Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Historically studies have focused on either the terrestrial or aquatic environments independently. However, these systems are inherently linked through numerous pathways including organisms with complex life cycles. Both abiotic factors and predators of these organisms can influence connections by changing the number of prey moving across habitat boundaries and by changing the phenotype of prey. When the focal organisms are primary consumers, these effects may have important implications for ecosystem processes. My study investigated how terrestrial predators and abiotic factors affect the number and phenotype of herbivorous tadpole inputs into a tropical forest pond. I found that predators and abiotic factors altered survival and timing of hatching and these effects varied temporally. Thus, temporal changes in the relative importance of these threats from abiotic sources and terrestrial predators on prey with complex life cycles may potentially have implications for connections with and food web dynamics in adjacent ecosystems.

Polyploidization is a key mechanism of rapid speciation, with many phenotypic consequences which extent, however, is poorly understood. A deeper understanding of the evolutionary implications of genome duplication is limited due to lack of knowledge of the links between changes in genome, the phenotype of the individual and environmental constraints. Natural lineages closely related to model species represent the ideal systems for addressing such questions. The thesis is thus focuses on highly promising yet overlooked di-polyploid system within Arabidopsis genus. In the western Carpathians morphologically distinct populations of diploid and tetraploid plants of Arabidopsis arenosa grow along a marked altitudinal gradient. Using high-throughput DNA sequencing, measuring morphological characteristics and collecting ecological data of high alpine and foothill populations I try to reveal main trends of genetic and morphological variability of these populations. Additionaly, using morphometrics of natural and experimentally planted populations we want to test the hypothesis whether morphological divergence of alpine and foothill populations has a genetic basis or is driven by phenotypic plasticity. The presented thesis is an important multidisciplinary combination of genetic research on natural related of model...

L'objectif de cette thèse est d'étudier la croissance et le développement du manguier (Mangifera indica L.) en lien avec un facteur exogène, la température, et plusieurs facteurs endogènes architecturaux de type structurel (topologie) et temporel. Les études sont menées à l'échelle de l'organe (l'axe végétatif, ses feuilles et l'axe florifère) et de la succession des axes (végétatifs ou florifères) sur plusieurs cycles de croissance. À la première échelle, l'étude met en évidence une relation allométrique négative entre la vitesse relative de croissance, positivement reliée à la température, et la durée de croissance de l'organe. Cette relation est commune entre les trois organes et les deux cultivars étudiés. Par ailleurs, des modèles de segmentation montrent que les stades phénologiques classiquement admis sont caractérisés par des vitesses absolues de croissance contrastées. Cette approche met en évidence des asynchronismes entre l'axe végétatif et ses feuilles. À la seconde échelle, l'effet des facteurs architecturaux sur le développement est analysé pour quatre cultivars. Nos résultats montrent de fortes interactions entre certains facteurs structurels (ex. position de l'axe, apicale vs. latérale) ou temporels (ex. date d'apparition), et des caractéristiques développementales qualitatives (ex. occurrence de la floraison), quantitatives (ex. nombre d'inflorescences) ou temporelles (ex. date de floraison). Ces résultats font ressortir une « mémoire de l'effet architectural » qui se propage d'un cycle de croissance aux suivants. Nos études multi-échelles permettent de quantifier les parts respectives des facteurs endogènes et exogènes contribuant aux variations phénotypiques (incluant la plasticité) du manguier. / The aim of this work is to study mango (Mangifera indica L.) growth and development in relation to an exogenous factor, temperature, and to endogenous factors, whether structural (topology) or temporal. The study is carried out at two scales: the organ (vegetative axis, its leaves, and reproductive axis) and the succession of axes (vegetative or reproductive). At the first scale, there was a common negative allometric relationship between the relative growth rate, positively related to temperature, and the duration of growth of the organ. This relationship is common between the three organs and the two studied cultivars. Otherwise, segmentation models reveal that the phenological stages classically studied are characterized by contrasted values of absolute growth rate. This approach shows asynchronisms between the axis and its leaves. At the second scale, the effect of endogenous factors on mango development is investigated for four cultivars. Our results reveal strong interplays between some structural (e.g. axis position, apical vs. lateral) or temporal (e.g. date of burst) factors, and qualitative (e.g. occurrence of flowering), quantitative (e.g. number of inflorescences) or temporal (e.g. date of flowering) developmental traits. These results highlight a “memory of the architectural effect” which spreads from one growing cycle to the followings. Our multiscale study enables to quantify the respective contributions of endogenous and exogenous factors to the phenotypic variations (including plasticity) of mango.

Résumé non transmis / Summary not transmitted

L'expression intracellulaire des anticorps (intracorps) est une approche qui permet l'étude et le ciblage des antigènes dans les compartiments intracellulaires. Néanmoins, l'expression d'anticorps entiers fonctionnels dans les cellules reste une tâche difficile en raison de leur grande taille et de leur structure, l'environnement réducteur du milieu intracellulaire étant défavorable à la formation des ponts disulfure. Notre groupe a une forte expertise dans le domaine de l'immunisation intracellulaire et son application pour l'identification de nouvelles cibles thérapeutiques. Pour cela, notre équipe a élaboré des banques de fragments d'anticorps scFv optimisés pour une meilleure expression intracellulaire. Nos travaux antérieurs ont démontré que ces intracorps peuvent cibler spécifiquement des domaines ou des modifications post-traductionnelles de protéines dans des cellules vivantes. Ceci est particulièrement important car il démontre l'un des avantages principaux des intracorps par rapport à l'approche basée sur l'ARNi. Cet avantage a été démontré par un criblage phénotypique dans un modèle d'allergie. En appliquant cette approche à l'étude de l'activation des mastocytes, nous avons pu identifier un nouvel acteur moléculaire impliqué dans la voie de signalisation mise en jeu. Ce travail a été protégé par un brevet européen en 2013 et est publié récemment. Dans le cadre de mon projet de thèse, j'ai construit une nouvelle banque synthétique (HUSCIv) optimisée pour la stabilité, la diversité et l'affinité des scFvs. Pour cela, le scFv 13R4 isolé dans notre équipe a servi de charpente pour le greffage des différentes boucles hypervariables, tout en respectant la diversité des régions CDR observée dans les anticorps naturels humains. Nous avons utilisé la protéine GFP en tant que rapporteur pour étudier le repliement et la solubilité des intracorps. Nos résultats ont clairement démontré que la plupart des intracorps issus de la banque HUSCIv sont soluble dans le cytoplasme des cellules mammifères. Mon projet de thèse décrit ici rapporte l'utilisation de la banque HUSCIv pour un criblage phénotypique dans des cellules de cancer colorectal portant une mutation du gène K-RAS et résistantes au traitement par l'anticorps chimérique Cetuximab. Le projet cherche à sélectionner des scFv capables de restaurer la sensibilité au Cetuximab, avec comme objectif l'identification des cibles intracellulaires impliquées.Pour ce criblage fonctionnel, la banque HUSCIv a été exprimée dans les cellules HCT116 par l'intermédiaire d'un système d'expression rétroviral. Le processus de sélection est basé sur la sélection directe de la prolifération des cellules en utilisant un colorant fluorescent (CMRA). Les cellules dont la prolifération est bloquée sont isolées et un séquençage à haut débit permet de suivre l'évolution des populations de scFv tout au long de l'expérience. Ainsi, ce projet a nécessité un séquençage profond d'un grand nombre de scFv afin de réaliser une analyse statistique. Nous avons réalisé à ce jour deux tours de sélection. Les tests de cytotoxicité réalisés sur les populations sélectionnées ont montré une inhibition significative de la prolifération en présence du Cetuximab d'environ 10%. Ces résultats indiquent l'évolution du phénotype qui tend vers une sélection de scFv inhibiteurs et suggèrent que nous devons réaliser au moins un ou plusieurs tours plus sélectifs avant de formuler des conclusions.L'approche introduite ici est différente de toutes les études existantes en ce qu'elle utilise des banques « naïves », et permet non seulement de répondre à la diversité du protéome, mais aussi d'étudier les messagers secondaires et le métabolisme des cellules. En tant que tel, et par rapport à d'autres approches à grande échelle, celle-ci représente une voie simple pour la découverte de molécules thérapeutiques potentielles. / Intracellular expression of antibodies (intrabodies) permitted the study and targeting of antigens in cellular compartments. However, the expression of functional intrabodies remains a difficult task due to their large size, structure, and the reducing intracellular environment. Our group has a strong expertise in the field of intracellular immunization and the identification of new therapeutic targets. For this purpose, we have developed an scFv library optimized for intracellular expression of scFv antibody fragments. Our previous works have shown the successful use of intrabodies for targeting specific domains or post-translational modifications in living cells. This is particularly important because it demonstrates one of the main advantages of intrabodies compared to the approaches using RNAi. This benefit was demonstrated by a phenotypic screen in a model of allergy. Applying this approach to the study of mast cell activation, we identified a new molecular player involved in the signaling pathway implemented. This work was protected by a European patent in 2013 and was recently published. As part of my thesis project, I designed a new synthetic library (HUSCIv) optimized for scFv stability, diversity and affinity. For this, a highly soluble and hyper-stable framework, scFv13R4 isolated in our group, was used as a scaffold for grafting different hypervariable loops, while respecting the diversity of CDRs observed in human natural antibodies. We used protein GFP as a reporter to study the folding and solubility of intrabodies. Our findings clearly demonstrated that most of the intrabodies from HUSCIv library are soluble in the cytoplasm of mammalian cells. My thesis project described here reports the use of HUSCIv in a phenotypic screen of colorectal cancer cells carrying a mutation in the K-RAS gene and resistant to the treatment with the chimeric antibody Cetuximab. The project seeks to select scFv fragments able to restore the sensitivity to Cetuximab, with the objective to identify the intracellular targets involved. For this functional screen, the HUSCIv library was expressed in HCT116 cells via a retroviral expression system. The selection process is based on the direct selection of cell proliferation using a fluorescent dye (CMRA). The cells whose proliferation is blocked are isolated and the evolution of scFv populations throughout the experiment are tracked via high-throughput sequencing. This sequencing requires a large number of scFvs to perform a statistical analysis. So far, we have achieved two rounds of selection. The cytotoxicity tests carried out on the selected populations showed a significant inhibition of proliferation (10%) in the presence of Cetuximab. These results indicate that the evolving phenotypes are tending towards a selection of scFv inhibitors and suggest that we need to perform at least one or more selective rounds before making conclusions. The approach introduced here is different from all existing studies in that it uses "naive" libraries not only to respond to the diversity of the proteome, but also to study secondary messengers and metabolism in cells. As such, and in comparison to other large-scale approaches, it is a simple way for the discovery of potential therapeutic molecules.

A doença de Chagas ou tripanossomíase americana é uma doença zoonótica, transmitida pelas fezes do inseto triatomíneo, pertencente à família Reduviidae, subfamília Triatominae. Estima-se que 8 milhões de pessoas estejam infectas por T. cruzi em todo o mundo e 25 milhões de pessoas estejam sob o risco de infecção. Durante a prenhez, a demanda de zinco no organismo é aumentada, razão pela qual as mulheres grávidas são mais suscetíveis a apresentarem déficit deste micronutriente. O zinco é um oligoelemento com função essencial no crescimento, desenvolvimento, diferenciação celular e sistema imunológico. Assim, o objetivo deste estudo foi avaliar a influência do zinco na ativação de diferentes compartimentos do sistema imune em ratas Wistar prenhas infectadas pela cepa Y de T. cruzi. Foram utilizados 4 grupos experimentais: prenhas infectadas sem tratamento (PI), prenhas infectadas tratadas com sulfato de zinco (PIZ), prenhas controles sem tratamento (PC), prenhas controles tratadas com sulfato de zinco (PCZ). Fêmeas do grupo infectado foram acasaladas trinta dias após a infecção e o tratamento com zinco (20 mg/kg/dia) foi efetuado durante 18 dias. Os animais foram eutanasiados no 18° dia da prenhez (48°dia da infecção). Foram avaliados os seguintes parâmetros: população de macrófagos, produção de nitrito e expressão de RT1B (MHC II) no lavado peritoneal; proliferação de linfócitos (CFSE), perfil apoptótico (anexina V e iodeto de propídeo), expressão fenotípica de linfócitos CD161+, CD3+CD161+, TCD3+CD4+, TCD3+CD8+, expressão de CD11a+, CD28+, células apresentadoras de antígenos CD11b/c+, moléculas coestimulatórias, CD80+,CD86+, linfócito B (CD45RA), células T reguladoras TCD3+CD4+CD25+Foxp3high, perfil de memória CD62L/CD44high. Também foram avaliadas citocinas intracelulares produzidas por linfócitos esplênicos (IL-4, IL- 10, IFN-?, TNF-? e quimiocina MCP-1), citocinas do soro (IL- 2, IL- 4, IL- 6, IL- 10, IL- 12, IL- 17, TGF- ?, INF-? e TNF-?), corticosterona plasmática, zinco sérico, e análise molecular de amostras fixadas (coração, placenta e fetos) para detecção de DNA ii genômico de T. cruzi. Nossos resultados mostraram que o tratamento com zinco aumentou as concentrações de nitrito e expressão de RT1B em macrófagos. Por outro lado, observou-se uma diminuição no perfil de proliferação de esplenócitos e no percentual de células em apoptose. Em relação às populações de linfócitos, o tratamento com zinco diminuiu o percentual de células NKT+, TCD4+, TCD8+ e Treg. Foi possível observar ainda, uma diminuição na produção de TNF-? e IFN-?, e aumento de IL-17 e TGF-?. Desta forma, os resultados mostraram que o zinco exerce papel modulador nos diferentes parâmetros imunológicos em ratas prenhas infectadas com T. cruzi / Chagas disease or American trypanosomiasis is a zoonotic disease, transmitted by the feces of the triatomine insect, belonging to the Reduviidae family, Triatominae subfamily. It is estimated that 8 million people worldwide are infected with T. cruzi and 25 million people are under risk of infection. During pregnancy, an increased demand for zinc is observed, reason why pregnant women are more likely to have a deficit in this micronutrient. Zinc is a trace element which plays an essential function during growth, development, cellular differentiation and immune response. Thus, the goal of this study was to evaluate the influence of zinc on the activation of different compartments of the immune system in pregnant Wistar rats infected with T. cruzi Y strain. Four experimental groups were used: infected pregnant without treatment (PI) and infected pregnant treated with zinc sulfate (PIZ), pregnant controls without treatment (PC), pregnant controls treated with zinc sulfate (PCZ). Females from infected group were mated 30 days post infection and treatment with zinc (20mg/kg/day) was performed for 18 days. Animals were euthanized on the 18th day of pregnancy (48th day of infection). The following parameters were evaluated: macrophages subsets, nitrite production and RT1B (MHC II) expression in the peritoneal exudate; Lymphocyte proliferation (CFSE), apoptotic profile (annexin V and propidium iodide), phenotype expression of CD161+ lymphocytes, CD3+ CD161+, TCD3+ CD4+, TCD3+ CD8+, CD11a+, CD28+, CD11b/c+, CD45RA, TCD3+ CD4+ CD25+ Foxp3high regulatory T cells, CD62L/CD44high memory profile. Intracellular cytokines produced by splenic lymphocytes (IL-4, IL-10, IFN-?, TNF-? and MCP- 1 chemokine), serum cytokines (IL-2, IL-4, IL- 10, IL-12, IL-17, TGF-?, INF-? and TNF-?), plasma corticosterone, serum zinc, and molecular analysis of fixed samples (heart, placenta and fetuses) for detection of T. cruzi genomic DNA. Our results demonstrated that zinc treatment increased nitrite concentrations and RT1B expression in macrophages. On the other hand, there was a decrease in splenocytes proliferation and in the percentage of apoptosis. Furthermore, zinc therapy decreased the percentage of NKT+, TCD4+, TCD8+ and Treg cells. Besides, zinc treatment decreases TNF-? and IFN-? and increase IL-17 and TGF-? production. Indeed, our results demonstrated that actually zinc exerts a modulatory role on the different immune responses as well as cellular subsets from T. cruzi infected and pregnant rats.

When an animal’s pigmentation matches the background across various types of environments, it is potentially an example of cryptic pigmentation, most likely as a response to natural selection by visually oriented predators. One example of cryptic pigmentation is phenotypic plasticity, meaning that an organism can exhibit different phenotypes in different environments. The freshwater isopod Asellus aquaticusliving in stands of reeds tends to have darker pigmentation than individuals living amongst lighter-coloured stoneworts, which has been suggested to result from visual predation. A recent study showed, however, that pigmentation in A. aquaticus is partly plastic, influenced by the nutritional composition in their diet. Here, I performed a laboratory experiment on A. aquaticusto see if the nutritional composition in stoneworts decreases pigmentation. Isopods were provided with a diet of either decaying leaves or stoneworts. The experiment took place over four weeks and pigmentation and growth were analysed at 0, 15 and 31 days. I found that pigmentation in A. aquaticusincreased significantly on both diets. And, there was no difference between both diets in amount of change in pigmentation. The fact that isopods that were feeding on stoneworts did not become lighter to match their background colour preferably depend on a high nutritional composition in the provided food, considering they also more than doubled their weight. In other words, phenotypic plasticity due to different diets between habitats is not the explanation to lighter coloured isopods living amongst stoneworts. However, these results do not exclude that differences can arise over a longer time or differs between different species of stoneworts.

Os hormônios sexuais desempenham um importante papel no metabolismo ósseo. Além disso, polimorfismos genéticos em genes expressos durante o desenvolvimento craniofacial estão associados com alterações dimensionais na maxila e na mandíbula. Desta forma, o objetivo deste trabalho foi avaliar, por meio de modelo animal e de genética humana, o papel do estrógeno e dos polimorfismos genéticos nos seus receptores (ERα e ERβ ), codificados pelos genes ESR1 e ESR2, nas alterações dimensionais da maxila e da mandíbula. Um total de 40 ratas Wistar foram divididas em 2 grupos: Ovariectomizadas (para estimular deficiência de estrógeno) e Sham. Foram realizadas 3 tomadas radiográficas da região craniofacial das ratas, sendo a primeira aos 21 dias, a segunda aos 45 dias e a última aos 63 dias de vida. Medidas cefalométricas da maxila e da mandíbula foram obtidas para avaliação das alterações verticais e sagitais. Após a eutanásia, peças anatômicas das regiões de interesse foram utilizadas para realização de imuno-histoquímica e PCR em tempo real para os genes ESR1 e ESR2. Paralelamente, na área da genética humana, um total de 143 amostras de DNA genômico e dados relativos à análise cefalométrica destes indivíduos foram avaliados quanto aos polimorfismos genéticos nos genes de interesse pelo método de discriminação alélica por PCR em tempo real. Os resultados foram agrupados de acordo com os diferentes testes; as variáveis contínuas foram avaliadas com testes paramétricos e/ou não paramétricos para comparação das médias entre os grupos. Os testes do qui-quadrado e exato de Fisher foram usados para avaliar a associação do padrão esquelético facial com as distribuições genotípica e alélica na população humana (alfa de 5%). No modelo animal, foi possível observar um aumento nas dimensões maxilares e mandibulares do grupo sob condição de deficiência de estrógeno (p0,05), onde não houve diferença na expressão do RNAm de ambos os genes (p>0,05). Para mais, a presença de ERβ foi confirmada nos sítios de crescimento mandibulares de ambos os grupos. Na população humana, foi possível identificar associação estatisticamente significante entre medidas cefalométricas e polimorfismos em ESR1 e ESR2 (p0,05). Desta forma, conclui-se que o estrógeno tem um papel importante no crescimento e desenvolvimento da maxila e da mandíbula em modelo animal e que polimorfismos genéticos nos seus receptores estão associados com o padrão esquelético facial / Sex hormones play an important role on bone metabolism. In addition, genetic polymorphisms in genes expressed during craniofacial development are associated with dimensional changes in maxilla and mandible. Thus, the aim of this study was to evaluate, using animal models and human genetics, the role of estrogen and genetic polymorphisms in its receptors (ERα and ERβ ), codified by ESR1 and ESR2, in the dimensional alterations of the maxilla and the mandible. A total of 40 Wistar rats were divided into 2 groups: Ovariectomized (to stimulate estrogen deficiency) and Sham. Radiographic exams of rats craniofacial region were performed, the first at 21 days, the second at 45 days and the last at 90 days old. Cephalometric measurements of maxilla and mandible were obtained to evaluate the vertical and sagittal skeletal alterations. After euthanasia, anatomical parts of interesting regions were used to performed immunohistochemistry and real time PCR for ESR1 and ESR2. In parallel, a total of 143 genomic DNA samples and cephalometric data of these individuals were evaluated for genetic polymorphisms by real time PCR (allele discrimination method). The results were grouped according to the different tests; continuous variables were evaluated with parametric and/or non-parametric test to compare means between groups. Chi-square and Fishers exact tests were used to evaluate the association of facial skeletal pattern with genotypic and allelic distributions in the human population (5% alpha). In the animal model, it was possible to detect an increase in the maxillary and mandibular dimensions of ovariectomized group (p0,05). There was no difference in the mRNA expression of both genes (p>0.05). Moreover, the ERβ presence was confirmed at mandibles growth sites of both groups. In the human population, it was possible to identify statistically significant association in cephalometric measures and polymorphisms in ESR1 and ESR2 (p0.05). Therefore, in conclusion, estrogen has an important role in the mandible and maxilla growth and development in the animal model, and genetic polymorphisms in estrogen receptors are associated with facial skeletal patterns

Le processus de biominéralisation confère aux organismes qui le développent une valeur adaptative. La coquille carbonatée des mollusques intègre les fonctions de protection biomécanique à différentes échelles. La coquille résulte de l'association de composés inorganiques et d'une matrice organique protéique, médiatrice du contrôle biologique de la minéralisation. L'analyse du protéome de la coquille chez 4 espèces de bivalves met en évidence deux patrons fonctionnels et leur degré de conservation phylogénétique : l'un lié au contrôle de la minéralisation stricto sensu ; l'autre à la protection immune. L'étude de populations vivant à l'état naturel en mer Baltique, dont les eaux présentent localement de fortes variations ioniques montre que le protéome intègre également l'impact de conditions environnementales limitantes. L'anthropocène impose un rythme adaptatif pressant aux organismes et la modification acido-basique des eaux océaniques est susceptible d'impacter sensiblement les organismes calcifiants. La signature de mécanismes adaptatifs du contrôle biologique de la biominéralisation se traduit dans le protéome de la coquille. Les implications sont particulièrement signifiantes dans un contexte d'intérêt de développement aquacole grandissant. / In this study, the SMPs from four commercially important and divergent bivalve species crassostrea gigas (pacific oyster), Mya truncata (soft shell clam), Mytilus edulis (blue mussel) and Pecten maximus (king scallop) were extracted and analysed using standardized extraction protocol and proteomic pipeline. This enables us to identify critical elements of basic biomineralization tool kit for calcification process irrespective of their shell morphology, mineralogy and microstructure. In addition, it enables the identification of SMPs that are specific to calcite and aragonite mineralogies. The signifiant numbers of SMPs found species-specific were hypothesized as adaptation to their modus vivendi. In fact, the latter proteins possess immunity-related functions and fit into specific pathway, phenoloxidase, suggesting their role in defense against pathogen. The comparative study of shell proteome of mussels living in full marine condition, North Sea and the Iow saline Baltic Sea showed the modulation of the SMPs that constitute the basic biomineralization tool kit. Higher modulation of chitin related proteins and non-modulated protein such as carbonic anhydrase, EGF and fibronectin domain containing proteins points out the impaired scaffold and mineral nucleation process in Baltic mussel. The modulation of immunity related proteins denote the influence of biotic components. These investigations show the functional diversity of SMPs and their roles beyond shell formation in the bivalvesand put forth the idea that shell is dynamic, endowed with both biochemical and mechanical protection.