadas por dois isótopos, nos extratos totais de taquizoítas submetidos ou não ao estímulo secretório. O resultado esperado seria com as proteínas secretadas diminuídas no parasita estimulado. Em ambas as abordagens foram utilizadas técnicas de espectrometria de massas de alta resolução (nanoLC-MS/MS), o que resultou num alto número de identificações; 615 proteínas no ESA e 2011 proteínas quantificadas. A comparação das duas abordagens permitiu o reconhecimento de proteínas com maior probabilidade de secreção. Uma rede de interação entre as proteínas diferencialmente expressas foi predita, gerando resultados que, associados às informações sobre as proteínas aumentadas, permitiram uma investigação sobre proteínas potencialmente envolvidas com a regulação do metabolismo relacionado à secreção. Os resultados obtidos por ambos os estudos aqui demonstrados somam conhecimento acerca do parasita N. caninum e demonstram ser úteis para guiar a busca e seleção de alvos a serem investigados para o desenvolvimento de terapêutica contra a neosporose. / Neospora caninum (Apicomplexa phylum) is an obligatory intracellular parasite like all members from this phylum, some causing diseases with relevant impact on human (Plasmodium and Toxoplasma) and veterinary (Babesia, Eimeria and Cryptosporidium) health. Causative agent of neosporosis, N. caninum has emerged as one of the leading causes of infectious abortion in cattle, generating huge economical losses in worldwide livestock. Due to its recent discovery, knowledge of N. caninum biochemical processes remains scarce, demanding new research for comprehending its survival mechanisms and, consequently, identifying new targets for therapeutic intervention. The invasion process has often been investigated in apicomplexans since their survival depends on the success of their entry into the host cell. Proteins secreted from phylum-specific organelles (micronemes, rhoptries and dense granules) are deeply involved with invasion. They are responsible for the initial interaction with the host cell; participate of the moving junction formed in the moment of invasion; and contribute for the stabilization of the parasitophorus vacuole. In this study, the proteins secreted by N. caninum tachyzoites were investigated in two ways: (1) the molecular characterization of Apple domaincontaining proteins; and (2) exploring the parasite secretome. The Apple protein domains are characterized by the ability to interact as protein-protein and proteincarbohydrate, and are present in some microneme proteins with adhesive properties. Here three N. caninum proteins containing Apple domains were characterized: MIC17A, MIC17B and MIC17C. Analyses of the Apple domains sequences and structures, obtained by molecular modeling, revealed high sequential and structural identities between MIC17A and MIC17C. Although being a paralog of the other two proteins, MIC17B presents significant differences in its sequence and structure. Experiments were performed for native MIC17B and MIC17C detection in the total and secreted tachyzoite extracts, suggesting different processing forms for these proteins in the parasite. For MIC17B, the microneme localization was confirmed, differently from the pattern observed for MIC17C. Invasion and localization assays indicated that these proteins are related to the cell invasion process; nevertheless, their functions remain unknown. The secretome is the set of proteins secreted by the parasite and, to explore this extract (ESA) composition in N. caninum, two complementary approaches were used. Firstly proteins present in ESA were identified by mass spectrometry. In the second approach, a relative quantification was performed on the proteomes of ethanol stimulated/non stimulated tachyzoites, expecting that the secreted proteins would be down regulated at the stimulated parasite. Both approaches were performed with high resolution mass spectrometry techniques (nanoLC-MS/MS), reaching a high number of identifications: 615 proteins iv in ESA and 2011 quantified proteins. The comparison between both approaches allowed the recognition of the most likely secreted proteins. An interaction network was predicted, involving the differentially expressed proteins. These results, associated with the information of up regulated proteins, allowed the investigation of proteins potentially involved with the secretion metabolism regulation. The findings from our two studies add up knowledge about N. caninum and demonstrate to be useful in guiding the search and selection for new targets for therapeutic development against neosporosis.

Der Transkriptionsfaktor CCAAT/enhancer-binding Protein β (C/EBPβ) reguliert die Expression zahlreicher Gene, welche die Proliferation, Differenzierung und Seneszenz in hämatopoietischen Zellen, Adipozyten und Leukämiezellen kontrollieren. Um diese mannigfaltigen Aufgaben zu erfüllen interagiert C/EBPβ mit zahlreichen Kofaktoren und Proteinen der Transkriptionsregulations-Maschinerie. Da das funktionale Netzwerk von C/EBPβ und seinen zahlreichen Kooperationspartnern bis heute nicht vollständig entziffert ist, ist es das Ziel dieser Arbeit das Netzwerk aus Interaktionspartnern und C/EBPβ regulierten Proteinen in Leukämiezelllinien und darüber hinaus zu erforschen und aufzudecken. Das Interaktom von C/EBPβ wurde mittels einer Kombination aus einem membranbasierten Peptid-Interaktions Testverfahrens (APS) und endogener Immunprezipitationen mit gekoppelter MS-Analyse untersucht. Außerdem wurde die Proteinmenge von C/EBPβ und von potentiell von C/EBPβ regulierten Proteinen mittels proteomischer MS-Analyse in C/EBPβ Knock-out- und Leukämiezelllinien untersucht. Die Protein-Interaktionsversuche ergaben epigenetische und allgemeine transkriptionsregulierende Proteine, sowie Chromatinstruktur modellierende Faktoren, die mit C/EBPβ interagieren. Zusätzlich konnten neue Interaktionen von C/EBPβ mit Kondensin- und Kinetochorproteinen beobachtet werden. Die Versuchsergebnisse eröffnen überdies neue Interaktionen von C/EBPβ mit DNA Reparatur und Apoptose assoziierten Proteinen. Interessanterweise konnten auch Komponenten des Spliceosomes und RNA-prozessierende Proteine als Interaktoren von C/EBPβ identifiziert werden. Zusammenfassend ermöglicht diese Studie nicht nur die Verifikation von bereits bekannten Proteininteraktionen von C/EBPβ, sondern eröffnet zahlreiche weitere zukünftige Forschungsfelder bezüglich des Interaktionsnetzwerkes von C/EBPβ in Leukämien, sowie anderen Zellarten und Geweben. / The basic leucine zipper transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) regulates the expression of various genes that control the proliferation, differentiation and senescence of haematopoietic cells, adipocytes and leukemia cells. To facilitate its multifaceted functions C/EBPβ interacts with a collection of cofactors and proteins of the transcription regulation machinery. As the functional network of C/EBPβ and its numerous cooperation partners is still incomplete this study attempted to analyze interaction partners and downstream proteins of C/EBPβ in leukemia cells and beyond. A combinatory approach of an array based peptide-interaction screening (APS) and endogenous shotgun IP-MS from leukemia cell lines was applied to elucidate the interactome of C/EBPβ. Moreover, C/EBPβ abundance and potential C/EBPβ regulated proteins were determined by MS proteomics in C/EBPβ knockout and leukemia cell lines. The interaction screenings revealed proteins associated with the general and epigenetic regulation of transcription, with chromatin remodeling and mitotic chromatin organization as well as cell cycle regulation. Additionally, new interactions of C/EBPβ with condensin and kinetochore proteins could be elucidated. The data reports of novel C/EBPβ interactors involved in DNA repair and apoptosis. In addition, components of the spliceosome and RNA-processing were detected. Altogether this study verifies known and reveals various novel interactions of the transcription factor C/EBPβ and augments the network of previous reported interactions and potential cooperation partners. The here collected data discloses new subjects for further research concerning the interaction network of C/EBPβ during cell differentiation and in leukemia.

Diante da abundância de informações e de compartilhamentos em sites de redes sociais e da proliferação de convites para o uso desses sites na Educação, esta tese propõe a pesquisa das estruturas relacionais que emergem das interações sociais em grupos no site de rede social Facebook. O objetivo geral é compreender como as interações sociais em rede e nas redes podem contribuir para uma Educação em Rede a partir da análise dos padrões de interação social em grupos no site Facebook. Analisamos dois casos distintos de redes sociais organizadas como parte de processos pedagógicos. Os recursos curtir, comentar e curtir comentários são os tipos de interação social em rede que deram origem aos grafos analisados. As temáticas das postagens e os tipos de suportes tecnológicos são identificados na análise dos padrões de interação social em rede e nas redes, nas quais destacamos a participação, o engajamento e o sentimento de pertencimento dos atores. A Análise de Redes Sociais é o método utilizado para compreender as dinâmicas das interações. O Modelo de Análise da Comunicação Assíncrona é adotado na análise de conteúdo das postagens e dos comentários dessas postagens com vistas a destacar as temáticas compartilhadas e discutidas. Nas redes sociais em foco, as pessoas se organizam e interagem, prioritariamente, com o professor ou com os atores sociais que assumem as funções de professor. Considerando que os recursos tecnológicos disponíveis permitem fluxo de informações de diferentes fontes, o que observamos é que o professor é a pessoa fundamental para articular as aprendizagens nas redes sociais. A configuração da sala de aula analógica se transfere para os comportamentos e às expectativas do que deve acontecer na sala de aula em redes sociais. Essa dinâmica fornece subsídios para propor orientações à formação de professores e diretrizes para a realização de Educação em Rede. A difusão das informações e a circulação de atores-ponte na rede contribuem para que haja presença social. A presença social é uma peça fundamental para a presença cognitiva. O uso de sites de redes sociais na educação tem valor pedagógico desde que haja presença social endereçada ao ensino. É necessário que um ator ou alguns atores ajam de modo a garantir relações de ensino e aprendizagem. / Faced with the abundance of information and shares on social networking sites and invitations proliferation to the use of these sites in education, this thesis proposes the research of relational structures that emerge from social interactions in groups on the Facebook social networking site. The overall goal is to understand how social interactions network and the networks can contribute to an Education Network based on the analysis of patterns of social interaction in groups on Facebook site. We analyzed two different cases of social networks organized as part of pedagogical processes. Resources Like, Comment and Like Comments are the types of social interaction network that gave rise to the analyzed graphs. The thematic of posts and the types of technological support are identified in the analysis of patterns of social interaction in the network and the networks in which we highlight the participation, engagement and sense of belonging of the actors. The Social Network Analysis is the method used to understand the dynamics of interactions. The Asynchronous Communications Analysis Model is adopted in the content analysis of posts and comments of these posts with a view to highlight the shared and discussed themes. In social networks in focus, people are organized and interact primarily with the teacher or with the social actors who assume the teacher functions. Whereas the available technological resources enable flow of information from different sources, which we observe is that the teacher is the key person to articulate learning in social networks. The configuration of the analog classroom is transferred to the behaviors and expectations of what should happen in the classroom in social networks. This dynamic provides subsidies to propose guidelines for teacher training and guidelines for conducting Education Network. The dissemination of information and the circulation of actors bridge on the network contribute to social presence there. Social presence is a key part of cognitive presence. The use of social networking sites in education has educational value as long as there is social presence addressed to education. It is necessary for an actor or some actors to act in order to ensure teaching and learning relations. / Frente a la abundancia de información y acciones en las redes sociales y la proliferación de las invitaciones a la utilización de estos sitios en la educación, esta tesis propone la investigación de las estructuras relacionales que surgen de las interacciones sociales en grupos en la red social Facebook. El objetivo general es entender cómo las interacciones de la red social y las redes puede contribuir a una red de educación basado en el análisis de los patrones de interacción social en grupos en Facebook. Se analizaron dos casos diferentes de redes sociales organizados en el marco de los procesos pedagógicos. Recursos Me gusta, Comentar y Me Gusta el Comentario son los tipos de red de interacción social que dieron origen a los gráficos analizados. La temática de los puestos y los tipos de apoyo tecnológico son identificados en el análisis de los patrones de interacción social en la red y las redes en las que podemos destacar la participación, el compromiso y el sentido de pertenencia de los actores. El análisis de redes sociales es el método utilizado para entender la dinámica de las interacciones. El Modelo de Análisis de Comunicaciones asíncronas se adopta en el análisis del contenido de los mensajes y los comentarios de estos mensajes con el fin de poner de relieve los temas compartidos y discutidos. En las redes sociales en el enfoque, las personas se organizan e interactúan principalmente con el profesor o con los actores sociales que asumen las funciones de los maestros. Considerando que los recursos tecnológicos disponibles permiten el flujo de información de diferentes fuentes, que observamos es que el maestro es la persona clave para articular el aprendizaje en las redes sociales. La configuración del clase analógica se transfiere a los comportamientos y expectativas de lo que debería ocurrir en el aula en las redes sociales. Esta dinámica proporciona subsidios a proponer directrices para la formación del profesorado y directrices para la realización de la Red de Educación. La difusión de la información y la circulación de los actores puente en la red contribuyen a la presencia social allí. La presencia social es una parte clave de la presencia cognitiva. El uso de las redes sociales en la educación tiene un valor educativo, siempre y cuando no hay presencia social dirigida a la educación. Es necesario que un actor o algunos actores para actuar con el fin de garantizar las relaciones de enseñanza y aprendizaje.

Occupé depuis près de 10 000 ans, le Témiscouata est un lieu clé pour mieux comprendre la préhistoire du Québec, en raison de son emplacement stratégique entre l’Atlantique et la vallée du Saint-Laurent et de la cinquantaine de sites archéologiques connus dans la région. Le site CjEd-8 est le seul site associé à l’Archaïque supérieur (6 000 à 4 000 ans avant aujourd’hui) connu dans la région. Cette recherche palethnographique porte sur l’analyse de la collection lithique du site archéologique CjEd-8. Nos questions de recherche sont les suivantes : quel était le mode de vie et les activités des occupants du site CjEd-8? Quel était leur environnement et comment s’y sont-ils adapté? Comment l’espace était-il utilisé sur le site? Comment ce site se compare-t-il aux autres sites de l’Archaïque supérieur au Québec et dans le nord-est américain? Est-il possible de relier l’occupation du site CjEd-8 à un plus vaste cadre régional ou culturel, comme un réseau d’interaction, par exemple? Nous avons effectué une analyse techno-morphologique individuelle de tous les outils et du débitage de la collection. Nous avons pu constater que tous les stades de réduction lithique (initial, intermédiaire et final) sont présents sur le site CjEd-8. Les matières premières locales sont représentées dans tous ces stades de réduction, bien que les matières premières exotiques soient surtout présentes au sein du stade initial de réduction, davantage que les matières premières locales. Ceci laisse croire que le dégrossissement initial des blocs et galets de matière première locale se faisait ailleurs que sur le site, fort probablement sur les carrières de chert de la région, et que des matières exotiques ont aussi été travaillées sur le site. Des activités de taille ont eu lieu sur les deux zones du site, probablement autour de deux foyers distincts. Les quelques individus présents y sont demeurés très brièvement, semblent avoir effectué un nombre limité d’activités et sont peut-être repassés quelques temps après. L’Archaïque supérieur est caractérisé par une augmentation de la population et par l’intensification des interactions et de l’appartenance régionale. Or, il semblerait que ce soit l’inverse que l’on observe archéologiquement au Témiscouata. L’assemblage de CjEd-8 ne présente aucune ressemblance particulière avec les sites associés à l’Archaïque supérieur au Québec, bien que pour presque tous ces sites, on constate une exploitation importante des matières premières locales (même si ces dernières sont de qualité moyenne), combinée à l’exploitation de matières premières exotiques de sources très éloignées. L’industrie du quartz, importante dans la plupart des sites de l’Archaïque supérieur, se reflète très peu sur CjEd-8. Bien qu’il nous soit impossible d’associer l’occupation du site CjEd-8 à une tradition culturelle précise, cela ne signifie pas que ses occupants n’étaient pas en interaction avec l’une ou l’autre des entités culturelles de la fin de l’Archaïque, que ce soit avec les groupes de la région de Quoddy, de la Gaspésie, de la Vallée du Saint-Laurent, de l’Outaouais, de la Haute Côte nord, du Vermont, de l’État de New York et de l’intérieur du Maine. La question des modes de subsistance des occupants de CjEd-8 n’a pas été soulevée lors de nos analyses. Nous savons toutefois que les occupants du Témiscouata avaient alors accès à une faune riche et diversifiée. / Continuously occupied for 10,000 years, and due to its strategic location between the Atlantic Ocean and the St. Lawrence Valley, the Temiscouata region is key to understand the prehistory of Quebec. Moreover, some fifty archaeological sites are known in the region. Of those, the site labelled CjEd-8 is the only one that has a Late Archaic occupation (6,000 to 4,000 BP). The present research consists in the analysis of the lithic assemblage recovered from CjEd-8. Our research questions are the following: What were the activities and lifeways of the site's inhabitants? What were the environmental characteristics and what cultural adaptations did they trigger? How was space used at the site? How does the site compare with other Late Archaic settlements in Quebec and the Northeast? Can we position the site's occupation within a broader cultural or regional framework, within an interaction sphere, for instance? We have performed an individual techno-morphological analysis of all tools and debitage from the collection. These demonstrate that all stages of lithic reduction (initial, intermediary, and final) are present at CjEd-8. All these reduction stages include local raw materials, but exotic raw materials are principally represented among the initial reduction stages, more so than local materials. This suggests that the initial reduction of local raw material blocks and cobbles was performed elsewhere than on the site, most likely directly in the vicinity of the region's chert quarries. It also indicates that exotic materials were worked at the site. Stone knapping activities have taken place on both the site's zones, probably around two distinct hearths. It appears that the few individuals present at the site remained there for a short period of time, performing a limited number of activities before leaving, and that they returned some time thereafter. The Late Archaic is characterized by population growth, intensification of inter-group interaction, and a rise in regional identity. However, we are witnessing the opposite to be happening in Temiscouata. The assemblage from CjEd-8 presents no obvious similarity with other Late Archaic sites in Quebec, although all these sites exhibit a preferred exploitation of local raw materials (even when they are of mediocre quality), combined with the use of exotic raw materials from very distant sources. The use of quartz, very common on most Late Archaic sites, is not well represented at CjEd-8. Although it is at present impossible to link the occupation of CjEd-8 with any specific cultural tradition, it does not immediately follow that its inhabitants were not interacting with one or more Late Archaic cultural entities, such as groups from the Quoddy region, Gaspesie, the St. Lawrence Valley, the Ottawa Valley, the Upper North Coast of the St. Lawrence River, Vermont, New York State, or inland Maine. The matter of subsistence patterns was not addressed as part of our analyses. We nonetheless know that the inhabitants of Temiscouata had access to a rich and diversified fauna.

Occupé depuis près de 10 000 ans, le Témiscouata est un lieu clé pour mieux comprendre la préhistoire du Québec, en raison de son emplacement stratégique entre l’Atlantique et la vallée du Saint-Laurent et de la cinquantaine de sites archéologiques connus dans la région. Le site CjEd-8 est le seul site associé à l’Archaïque supérieur (6 000 à 4 000 ans avant aujourd’hui) connu dans la région. Cette recherche palethnographique porte sur l’analyse de la collection lithique du site archéologique CjEd-8. Nos questions de recherche sont les suivantes : quel était le mode de vie et les activités des occupants du site CjEd-8? Quel était leur environnement et comment s’y sont-ils adapté? Comment l’espace était-il utilisé sur le site? Comment ce site se compare-t-il aux autres sites de l’Archaïque supérieur au Québec et dans le nord-est américain? Est-il possible de relier l’occupation du site CjEd-8 à un plus vaste cadre régional ou culturel, comme un réseau d’interaction, par exemple? Nous avons effectué une analyse techno-morphologique individuelle de tous les outils et du débitage de la collection. Nous avons pu constater que tous les stades de réduction lithique (initial, intermédiaire et final) sont présents sur le site CjEd-8. Les matières premières locales sont représentées dans tous ces stades de réduction, bien que les matières premières exotiques soient surtout présentes au sein du stade initial de réduction, davantage que les matières premières locales. Ceci laisse croire que le dégrossissement initial des blocs et galets de matière première locale se faisait ailleurs que sur le site, fort probablement sur les carrières de chert de la région, et que des matières exotiques ont aussi été travaillées sur le site. Des activités de taille ont eu lieu sur les deux zones du site, probablement autour de deux foyers distincts. Les quelques individus présents y sont demeurés très brièvement, semblent avoir effectué un nombre limité d’activités et sont peut-être repassés quelques temps après. L’Archaïque supérieur est caractérisé par une augmentation de la population et par l’intensification des interactions et de l’appartenance régionale. Or, il semblerait que ce soit l’inverse que l’on observe archéologiquement au Témiscouata. L’assemblage de CjEd-8 ne présente aucune ressemblance particulière avec les sites associés à l’Archaïque supérieur au Québec, bien que pour presque tous ces sites, on constate une exploitation importante des matières premières locales (même si ces dernières sont de qualité moyenne), combinée à l’exploitation de matières premières exotiques de sources très éloignées. L’industrie du quartz, importante dans la plupart des sites de l’Archaïque supérieur, se reflète très peu sur CjEd-8. Bien qu’il nous soit impossible d’associer l’occupation du site CjEd-8 à une tradition culturelle précise, cela ne signifie pas que ses occupants n’étaient pas en interaction avec l’une ou l’autre des entités culturelles de la fin de l’Archaïque, que ce soit avec les groupes de la région de Quoddy, de la Gaspésie, de la Vallée du Saint-Laurent, de l’Outaouais, de la Haute Côte nord, du Vermont, de l’État de New York et de l’intérieur du Maine. La question des modes de subsistance des occupants de CjEd-8 n’a pas été soulevée lors de nos analyses. Nous savons toutefois que les occupants du Témiscouata avaient alors accès à une faune riche et diversifiée. / Continuously occupied for 10,000 years, and due to its strategic location between the Atlantic Ocean and the St. Lawrence Valley, the Temiscouata region is key to understand the prehistory of Quebec. Moreover, some fifty archaeological sites are known in the region. Of those, the site labelled CjEd-8 is the only one that has a Late Archaic occupation (6,000 to 4,000 BP). The present research consists in the analysis of the lithic assemblage recovered from CjEd-8. Our research questions are the following: What were the activities and lifeways of the site's inhabitants? What were the environmental characteristics and what cultural adaptations did they trigger? How was space used at the site? How does the site compare with other Late Archaic settlements in Quebec and the Northeast? Can we position the site's occupation within a broader cultural or regional framework, within an interaction sphere, for instance? We have performed an individual techno-morphological analysis of all tools and debitage from the collection. These demonstrate that all stages of lithic reduction (initial, intermediary, and final) are present at CjEd-8. All these reduction stages include local raw materials, but exotic raw materials are principally represented among the initial reduction stages, more so than local materials. This suggests that the initial reduction of local raw material blocks and cobbles was performed elsewhere than on the site, most likely directly in the vicinity of the region's chert quarries. It also indicates that exotic materials were worked at the site. Stone knapping activities have taken place on both the site's zones, probably around two distinct hearths. It appears that the few individuals present at the site remained there for a short period of time, performing a limited number of activities before leaving, and that they returned some time thereafter. The Late Archaic is characterized by population growth, intensification of inter-group interaction, and a rise in regional identity. However, we are witnessing the opposite to be happening in Temiscouata. The assemblage from CjEd-8 presents no obvious similarity with other Late Archaic sites in Quebec, although all these sites exhibit a preferred exploitation of local raw materials (even when they are of mediocre quality), combined with the use of exotic raw materials from very distant sources. The use of quartz, very common on most Late Archaic sites, is not well represented at CjEd-8. Although it is at present impossible to link the occupation of CjEd-8 with any specific cultural tradition, it does not immediately follow that its inhabitants were not interacting with one or more Late Archaic cultural entities, such as groups from the Quoddy region, Gaspesie, the St. Lawrence Valley, the Ottawa Valley, the Upper North Coast of the St. Lawrence River, Vermont, New York State, or inland Maine. The matter of subsistence patterns was not addressed as part of our analyses. We nonetheless know that the inhabitants of Temiscouata had access to a rich and diversified fauna.

La Cirrhose Amérindienne Infantile (CAI, NAIC) est une forme de cholestase non-syndromique héréditaire à transmission autosomique récessive, décrite uniquement chez les enfants autochtones du Nord-Ouest québécois et issue d’un effet fondateur. La maladie se présente d’abord sous la forme d’une jaunisse néonatale chez un enfant autrement en bonne santé, qui progresse en cirrhose de type biliaire dans l’enfance et dans l’adolescence. Le taux de survie à l’âge adulte est inférieur à 50% et la seule thérapie efficace à ce jour pour les patients avancés dans la maladie demeure la transplantation hépatique. Les recherches antérieures menées par le groupe ont permis d’identifier le locus ainsi que le gène responsable de NAIC, qui encode la protéine nucléolaire Cirhin. Cirhin est exprimée uniquement dans le foie et tous les patients sont homozygotes pour la mutation R565W. La fonction de Cirhin est inconnue, mais les motifs WD40 retrouvés dans sa séquence indiquent qu’elle participerait à des interactions protéine-protéine et serait impliquée dans un mécanisme moléculaire de base. Cirhin interagit avec la protéine nucléaire Cirip, qui a un effet positif important sur la transcription de l’élément activateur HIV-1 LTR et qui a un rôle dans la prolifération cellulaire. L’interaction de Cirhin et Cirip est affectée par la mutation R565W. À l’aide de la technique du double hybride chez la levure, la protéine nucléolaire Nol11 a été identifiée comme étant un partenaire d’interaction de Cirhin. Par son interaction avec MARK3 et c-Myc, Nol11 serait impliquée dans des processus cellulaires tels que le contrôle du cycle cellulaire, la polarité, la croissance cellulaire et possiblement la biogenèse des ribosomes. La portion C-terminale de Nol11 interagirait avec Cirhin, et la mutation R565W abolit cette interaction. Le résidu R565 serait donc important pour la fonctionnalité de Cirhin. / North American Indian childhood cirrhosis (NAIC) is a nonsyndromic form of hereditary autosomal recessive cholestasis, described only in children of Algonquin origin from the Abitibi region of north-western Québec and issued from a founder effect. NAIC typically presents with transient neonatal jaundice in a child who is otherwise healthy, but progresses to biliary fibrosis in childhood or young adulthood. The survival rate at adulthood is lower than 50% and liver transplantation is currently the only effective therapy for patients with advanced disease. Previous research by the group allowed the identification of the locus and the gene responsible for NAIC, which encodes the nucleolar protein Cirhin. Cirhin is expressed only in the liver and all patients are homozygous for the R565W mutation. Cirhin’s function is unknown, but it is a WD40-repeat containing protein, which indicates that it would participate in protein-protein interactions and would be involved in a basic molecular mechanism. Cirhin interacts with the nuclear protein Cirip, which has a significant positive effect on the transcription of the HIV-1 LTR enhancer element and has a role in cellular proliferation. The interaction between Cirhin and Cirip is affected by the R565W mutation. Using the yeast two-hybrid technique, the nucleolar protein Nol11 was identified as an interacting partner of Cirhin. By its interaction with MARK3 and c-Myc, Nol11 may be involved in cellular processes such as cell cycle control, polarity, cell growth and possibly ribosome biogenesis. The C-terminal portion of Nol11 interacts with Cirhin and the R565W mutation abolishes the interaction. The residue R565 may thus be important for Cirhin’s functionality.

Neospora caninum (filo Apicomplexa) é um parasita obrigatório intracelular como todos os membros deste filo, alguns reconhecidos por causarem doenças com impacto relevante na saúde humana (Plasmodium e Toxoplasma) e veterinária (Babesia, Eimeria e Cryptosporidium). Causador da neosporose, N. caninum vem emergindo como um dos maiores causadores de abortos infecciosos em bovinos, levando a consideráveis perdas econômicas na bovinocultura mundial. Devido à sua recente descoberta, o conhecimento sobre diversos processos bioquímicos de N.caninum ainda é limitado, demandando novas pesquisas para a compreensão de seus mecanismos de sobrevivência e consequente identificação de alvos para intervenção terapêutica. O processo de invasão celular é bastante investigado em pesquisas envolvendo apicomplexas, uma vez que a sobrevivência desses parasitas depende do sucesso de sua entrada na célula hospedeira. Proteínas secretadas de organelas filo-específicas (micronemas, roptrias e grânulos densos) estão intimamente envolvidas com a invasão celular. Elas são responsáveis pela interação inicial com a célula hospedeira, participam da junção de movimento formada no momento da invasão, e contribuem para a estabilização do vacúolo parasitóforo. Neste trabalho as proteínas secretadas por taquizoítas de N. caninum foram investigadas de duas formas: (1) por caracterização molecular de proteínas com domínio Apple; e (2) por estudo do secretoma do parasita. Os domínios proteicos do tipo Apple são caracterizados pela capacidade de interação proteína-proteína e proteína-carboidrato, e estão presentes em algumas proteínas micronêmicas com propriedades adesivas. Neste trabalho três proteínas de N. caninum contendo domínios Apple foram caracterizadas: MIC17A, MIC17B e MIC17C. A análise das sequências proteicas e das estruturas dos domínios Apple, obtidas por modelagem molecular, mostraram alta identidade sequencial e estrutural entre MIC17A e MIC17C. Apesar de ser paráloga às outras duas, MIC17B apresenta diferenças importantes em sua sequência e estrutura. Para MIC17B e MIC17C foram realizados experimentos de detecção das proteínas nativas nos extratos total e secretado do taquizoíta que sugerem diferentes formas de processamento entre essas proteínas no parasita. Para MIC17B foi confirmada a localização em micronemas, num padrão diferente do observado para MIC17C. Os ensaios de invasão combinados aos de localização indicam que estas proteínas estejam relacionadas ao processo de invasão celular, porém, suas funções permanecem desconhecidas. O secretoma é o conjunto de proteínas secretadas pelo parasita e, para explorar a composição deste extrato (ESA) no taquizoíta de N. caninum, duas abordagens complementares foram utilizadas. Na primeira abordagem foram identificadas as proteínas presentes no ESA por espectrometria de massas. Na segunda abordagem realizou-se uma ii quantificação relativa das proteínas, marcadas por dois isótopos, nos extratos totais de taquizoítas submetidos ou não ao estímulo secretório. O resultado esperado seria com as proteínas secretadas diminuídas no parasita estimulado. Em ambas as abordagens foram utilizadas técnicas de espectrometria de massas de alta resolução (nanoLC-MS/MS), o que resultou num alto número de identificações; 615 proteínas no ESA e 2011 proteínas quantificadas. A comparação das duas abordagens permitiu o reconhecimento de proteínas com maior probabilidade de secreção. Uma rede de interação entre as proteínas diferencialmente expressas foi predita, gerando resultados que, associados às informações sobre as proteínas aumentadas, permitiram uma investigação sobre proteínas potencialmente envolvidas com a regulação do metabolismo relacionado à secreção. Os resultados obtidos por ambos os estudos aqui demonstrados somam conhecimento acerca do parasita N. caninum e demonstram ser úteis para guiar a busca e seleção de alvos a serem investigados para o desenvolvimento de terapêutica contra a neosporose. / Neospora caninum (Apicomplexa phylum) is an obligatory intracellular parasite like all members from this phylum, some causing diseases with relevant impact on human (Plasmodium and Toxoplasma) and veterinary (Babesia, Eimeria and Cryptosporidium) health. Causative agent of neosporosis, N. caninum has emerged as one of the leading causes of infectious abortion in cattle, generating huge economical losses in worldwide livestock. Due to its recent discovery, knowledge of N. caninum biochemical processes remains scarce, demanding new research for comprehending its survival mechanisms and, consequently, identifying new targets for therapeutic intervention. The invasion process has often been investigated in apicomplexans since their survival depends on the success of their entry into the host cell. Proteins secreted from phylum-specific organelles (micronemes, rhoptries and dense granules) are deeply involved with invasion. They are responsible for the initial interaction with the host cell; participate of the moving junction formed in the moment of invasion; and contribute for the stabilization of the parasitophorus vacuole. In this study, the proteins secreted by N. caninum tachyzoites were investigated in two ways: (1) the molecular characterization of Apple domaincontaining proteins; and (2) exploring the parasite secretome. The Apple protein domains are characterized by the ability to interact as protein-protein and proteincarbohydrate, and are present in some microneme proteins with adhesive properties. Here three N. caninum proteins containing Apple domains were characterized: MIC17A, MIC17B and MIC17C. Analyses of the Apple domains sequences and structures, obtained by molecular modeling, revealed high sequential and structural identities between MIC17A and MIC17C. Although being a paralog of the other two proteins, MIC17B presents significant differences in its sequence and structure. Experiments were performed for native MIC17B and MIC17C detection in the total and secreted tachyzoite extracts, suggesting different processing forms for these proteins in the parasite. For MIC17B, the microneme localization was confirmed, differently from the pattern observed for MIC17C. Invasion and localization assays indicated that these proteins are related to the cell invasion process; nevertheless, their functions remain unknown. The secretome is the set of proteins secreted by the parasite and, to explore this extract (ESA) composition in N. caninum, two complementary approaches were used. Firstly proteins present in ESA were identified by mass spectrometry. In the second approach, a relative quantification was performed on the proteomes of ethanol stimulated/non stimulated tachyzoites, expecting that the secreted proteins would be down regulated at the stimulated parasite. Both approaches were performed with high resolution mass spectrometry techniques (nanoLC-MS/MS), reaching a high number of identifications: 615 proteins iv in ESA and 2011 quantified proteins. The comparison between both approaches allowed the recognition of the most likely secreted proteins. An interaction network was predicted, involving the differentially expressed proteins. These results, associated with the information of up regulated proteins, allowed the investigation of proteins potentially involved with the secretion metabolism regulation. The findings from our two studies add up knowledge about N. caninum and demonstrate to be useful in guiding the search and selection for new targets for therapeutic development against neosporosis.

Diante da abundância de informações e de compartilhamentos em sites de redes sociais e da proliferação de convites para o uso desses sites na Educação, esta tese propõe a pesquisa das estruturas relacionais que emergem das interações sociais em grupos no site de rede social Facebook. O objetivo geral é compreender como as interações sociais em rede e nas redes podem contribuir para uma Educação em Rede a partir da análise dos padrões de interação social em grupos no site Facebook. Analisamos dois casos distintos de redes sociais organizadas como parte de processos pedagógicos. Os recursos curtir, comentar e curtir comentários são os tipos de interação social em rede que deram origem aos grafos analisados. As temáticas das postagens e os tipos de suportes tecnológicos são identificados na análise dos padrões de interação social em rede e nas redes, nas quais destacamos a participação, o engajamento e o sentimento de pertencimento dos atores. A Análise de Redes Sociais é o método utilizado para compreender as dinâmicas das interações. O Modelo de Análise da Comunicação Assíncrona é adotado na análise de conteúdo das postagens e dos comentários dessas postagens com vistas a destacar as temáticas compartilhadas e discutidas. Nas redes sociais em foco, as pessoas se organizam e interagem, prioritariamente, com o professor ou com os atores sociais que assumem as funções de professor. Considerando que os recursos tecnológicos disponíveis permitem fluxo de informações de diferentes fontes, o que observamos é que o professor é a pessoa fundamental para articular as aprendizagens nas redes sociais. A configuração da sala de aula analógica se transfere para os comportamentos e às expectativas do que deve acontecer na sala de aula em redes sociais. Essa dinâmica fornece subsídios para propor orientações à formação de professores e diretrizes para a realização de Educação em Rede. A difusão das informações e a circulação de atores-ponte na rede contribuem para que haja presença social. A presença social é uma peça fundamental para a presença cognitiva. O uso de sites de redes sociais na educação tem valor pedagógico desde que haja presença social endereçada ao ensino. É necessário que um ator ou alguns atores ajam de modo a garantir relações de ensino e aprendizagem. / Faced with the abundance of information and shares on social networking sites and invitations proliferation to the use of these sites in education, this thesis proposes the research of relational structures that emerge from social interactions in groups on the Facebook social networking site. The overall goal is to understand how social interactions network and the networks can contribute to an Education Network based on the analysis of patterns of social interaction in groups on Facebook site. We analyzed two different cases of social networks organized as part of pedagogical processes. Resources Like, Comment and Like Comments are the types of social interaction network that gave rise to the analyzed graphs. The thematic of posts and the types of technological support are identified in the analysis of patterns of social interaction in the network and the networks in which we highlight the participation, engagement and sense of belonging of the actors. The Social Network Analysis is the method used to understand the dynamics of interactions. The Asynchronous Communications Analysis Model is adopted in the content analysis of posts and comments of these posts with a view to highlight the shared and discussed themes. In social networks in focus, people are organized and interact primarily with the teacher or with the social actors who assume the teacher functions. Whereas the available technological resources enable flow of information from different sources, which we observe is that the teacher is the key person to articulate learning in social networks. The configuration of the analog classroom is transferred to the behaviors and expectations of what should happen in the classroom in social networks. This dynamic provides subsidies to propose guidelines for teacher training and guidelines for conducting Education Network. The dissemination of information and the circulation of actors bridge on the network contribute to social presence there. Social presence is a key part of cognitive presence. The use of social networking sites in education has educational value as long as there is social presence addressed to education. It is necessary for an actor or some actors to act in order to ensure teaching and learning relations. / Frente a la abundancia de información y acciones en las redes sociales y la proliferación de las invitaciones a la utilización de estos sitios en la educación, esta tesis propone la investigación de las estructuras relacionales que surgen de las interacciones sociales en grupos en la red social Facebook. El objetivo general es entender cómo las interacciones de la red social y las redes puede contribuir a una red de educación basado en el análisis de los patrones de interacción social en grupos en Facebook. Se analizaron dos casos diferentes de redes sociales organizados en el marco de los procesos pedagógicos. Recursos Me gusta, Comentar y Me Gusta el Comentario son los tipos de red de interacción social que dieron origen a los gráficos analizados. La temática de los puestos y los tipos de apoyo tecnológico son identificados en el análisis de los patrones de interacción social en la red y las redes en las que podemos destacar la participación, el compromiso y el sentido de pertenencia de los actores. El análisis de redes sociales es el método utilizado para entender la dinámica de las interacciones. El Modelo de Análisis de Comunicaciones asíncronas se adopta en el análisis del contenido de los mensajes y los comentarios de estos mensajes con el fin de poner de relieve los temas compartidos y discutidos. En las redes sociales en el enfoque, las personas se organizan e interactúan principalmente con el profesor o con los actores sociales que asumen las funciones de los maestros. Considerando que los recursos tecnológicos disponibles permiten el flujo de información de diferentes fuentes, que observamos es que el maestro es la persona clave para articular el aprendizaje en las redes sociales. La configuración del clase analógica se transfiere a los comportamientos y expectativas de lo que debería ocurrir en el aula en las redes sociales. Esta dinámica proporciona subsidios a proponer directrices para la formación del profesorado y directrices para la realización de la Red de Educación. La difusión de la información y la circulación de los actores puente en la red contribuyen a la presencia social allí. La presencia social es una parte clave de la presencia cognitiva. El uso de las redes sociales en la educación tiene un valor educativo, siempre y cuando no hay presencia social dirigida a la educación. Es necesario que un actor o algunos actores para actuar con el fin de garantizar las relaciones de enseñanza y aprendizaje.