Background Primary brain tumors remain the most intractable form of childhood cancer. Pediatric glioblastomas (pGBM), WHO grade IV astrocytomas, are rare but devastating neoplasms in children, accounting for 15% of all brain tumors and yielding a dismal three-year survival of less than 20%, despite aggressive treatments. While considerable information is available on adult GBM (aGBM), substantially fewer genetic/molecular data exist for pGBM. We previously established that pGBM are molecularly distinct from aGBM and showed that overexpression of Y-Box binding protein 1 (YB-1) may help drive oncogenesis in this tumor. YB-1 is broadly expressed throughout development and is mandated for late embryonic development. Its expression level correlates with the cell proliferation state and is turned down in quiescent cells including in normal brain. This protein is a major structural component of messenger ribonucleoprotein complexes in the cytoplasm where it acts as a translational regulator. Several studies proposed that Akt-dependent phosphorylation of YB-1 at Ser102 may induce nuclear translocation in the presence of functional p53 and the transcription of genes including EGFR. Nuclear YB-1 in turn interacts with p53 inhibiting p53-induced cell death and has been involved in the oncogenesis of several epithelial cancers but our study is the first that associates it with brain cancer. We hypothesize that dysregulated YB-1 expression is driving oncogenesis in a subset of pGBM. The objectives of this thesis are to further define the role of YB-1 dysregulated expression in pGBM oncogenesis in vitro and in vivo. Methods (i) We stably overexpressed HA-tagged wild type (WT) and mutant S102A YB-1 (preventing nuclear shuttling) in pGBM (SF188) and aGBM (U251) cell lines. In parallel, we stably knocked-down YB-1 expression using shRNA in these cell lines. We investigated the effects of overexpression and silencing of YB-1 on cell signalling (Ras and Akt pathways) and EGFR lev / Les tumeurs cérébrales primaires demeurent la forme de cancer la plus incurable de l'enfance. Les glioblastomes pédiatriques (pGBM), astrocytes de grade IV selon l'OMS, sont des néoplasmes rares mais dévastateurs. Ils représentent 15% de toutes les tumeurs cérébrales et ont un taux de survie à 3 ans de seulement 20%. Contrairement aux GBM de l'adulte (aGBM), peu de données sont disponibles concernant les pGBM. Nous avons précédemment établi que les pGBM sont distincts du point de vue moléculaire des aGBM et avons montré que la surexpression de Y-Box binding protein 1 (YB-1) pourrait contribuer à leur oncogénèse. YB-1 est largement exprimée au cours du développement, son expression est requise pour le développement tardif de l'embryon. Son niveau d'expression est corrélé à l'état de prolifération cellulaire, s'éteignant dans les cellules au repos. Cette protéine est un composant structural majeur des complexes ribonucléoprotéiques messagers dans le cytoplasme où elle agit comme régulateur de transcription. Plusieurs études ont proposé que la phosphorylation de YB-1 sur le résidu sérine 102, dépendante d'Akt, pourrait induire la translocation nucléaire de YB-1, en présence d'un p53 fonctionnel, et la transcription de différents gènes, dont EGFR. YB-1 nucléaire interagit avec p53, inhibant la mort cellulaire induite par p53, et a été impliquée dans l'oncogénèse de plusieurs cancers épitheliaux; toutefois notre étude est la première à associer cette protéine et les GBM. Notre hypothèse est que la dérégulation de l'expression de YB-1 actionne l'oncogénèse dans un sous-ensemble de pGBM. Les objectifs de mon mémoire sont de mieux définir le rôle de la dérégulation de l'expression de YB-1 dans l'oncogénèse des pGBM in vitro et in vivo. Pour cela, nous avons (i) surexprimé de facon stable la protéine YB-1 wild type et mutée S102A (mutation empêchant la translocation nucléaire de YB-1) dans des lignées c

We are interested in genes that regulate lung development in late gestation. Using a probe identified by differential display-PCR, we cloned a 3.1 kb cDNA LGL1 (AF109674) encoding a deduced polypeptide of 480 amino acids. Northern blot analysis showed that LGL1 is induced by glucocorticoid and is expressed in fetal lung adjacent fibroblasts but not in the epithelium. LGL1 is detectable in fetal rat heart, kidney and intestine, and is conserved across human, rat and mouse adult lungs. RT-PCR analysis detected LGL1 expression in human fetal lung, kidney, heart and intestine. In situ hybridization studies demonstrated that LGL1 expression is restricted to the mesenchyme, while FISH mapped LGL1 to human chromosome 16824. LGL1 demonstrated homology to P25TI, a polypeptide with weak trypsin inhibitor activity, as well as the CRISP family of androgen-regulated, cysteine rich secretory proteins. We expressed a hexahistidine tagged LGL1 in an E. coli expression system in order to facilitate characterization of the protein and determine its role in fetal lung maturation. The data gathered thus far are consistent with LGL1 having a role in lung development via the regulation of extracellular matrix degradation.

Maternal genetic and nutritional influences have a vast impact on developmental outcome. In particular, disturbances in folate metabolism have been well-studied since the finding that dietary folate dramatically reduces the incidence of neural tube defects (NTD). A mild deficiency in the folate-metabolizing enzyme, methylenetetrahydrofolate reductase (MTHFR), and altered dietary folate are clearly associated with NTD, however, their effects on other congenital defects and pregnancy complications are not conclusive. In this thesis, the impact of MTHFR deficiency and altered dietary folate on embryonic and placental development were investigated in our mouse model of MTHFR deficiency. Additional studies characterizing Mthfr promoter activity in vitro and in vivo will help better understand the role of MTHFR deficiency in the associated human disorders. Maternal MTHFR and folate deficiencies increased embryonic delay and growth retardation and resulted in a low incidence of embryonic defects at 10.5 days post coitum (dpc). Folate deficiency increased embryonic loss and abnormal placental phenotypes, including abruption and disturbed patterning of placental layers. Folate-deficient placentae also had decreased ApoA-I staining suggesting that a deficiency in cholesterol may contribute to the embryonic and placental abnormalities observed. A high folate diet increased embryonic delay, growth retardation, and non-significantly of embryonic defects, at 10.5 and/or 14.5 dpc. Maternal MTHFR deficiency appeared to improve some of the adverse outcomes due to high dietary folate. Characterization of the two Mthfr promoters revealed temporal and tissue-specific regulation of Mthfr. The downstream promoter had specific activity in 10.5-dpc embryos and placentae, while the upstream promoter had strongest activity in cultured neuronal cells and in the adult brain and testis. Activity from both promoters was observed in neonatal epididymis. NF-κB was identifi / La nutrition de la mère ainsi que des facteurs génétiques maternels ont des effets importants sur le développement de l’embryon. En particulier, la perturbation du métabolisme des folates a été bien étudiée puisque la consommation de folates réduit considérablement les risques de d’anomalis du tube neural (ATN). Une déficience en méthylènetétrahydrofolate réductase (MTHFR) ou une diète modifiée en folates est associée avec les ATN, mais leurs effets sur d’autres défauts congénitaux et complications de grossesse sont encore controversés. Nous avons étudié l’impact d’une déficience en MTHFR et d’une diète modifiée en folates sur le développement embryonnaire et placentaire en utilisant notre modèle de souris modérément déficiente en MTHFR. Des recherches additionnelles caractérisant les promoteurs de Mthfr in vitro et in vivo on permis de mieux comprendre le rôle de MTHFR dans les maladies s’y rattachant. Nous avons observé que les déficiences maternelles en MTHFR et en folates augmentent la fréquence des retards du développement et de la croissance embryonnaire, et possiblement des défauts congénitaux, à dix jours et demi post coitum. De plus, un apport inadéquat en folates augmente la fréquence des pertes d’embryons et les anomalies placentaires, incluant une réduction d’expression d’ApoA-I. Une diète enrichie en folates augmente aussi le retard de développement, et affecte la croissance embryonnaire et les défauts congénitaux, mais cette dernière observation n’est pas significativement appréciable. Nos résultats montrent aussi qu’une déficience maternelle en MTHFR diminue l’impact de certains effets défavorables causé par des niveaux élevés de folates. La caractérisation des deux promoteurs de Mthfr a démontré la régulation temporale et tissulaire de Mthfr. Le promoteur de Mthfr en aval est actif dans les embryons et dans le placenta à dix jours et demi pos

The serotonin transporter has long been implicated as a key modulator of mood, with deficits of central nervous system serotonin implicated in many psychiatric disorders (anxiety, depression, alcoholism, etc.). The serotonin transporter is responsible for the high-affinity reuptake of serotonin from the synapse, effectively terminating the potentiation of the serotonergic signal. The objectives of this work were to sequence the serotonin transporter promoter region in the vervet monkey (C. aethiops), identify polymorphisms and determine whether they were associated with any of the previously characterized behavioural dimensions of the vervet. 2.8kB of previously unknown sequence was obtained and one novel polymorphism was discovered (V-SERT-Alu). 199 vervet monkeys were screened for the V-SERT-Alu polymorphism and a significant association (p < 0.05) was found to behavioural factor three (fearfulness) which is the monkey analogue of human anxiety.

In mice, H2 and Ly49h genes determine natural resistance to cytomegalovirus infection by alternate mechanisms. We are interested in MA/My, an inbred strain that is resistant to MCMV infection despite the absence of Ly49H receptor and the presence of a haplotype highly related to the MCMV-susceptible strains 129 and FVB/N. Therefore, it is interesting to study its genetic basis of resistance to MCMV infection. In this study, we have identified Cmv1rm, a new resistance allele conferring resistance in MA/My. We have demonstrated by statistical analyses that both H2 and NKC genes are important and that their genetic interaction is necessary to confer resistance in MA/My. By the characterization of the Ly49 gene repertoire of MA/My, we identified 3 potential activating Ly49 gene candidates. Finally, we have confirmed the presence of an important additive effect of H2 and NKC in a FVB/N x BALB.K cross, indicating that Cmv1 rm resistance mechanism may be present in other inbred strains.

Using strain distribution pattern in a series of recombinant inbred strains derived from malaria susceptible A/J and resistant C57BL/6J (B6) inbred strains of mice, it was established that the genetic control of infection with Plasmodium chabaudi AS is multigenic. Quantitative trait linkage analysis in backcross and F2 crosses derived from A/J and B6 mice identified a major locus on mouse chromosome 8 (Pchr/Char2) controlling the extend of blood-stage parasite replication. The Chr.8 interval contains several candidate genes, one of them being the macrophage scavenger (Scvr) locus encoding for the scavenger receptor type A (SR-AI/AII) expressed at the surface of monocytes and macrophages. The possible role of the SR-A in host defense, and particularly as a candidate for being Char2, was investigated. Sequencing of the coding region and immunoprecipitation of the receptors from A/J and B6 mice, as well as functional studies in transfected CHO cells showed that the B6 strain has a particular haplotype at the Scvr locus. Despite the fact that a link between differential function of SR-A and resistance/susceptibility to P. chabaudi AS was not established, results have shown that the conformation of B6 SR-A at the cell surface differ from one of the known haplotype. Novel loci regulating response to P. chabaudi AS infection were investigated using an alternative strategy based on a newly derived set of AcB/BcA recombinant congenic strains (RCS) bred from A/J and B6 strains. One of the AcB strains, AcB55, was shown to be highly resistant to infection despite having 83% susceptible A/J genomic composition, which includes susceptibility alleles at Char2. Linkage analysis in an informative (AcB55 X A) F2 population located a new resistance locus on chromosome 3 (Char4). Early onset of parasite clearance in AcB55 is associated with lower peak parasitemia and absence of mortality. Investigation of possible mechanisms mediating P. chabaudi AS resistanc

The MMACHC gene is responsible for cblC, the most common inborn error of cobalamin metabolism in man. We created a knockout mouse model for its ortholog, Mmachc. Embryonic stem cells from 129 strain mice heterozygous for Mmachc containing a gene trap in intron 1 were injected into blastocysts from c57B6 mice to generate chimeras. Crossing chimeric males to c57B6 females generated heterozygous F1 mice. Geonotyping of F2 mice showed 36 wild type, 71 heterozygous and 3 homozygous. The three homozygous mutant F2 mice had a normal phenotype possibly due to alternative splicing causing expression of wild type Mmachc. Genotyping of embryos showed absence of homozygous mutants at e17.5, suggesting they died earlier. We observed phenotypes including open neural folds, amnionless-like, holoprosencephaly and abnormal limbs and face in individual homozygous mutant embryos. This work enables us to further clarify functions of Mmachc and the role of it in birth defects. / Chez l'homme, le gène MMACHC est responsable de la maladie cblC qui est l'erreur innée la plus commune de sentier de métabolique de la cobalamine. Nous avons créé un modèle de souris knockout pour son orthologue--Mmachc. Des cellules souches embryonnaires de type 129 heterozygotes pour un gène piégé dans l'intron 1 de Mmachc ont été injectées dans des blastocystes de type c57B6 afin de produire des chimères. Le croisement de mâles chimériques et de femelles de type c57B6 a produit des mutants hétérozygotes F1. Le génotypage a démontré 36 souris de type sauvage, 71 hétérozygotes et 3 homozygotes. Le génotypage a démontré l'absence d'embryons homozygotes mutants à e17.5, suggérant que les embryons homozygotes mutants sont morts avant e17.5. Nous avons observé des dysmorphologies telles que des plis neuronaux ouverts, un phenotype similaire à 'amnionless', de l'holoprosencephaly et des membres et visages anormaux dans les embryons homozygotes mutants. Les trois souris homozygotes mutantes qui ont survécues à la naissance avaient un phenotype normal. Elles exprimaient Mmachc de type sauvage et mutant, possiblement en raison d'un épissage alternatif.

For the development of a healthy baby, the regulation of gene expression during development is critical. DNA methylation is an epigenetic mechanism that regulates gene expression, is established during gametogenesis and must be properly maintained throughout development. DNA methylation also establishes the parent-of-origin expression of imprinted genes, which play key roles in embryo and placenta development, cancer, postnatal behaviour and are implicated in human disease. The primary objective of this work was to determine the effects on the embryo and placenta of perturbing epigenetic events during oocyte or preimplantation development. The administration of exogenous hormones to increase the numbers of oocytes ovulated (superovulation) was hypothesized to affect the establishment of imprints in the oocyte and was thus employed as a model to perturb this key event in development. Using this model, we demonstrated an effect on the maintenance of imprints that resulted in aberrant expression of imprinted genes specifically in the placenta and further identified the dysregulation of a key mitogen, Insulin-like growth factor II (IGF2), in the placenta as a potential contributor to the intrauterine growth restriction observed following superovulation. In order to perturb methylation during the peri-implantation stage, we identified betaine homocysteine methyltransferase (BHMT) as the enzyme likely to be responsible for the provision of one-carbon units during this critical stage of development. The expression of BHMT was knocked down using morpholino technologies, resulting in a significant increase in postimplantation loss. Further, the methylation of imprinted genes was not affected following BHMT knockdown, however hypomethylation of non-imprinted regions was observed. Taken together, the results of this thesis highlight the importance of appropriate DNA methylation for the growth and survival of the embryo, and have important implications for the use of / Afin d'assurer le sain développement d'un bébé, la régulation de l'expression génique pendant le développement embryonnaire est cruciale. La méthylation de l'ADN est l'un des mécanismes épigénétiques qui contrôlent l'expression génique. Cette modification épigénétique est instaurée pendant la gamétogenèse et doit être correctement maintenue pendant les étapes subséquentes du développement. La méthylation de l'ADN dicte aussi l'origine parentale de l'expression des gènes à empreinte; gènes qui jouent un rôle essentiel dans le développement de l'embryon, du placenta, du cancer, des comportements postnataux et qui sont aussi impliqués dans diverses maladies humaines. L'objectif principal de cette thèse était de déterminer les effets, sur l'embryon et le placenta, qu'ont des événements de perturbation épigénétique pendant le développement de l'ovocyte ou de l'embryon préimplantatoire. Nous avons émis l'hypothèse que l'administration d'hormones exogènes dans le but d'augmenter le nombre d'ovocytes ovulés (superovulation) aura pour effet d'altérer l'établissement des empreintes dans l'ovocyte. La superovulation fut donc utilisée comme modèle afin de perturber cet événement fondamental du développement. Nous démontrons dans nos travaux que la conservation des empreintes est bel et bien affectée, ce qui résulte en une expression erronée des gènes à empreinte, spécifiquement au niveau du placenta. De plus, nous avons découvert que la dérégulation d'un gène mitogène clé dans le placenta, le facteur de croissance analogue à l'insuline II (Igf2), pourrait contribuer à une croissance intra-utérine restreinte suite au processus de superovulation. Afin de perturber la méthylation pendant le stade péri-implantatoire, nous avons identifié BHMT (bétaïne-homocystéine méthyltransférase) comme l'enzyme potentiellement responsable de l'accumulation d'unités à un carbone pendant cette période critique du d

This thesis examines the functional and genetic factors that control the inbred mouse host response to Cryptococcus neoformans. The inbred mouse strains C57BL/6J and C3H/HeN were susceptible, and the inbred mouse strains CBA/J and SJL/J were resistant, to experimental cryptococcal pneumonia. This observation led to three general hypotheses: (1) early innate immune differences between inbred strains cause differential susceptibility to cryptococcal pneumonia; (2) natural variation in susceptibility among inbred strains is a complex trait under the control of specific genetic loci; and (3) allergic airway disease is a common phenotype among susceptible inbred strains. Functional characterization of the innate immune response in C57BL/6J and SJL/J mice revealed a heightened pro-inflammatory response in SJL/J as early as three hours post-infection. This polarization continued throughout the course of infection; C57BL/6J mice presented an allergic, Th2 immune response, and SJL/J mice presented a Th1 immune response. Intracellular signaling analysis in vitro revealed that the enhanced pro-inflammatory response observed in SJL/J mice was dependent on the prolonged activation of the NF-κB and phosphatidylinositol 3 kinase pathways. Next, a quantitative trait loci (QTL) analysis for cryptococcal pneumonia susceptibility in a segregating F2 population bred from the parental inbred strains C57BL/6J and CBA/J was performed. This revealed a sex-effect in the quantitative lung fungal burden that warranted a stratified QTL analysis approach. Two significant novel QTL were identified in the female F2 intercross: Cnes1 on chromosome 1, and Cnes2 on chromosome 17. Cnes3 was identified in the male F2 intercross as a unique QTL distal to Cnes2. Furthermore, a genome-wide pairwise analysis revealed significant QTL interactions in both the female and male F2 intercrosses that collectively explained 43.8 and 19.5% of the phenotypic variance in each sex, respectively. Finally, characte / Cette thèse étudie les facteurs fonctionnels et génétiques contrôlant la réponse de l'hôte aux infections fongiques causées par la levure Cryptococcus neoformans. Les lignées de souris consanguines C57BL/6J et C3H/HeN sont sensibles à la pneumonie expérimentale à cryptocoques, alors que les lignées CBA/J et SJL/J y sont résistantes. En se basant sur ces observations, trois hypothèses ont été formulées: (1) des différences du système immunitaire inné expliquent la sensibilité différentielle à la pneumonie à cryptocoques des lignées de souris consanguines; (2) la variation naturelle de la sensibilité entre lignées consanguines est un trait génétique complexe sous le contrôle de locus génétiques spécifiques; et (3) la maladie allergique respiratoire est un phénotype commun associé à une sensibilité à la pneumonie à cryptocoques progressive chez les lignées consanguines. La caractérisation fonctionnelle de la réaction immunitaire innée chez les souris C57BL/6J et SJL/J a révélé une réponse pro-inflammatoire accrue chez les souris SJL/J trois heures après l'infection. Cette polarisation a continué tout au long de l'infection, les souris C57BL/6J développant une réponse immunitaire allergique Th2, alors que les souris SJL/J ont présenté une réponse immunitaire Th1. Une analyse in vitro de la signalisation intracellulaire a révélé que la réaction pro-inflammatoire observée chez les souris SJL/J dépend de l'activation prolongée des cascades de signalisation NF-κB et PI3K (phosphatidylinositol 3 kinase). L'étude des liaisons de traits complexes (QTL) chez des souris F2 (C57BL/6J x CBA/J) a révélé un effet du sexe sur le phénotype, justifiant ainsi une analyse séparée. Deux locus de susceptibilité ont été identifiés chez les femelles: Cnes1 (chromosome 1), et Cnes2 (chromosome 17). Chez les mâles, seul le locus Cnes3 (chromosome 17, en aval de Cnes2) a été identifié. Une analyse QTL sur 2 locus a

Genetic control of susceptibility to tuberculosis in resistant C57BL/6 (B6) and susceptible DBA/2 (D2) mice is multigenic. Susceptibility in D2 is associated with a unique phenotype which includes unrestricted pulmonary replication, severe lung pathology, extensive tissue necrosis and early death. Quantitative trait linkage mapping in 2 genome wide scans was used to determine the number and location of genes controlling differential susceptibility to pulmonary tuberculosis in D2 and B6. In a first scan, 95 informative (B6XD2) F2 mice were infected i.v with 105 virulent M. tuberculosis H37Rv, and survival time was used as a phenotypic measure of susceptibility. These studies identified 3 significant linkages on chromosomes 1,3 and 7 designated Trl-1/2/3 respectively. In a second genome scan, 104 (B6XD2) F2 mice were infected with 10 2 M. tuberculosis H37Rv by the aerosol route, and the extent of bacterial replication in the lungs (CFU) at 90 days was used as a phenotypic measure of susceptibility. Results from this analysis identified a major linkage on chromosome 19 (Trl-4) accounting for 25% of the phenotypic variance. In this scan Trl-3 was confirmed as not only playing a role in overall survival to infection but also bacterial growth in the lungs. Our results suggest a strong genetic interaction between Trl-3 and Trl-4 in regulating pulmonary replication of M. tuberculosis. To further characterize the mechanistic basis of action of the Trl-1/2/3/4 effect on M. tuberculosis infection, global expression analysis of approximately 15 250 genes was utilized to identify genes differentially expressed between the lungs of B6 and D2 mice prior to and during infection with M. tuberculosis . Our data suggests that the classical complement pathway and the apoptotic pathway plays a role in the differential susceptibility between B6 and D2 mice, with these pathways being upregulated in B6. Moreover the increase in neutrophil associated gene expression corroborates with our histological finding of increased neutrophil cell counts in D2 mice. Finally these studies have provided candidate genes that map in the Trl-1 and Trl-4 regions (Cfh and Scd2 respectively).