Major mental illness is now recognised as one of the leading causes of adult morbidity. Of the adult onset psychiatric disorders, the functional psychoses (schizophrenia, bipolar disorder and recurrent major depression) are the most severe and most common in the general population. Evidence suggests that certain genetic factors influence an individual’s susceptibility to developing these disorders when combined with appropriate social and environmental conditions. Several good candidate genes have been identified. Of relevance to this study is Disrupted in Schizophrenia 1 (DISC1) which was identified in a large Scottish family that carried a balanced translocation (t1:11) and had a history of major mental illness. In 2008, two ENU mutant mouse models with missense mutations in exon 2 of Disc1 were characterised and found to have behavioural and neuroanatomical phenotypes consistent with schizophrenia and major depression. The primary aim of this thesis is to further analyse these mouse models by performing whole genome gene expression studies and secondary protein analysis to identify genes involved in the aetiology of schizophrenia and major depression. My initial analysis used Illumina BeadChip microarray technology to identify 368 genes that were differentially expressed in ENU mutant animals under different biological conditions, compared to appropriate control animals. Nine biological groups were compared including one embryonic group at E13, and three groups treated with appropriate anti-psychotic or anti-depressant drugs. Of the 368 genes identified as differentially expressed, 46 were chosen for validation by qRT-PCR based on fold-change, p-value, functional significance, overenrichment of GO terms, pathway analysis and previous implications in major mental illness. NRXN1, NRXN3 and CDH11 were found to be significantly up-regulated in the schizophrenia mouse model with EGR4 significantly down-regulated compared to C57BL/6J wild-type controls. These findings were also replicated in an independent sample using wildtype littermates. The mental retardation gene PAK3 was up-regulated in the schizophrenia mouse model and expression levels were corrected to a level not significantly different to wild-type, when treated with the PDE4 inhibitor Rolipram. Semi-quantitative western blotting also confirmed the disregulation of EGR4 and PAK3 at the protein level in these animals. RNA expression profiles were also characterised for each of the genes above, and DISC1, through development. In summary this thesis describes the striking disregulation of four prominent genetic candidates of major mental illness in an independent animal model. A first functional link between DISC1 and NRXN1 is described suggesting, for the first time, a DISC1- dependant mechanism for regulating neurexin gene expression.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

No description available.

La reconnaissance d'antigènes par le TCR active des protéines tyrosine kinases qui phosphorylent d'autres substrats intracellulaires dont LAT. Ceci engendre l'activation de molécules telles que PKCθ et CARMA-1. La mutation LatY136F associe des TCR "estropiés" dans le développement de cellules T effectrices générant des désordres lymphoprolifératifs. Nous avons essayé de comprendre les gènes aggravant ou empêchant cette lymphoprolifération en utilisant la mutagénèse ENU. Nous avons identifié une mutation appelée Basilic empêchant le déroulement de la pathologie LatY136F. Basilic est une mutation du gène Rltpr qui constitue une phénocopie de Cd28-/- sur fond sauvage et sur fond LatY136F. Rltpr est un une nouvelle protéine ayant de multiples domaines, qui appartient à la famille CARMIL et qui est exprimée dans les cellules T et B. L'objectif de ce travail était d'élucider les mécanismes au cours desquels CD28 et Rltpr coopèrent avec le TCR pour différencier des cellules T naïves en cellules T effectrices. Ce travail visait aussi à caractériser Rltpr, dont la structure/fonction et l'interactome sont encore inconnus. En utilisant des techniques de microscopie confocale, nous avons montré que la localisation et le recrutement de Rltpr et de RltprBas à la synapse immunologique sont tous deux CD28-dépendants. Les deux molécules colocalisent avec CD28 tout au long du processus d'activation. En outre, Rltpr est essentiel pour la translocation à la synapse de PKCθ et CARMA-1, qui sont induits lors de la co-stimulation par CD28. Ces résultats permettent une meilleure compréhension du fin réglage du système immunitaire adaptatif qui est mis en place lors de l'activation. / TCR recognition of antigens triggers the activation of protein tyrosine kinases that phosphorylate other intracellular substrates including LAT. LAT phosphorylation leads to the activation of PKCθ and CARMA-1. The point mutation LatY136F associates TCRs with crippled signaling abilities to the development of effector T cells generating lymphoproliferative disorders (LPDs). We tried to shed light on genes exacerbating or preventing the LatY136F LPD by using an ENU mutagenesis screening. We identified one point mutation called Basilic that prevents the unfolding of the LatY136F pathology. Basilic is a point mutation of the Rltpr gene and is a phenocopy of a Cd28-/- mutation both on a wild-type and on a LatY136F background. Rltpr is a newly-discovered, multidomain protein belonging to the CARMIL family that is expressed in T and B cells. The objective of the present work was to elucidate the mechanisms during which CD28 and Rltpr cooperate withthe TCR to differentiate naïve into effector T cells. I also aimed at characterizing the Rltpr molecule, whosestructure/function and interactome are still largely unknown. Using confocal microscopy in collaborationwith Takashi Saito's group and Christoph Wülfing we showed that the localization and the recruitment ofboth Rltpr and RltprBas at the immune synapse are CD28-dependent. The two molecules colocalize with CD28all along the activation process. Moreover, Rltpr is essential for the synapse translocation of PKCθ andCARMA-1, which are induced upon CD28 costimulation. Those results allow a better understanding of theadaptive immune system fine tuning upon activation.

Orientador: Ana Cláudia Ruggieri / Resumo: O setor agropecuário consiste em um pilar da economia nacional, no entanto está entre os principais responsáveis pelas emissões de gases de efeito estufa (GEE) no mundo, contribuindo com 38% de metano (CH4) proveniente da fermentação entérica. Frente a essa situação, o desafio no sistema produtivo de ruminantes é desenvolver dietas e estratégias de manejo que minimizem a produção de CH4, de modo a reduzir os impactos do aquecimento global e aumentar a eficiência produtiva dos pastos e dos animais. O estudo teve como objetivo avaliar a produção de massa, a composição química, o consumo, a digestibilidade, o balanço de nitrogênio (N) e a emissão de CH4 entérico por novilhas leiteiras mestiças mantidas em pastos de capim-marandu, fertilizados com N ou em consórcio com amendoim forrageiro. O experimento foi conduzido em delineamento inteiramente casualizado, com 3 tratamentos e 7 repetições para as variáveis relacionadas ao animal e 4 para as variáveis relacionadas à forragem. Os tratamentos constaram de pastos de capim-marandu sem fonte nitrogenada (controle), fertilizados com ureia (adubado) e em consórcio com amendoim forrageiro (consorciado). Os dados foram coletados durante os períodos experimentais da estação chuvosa de dois anos de avaliação (2016/2017 e 2017/2018). O consórcio de capim-marandu com amendoim forrageiro e a adubação nitrogenada em pastos de capim-marandu não mitigaram a emissão de CH4 entérico por novilhas mestiças. A adubação nitrogenada aumentou o valor nu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The agricultural sector is a pillar of the Brazilian economy, but it also serves as one of the main causes of emissions of greenhouse gases (GHG) in the world, contributing 38% of methane (CH4) from enteric fermentation. Faced with this situation, the challenge in the ruminant production system is to develop diets and management strategies that minimize CH4 production in order to reduce the impacts of global warming and increase the productive efficiency of pastures and animals. The objective of the study was to evaluate the forage mass production, chemical composition, intake, digestibility, balance of nitrogen (N) and enteric CH4 emission by crossbred dairy heifers kept in marandu grass pastures fertilized with N or mixed with forage peanut. The experiment was conducted in a completely randomized design with three treatments containing seven replications for variables related to the animals and four replications for variables related to the forage. The treatments consisted of pastures of marandu grass without nitrogen source (control), fertilized with urea (fertilized), and marandu grass mixed with forage peanut (mixed). Data were collected during two experimental periods consisting of the 2016/2017 and the 2017/2018 rainy seasons. The mixed and fertilized pastures did not mitigate enteric CH4 emission in crossbred heifers. Nitrogen fertilization did however increase the forage nutritive value as shown by increased values of crude protein, and B1 + B2 protein fraction, and ... (Complete abstract click electronic access below) / Mestre

No description available.

Les anomalies du tube neural (ATN) sont des malformations congénitales très fréquentes chez l’humain en touchant 1-2 nouveau-nés sur 1000 naissances. Elles résultent d’une fermeture incomplète du tube neural lors de l’embryogenèse. L’étiologie des ATN est complexe impliquant des facteurs environnementaux et des facteurs génétiques. La souris représente un outil puissant afin de mieux comprendre la génétique des ATN. Particulièrement, la souris modèle a impliqué fortement la voie de la polarité cellulaire planaire (PCP) dans ces malformations. Dans cette étude, nous avons identifié et caractérisé une nouvelle souris mutante, Skam26Jus dans le but d’identifier un nouveau gène causant les ATN. Skam26Jus a été générée par l’agent mutagène N-Ethyl-N-Nitrosuera. Cette souris est caractérisée par une queue en forme de boucle ou de crochet, soit un phénotype associé aux ATN. La complémentation génétique de la souris Skam26Jus avec une souris mutante d’un gène de la voie PCP Vangl2 (Looptail) a montré une interaction génétique entre le gène muté chez Skam26Jus et Vangl2, suggérant que ces deux gènes fonctionnent dans des voies de signalisation semblables ou parallèles. Un total de 50% des embryons doubles hétérozygotes avec un phénotype de la queue présentent un spina bifida. La cartographie par homozygotie du génome entier suivie par un clonage positionnel a permis d’identifier Lrp6 comme le gène muté chez Skam26Jus. Une mutation homozygote, p.Ile681Arg, a été identifiée dans Lrp6 chez les souris ayant une queue en boucle/crochet. Cette mutation était absente dans 30 souches génétiques pures indiquant que cette mutation est spécifique au phénotype observé. Une étude de phénotype-génotype évalue la pénétrance à 53 % de la mutation Ile681Arg. Lrp6 est connu pour activer la voie canonique Wnt/β-caténine et inhiber la voie non canonique Wnt/PCP. Le séquençage de la région codante et de la jonction exon-intron de LRP6 chez 268 patients a mené à l’identification de quatre nouvelles rares mutations faux sens absentes chez 272 contrôles et de toutes les bases de données publiques. Ces mutations sont p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile; p.Tyr1541Cys et leur pathogénicité prédite in silico indiquent que p.Val1386Ile est bénigne, et que p.Tyr306Hiset p.Tyr373Cys et p.Tyr1541Cys sont i possiblement dommageables. Les mutations p.Tyr306His, p.Tyr373Cys et p.Tyr1541Cys ont affecté l’habilité de LRP6 d’activer la voie Wnt/β-caténine en utilisant le système rapporteur luciférase de pTOPflash. Nos résultats suggèrent que LRP6 joue un rôle dans le développement des ATN chez une petite fraction de patients ayant une ATN. Cette étude présente aussi Skam26Jus comme un nouveau modèle pour étudier les ATN chez l’humain et fournit un outil important pour comprendre les mécanismes moléculaires à l’origine des A TN. / Neural tube defects (NTDs) are among the most common congenital malformations in humans affecting 1–2 infants per 1000 births. NTDs are caused by failure of the neural tube to close during embryogenesis. The most common forms of NTDs in humans are anencephaly and spina bifida. Their etiology is complex implicating both environmental and genetic factors. The mouse model represents a powerful tool to investigate the genetics of NTDs. Particularly, mouse mutants at genes belonging to the planar polarity pathway (PCP) developed severe forms of NTDs strongly implicating this pathway in the pathogenesis of NTDs. In this study, we identified and characterized a novel mouse mutant, Skam26Jus, as a model for NTDs. Skam26Jus was generated by N-Ethyl-N-Nitrosuera mutagenesis and displayed a characteristic kinky or loop tail that is considered as the minimal sign if NTDs. Complementation of Skam26Jus mutant with a PCP mouse mutant called Looptail (Lp) showed a genetic interaction between Skam26Jus and Vangl2, the gene mutated in Lp. This led to spina bifida in 50% of double heterozygotes with a kinky or looptail phenotype. Homozygosity mapping followed by a positional candidate gene approach led to the identification of Lrp6 as the gene mutated in Skam26Jus. We detected a homozygous mutation, p.Ile681Arg, in Lrp6 in Skam26Jus mice having loop/kinky tail phenotype. This mutation was absent in 30 inbred strains analyzed indicating that it is disease specific. Genotype-phenotype studies indicated a 52 % penetrance of the p.Ile681Arg mutation. Lrp6 is known to activate Wnt canonical β-catenin pathway and inhibit Wnt non canonical PCP pathway. Sequencing analysis of the open reading frame and exon-intron junctions of human LRP6 in 268 NTD patients led to the identification of 4 novel rare missense mutations that were absent in 272 controls analyzed and in all public databases. These mutations were p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile ; p.Tyr1541Cys, and of these, p.Val1386Iso was predicted to be benign, and p.Tyr306His ; p.Tyr373Cys and p.Tyr1541Cys were predicted to be possibly pathogenic using bioinformatics tools. Functional validation of these mutations with the luciferase reporter system pTOPflash assay demonstrated that mutation p.Tyr306His, p.Tyr373Cys and iii p.Tyr1541Cys reduced the ability of LRP6 to activate the Wnt canonical β-catenin pathway. Our data suggest that LRP6 could play a role in the development of NTDs in a small fraction of NTD patients. Our study also presents Skam26Jus as a new mouse model for the study of human NTDs and provides an important tool for better understanding of the molecular pathogenic mechanisms underlying NTDs.

Les anomalies du tube neural (ATN) sont des malformations congénitales très fréquentes chez l’humain en touchant 1-2 nouveau-nés sur 1000 naissances. Elles résultent d’une fermeture incomplète du tube neural lors de l’embryogenèse. L’étiologie des ATN est complexe impliquant des facteurs environnementaux et des facteurs génétiques. La souris représente un outil puissant afin de mieux comprendre la génétique des ATN. Particulièrement, la souris modèle a impliqué fortement la voie de la polarité cellulaire planaire (PCP) dans ces malformations. Dans cette étude, nous avons identifié et caractérisé une nouvelle souris mutante, Skam26Jus dans le but d’identifier un nouveau gène causant les ATN. Skam26Jus a été générée par l’agent mutagène N-Ethyl-N-Nitrosuera. Cette souris est caractérisée par une queue en forme de boucle ou de crochet, soit un phénotype associé aux ATN. La complémentation génétique de la souris Skam26Jus avec une souris mutante d’un gène de la voie PCP Vangl2 (Looptail) a montré une interaction génétique entre le gène muté chez Skam26Jus et Vangl2, suggérant que ces deux gènes fonctionnent dans des voies de signalisation semblables ou parallèles. Un total de 50% des embryons doubles hétérozygotes avec un phénotype de la queue présentent un spina bifida. La cartographie par homozygotie du génome entier suivie par un clonage positionnel a permis d’identifier Lrp6 comme le gène muté chez Skam26Jus. Une mutation homozygote, p.Ile681Arg, a été identifiée dans Lrp6 chez les souris ayant une queue en boucle/crochet. Cette mutation était absente dans 30 souches génétiques pures indiquant que cette mutation est spécifique au phénotype observé. Une étude de phénotype-génotype évalue la pénétrance à 53 % de la mutation Ile681Arg. Lrp6 est connu pour activer la voie canonique Wnt/β-caténine et inhiber la voie non canonique Wnt/PCP. Le séquençage de la région codante et de la jonction exon-intron de LRP6 chez 268 patients a mené à l’identification de quatre nouvelles rares mutations faux sens absentes chez 272 contrôles et de toutes les bases de données publiques. Ces mutations sont p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile; p.Tyr1541Cys et leur pathogénicité prédite in silico indiquent que p.Val1386Ile est bénigne, et que p.Tyr306Hiset p.Tyr373Cys et p.Tyr1541Cys sont i possiblement dommageables. Les mutations p.Tyr306His, p.Tyr373Cys et p.Tyr1541Cys ont affecté l’habilité de LRP6 d’activer la voie Wnt/β-caténine en utilisant le système rapporteur luciférase de pTOPflash. Nos résultats suggèrent que LRP6 joue un rôle dans le développement des ATN chez une petite fraction de patients ayant une ATN. Cette étude présente aussi Skam26Jus comme un nouveau modèle pour étudier les ATN chez l’humain et fournit un outil important pour comprendre les mécanismes moléculaires à l’origine des A TN. / Neural tube defects (NTDs) are among the most common congenital malformations in humans affecting 1–2 infants per 1000 births. NTDs are caused by failure of the neural tube to close during embryogenesis. The most common forms of NTDs in humans are anencephaly and spina bifida. Their etiology is complex implicating both environmental and genetic factors. The mouse model represents a powerful tool to investigate the genetics of NTDs. Particularly, mouse mutants at genes belonging to the planar polarity pathway (PCP) developed severe forms of NTDs strongly implicating this pathway in the pathogenesis of NTDs. In this study, we identified and characterized a novel mouse mutant, Skam26Jus, as a model for NTDs. Skam26Jus was generated by N-Ethyl-N-Nitrosuera mutagenesis and displayed a characteristic kinky or loop tail that is considered as the minimal sign if NTDs. Complementation of Skam26Jus mutant with a PCP mouse mutant called Looptail (Lp) showed a genetic interaction between Skam26Jus and Vangl2, the gene mutated in Lp. This led to spina bifida in 50% of double heterozygotes with a kinky or looptail phenotype. Homozygosity mapping followed by a positional candidate gene approach led to the identification of Lrp6 as the gene mutated in Skam26Jus. We detected a homozygous mutation, p.Ile681Arg, in Lrp6 in Skam26Jus mice having loop/kinky tail phenotype. This mutation was absent in 30 inbred strains analyzed indicating that it is disease specific. Genotype-phenotype studies indicated a 52 % penetrance of the p.Ile681Arg mutation. Lrp6 is known to activate Wnt canonical β-catenin pathway and inhibit Wnt non canonical PCP pathway. Sequencing analysis of the open reading frame and exon-intron junctions of human LRP6 in 268 NTD patients led to the identification of 4 novel rare missense mutations that were absent in 272 controls analyzed and in all public databases. These mutations were p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile ; p.Tyr1541Cys, and of these, p.Val1386Iso was predicted to be benign, and p.Tyr306His ; p.Tyr373Cys and p.Tyr1541Cys were predicted to be possibly pathogenic using bioinformatics tools. Functional validation of these mutations with the luciferase reporter system pTOPflash assay demonstrated that mutation p.Tyr306His, p.Tyr373Cys and iii p.Tyr1541Cys reduced the ability of LRP6 to activate the Wnt canonical β-catenin pathway. Our data suggest that LRP6 could play a role in the development of NTDs in a small fraction of NTD patients. Our study also presents Skam26Jus as a new mouse model for the study of human NTDs and provides an important tool for better understanding of the molecular pathogenic mechanisms underlying NTDs.

La mutagenèse chimique aléatoire par l’Ethyl-Nitroso-Urée (ENU), dont la puissance a été largement démontrée au cours de ces dernières décennies dans la création de modèles murins constitue un outil remarquable et essentiel en génomique fonctionnelle. Cette approche est en effet d’un apport considérable pour la compréhension de la fonction des gènes et de leur régulation et constitue un accélérateur pour identifier des éléments clés dans une voie de signalisation. Cette approche systématique, basée sur le criblage simultané d’un grand nombre de souris ne nécessite aucune connaissance préalable sur l’identité et la fonction des gènes étudiés. Les mutants sont identifiés au travers de cribles phénotypiques spécifiques, hiérarchisés et non-invasifs. L’identification du gène et de la mutation causale responsable du phénotype sont réalisés par un travail de cartographie génique en utilisant une série de marqueurs polymorphes ou par séquençage nouvelle génération. L’objectif de ma thèse a été de caractériser au niveau phénotypique et moléculaire, 3 lignées murines indépendantes issues de deux cribles ENU, un crible récessif et un crible dominant sensibilisé. Le premier crible visait à développer des modèles de pathologies humaines, à partir duquel a été isolée la lignée vdb, qui présente des défauts du système vestibulaire dus à une mutation dans le gène otog codant pour l’otogeline, et constitue un modèle de surdité chez l’homme. Le deuxième crible avait été mis en place dans le but d’approfondir nos connaissances fondamentales sur le développement du système squeletto-musculaire chez les mammifères, plus particulièrement chez l’homme et le bovin. Les analyses de deux lignées issues de ce crible, GMA24 et GMA06, ont permis d’identifier la mutation dans le gène Phex pour les souris GMA24 qui présentent un retard de croissance et constituent un modèle pour la maladie XLH (X-Linked Hypophosphatemic Rickets) chez l’homme. Pour les souris GMA06 présentant une hypermusculature, la mutation a été localisée sur le chromosome 2 et son identification est en cours. Ces trois modèles murins constituent des outils intéressants qui peuvent s’ajouter aux modèles déjà existants pour la surdité, la maladie XLH et les maladies type myopathies pour mieux appréhender les mécanismes moléculaires impliqués dans ces pathologies et les interactions génétiques mises en jeu dans l’objectif de tester de nouvelles thérapies. / The random chemical mutagenesis with the Ethyl-Nitroso-Urea (ENU), whose power has been widely demonstrated during these last decades in the murine models creation, is a remarkable and essential tool in functional genomics. This approach is indeed a significant contribution to the understanding of the genes’ function and regulation; it also establishes an accelerator to identify the key elements in a signaling pathway. This systematic approach, based on the simultaneous screening of a large number of mice, requires no prior knowledge on the identity and the function of the studied genes. The mutants are identified through specific, hierarchical and non-invasive phenotypic screens. The identification of the gene and the causal mutation responsible for the mutant phenotype are achieved by gene mapping by using a series of polymorphic markers or by new generation sequencing. The objective of my thesis has been to characterize at the phenotypic and molecular level 3 independent murine lines from two ENU screens, a recessive and a sensitized dominant one. The first screen aimed to develop models for human diseases, from which has been isolated the vbd murine line, presenting vestibular system defects due to a mutation in the Otog gene coding for the otogelin. This mouse line presents a model for human deafness. The second screen had been established in order to deepen our fundamental knowledge on the skeletto-muscular system development in mammals, more particularly in humans and cattle. Analyzes of two lines from this screen, GMA24 and GMA06, have allowed to identify the mutation in Phex gene for GMA24 mice showing a growth retardation and modeling XLH (X-linked Hypophosphatemic rickets) disease in humans. For the GMA06 mice presenting an increase muscle mass, the mutation has been localized on chromosome 2 and its identification is in progress. These three murine models are interesting added tools to the existing models for deafness, XLH and myopathies diseases for a better understanding of the molecular mechanisms and genetic interactions involved in these pathologies and so testing new therapies.