Background: The effect of common polymorphisms on transcription levels of a large number of genes has been well documented and defined genome-wide in numerous studies. However, the effects of common mRNA polymorphisms on translation have received little attention. RPS26 exhibited one of the strongest associations (p = 10-10) in our group's genome-wide search to identify transcripts whose relative distribution between heavy polysomes (active translation) and soluble RNA (inactive) was skewed by single-nucleotide polymorphisms. The RPS26 SNPs most strongly associated with ribosomal distribution, rs17118262 (C/G) and rs1131017(C/G), are located 9 bases apart, are in strong linkage disequilibrium (D'=1) but have different allele frequencies (r2=0.54). The goal of the present study was to confirm and quantify the effect of those RPS26 SNPs on translational efficiency at the protein level. RPS26 was selected for this purpose because rs1131017 is also associated with type 1 diabetes (T1D), the second strongest novel association in our genome-wide association study (GWAS).Methods: To independently confirm the translational imbalance found by ribosomal distribution, we studied allelic ribosomal distribution in single heterozygous samples (allelic translation) with Single Nucleotide Primer Extension (SNuPE) and Pyrosequencing. To test the hypothesis that mRNA with the allele most associated with ribosomes also produces more RPS26 protein, we applied both an ex vivo direct measurement and an in vitro translation approach.Results: Pyrosequencing and SNuPE confirmed the translational imbalance favouring the G allele of the rs17118262. Both ex vivo and in vitro translation showed that the same allele that was found at a higher proportion in the polyribosomal fraction than in the soluble RNA produces more protein product under the same translational environment. Conclusion: Both SNPs (the common rs1131017 and the rare rs17118262 variant) exert a significant effect on the amount of RPS26 protein that is produced. Adding to the well-studied polymorphic transcriptional effects, the effects of exonic single-nucleotide polymorphisms (SNPs) on translational efficiency may be equally important in determining protein expression levels and therefore contributing significantly to the disease risk. RPS26 in type 1 diabetes is an excellent example of such an association. / L'effet des polymorphismes fréquents (SNPs) sur les niveaux de transcription d'un grand nombre de gènes a été bien documenté et défini à l'échelle du génome dans de nombreuses études. Toutefois, les effets des polymorphismes communs sur la traduction de l'ARN messager (ARNm) ont reçus peu d'attention. Dans notre étude à l'échelle du génome qui étudiait des polymorphismes donnant une distribution différentielle entre les polysomes lourds (traduction active) et de l'ARN soluble (inactif), le gène RPS26 a montré une des plus fortes associations (p = 10-10). Les SNPs dans RPS26 les plus fortement associés à une différence de distribution des ribosomes, rs17118262 (C / G) et rs1131017 (C / G), sont séparés par 9 bases et sont en fort déséquilibre de liaison (D '= 1) mais ont des fréquences alléliques différentes (r2=0.54). L'objectif de la présente étude était de confirmer et de quantifier l'effet de ces SNP dans RPS26 sur son efficacité de traduction au niveau protéique.Méthodes: Pour confirmer de manière indépendante le déséquilibre constaté au niveau de la distribution des ribosomes, nous avons étudié la distribution des allèles dans des échantillons hétérozygotes pour chaque un des 2 SNPs avec à l'aide des techniques d'extension nucléotidique simple d'amorces (SNuPE) et de pyroséquençage. Pour tester l'hypothèse que l'ARNm avec un allèle particulier produit plus de protéines RPS26, nous avons appliqué à la fois une approche de quantification ex vivo et traduction in vitro.Résultats: Le SNuPE et le pyroséquençage ont confirmé le déséquilibre au niveau de la traduction montrant une supériorité de l'allèle G du SNP rs17118262. Les approches ex vivo et in vitro ont montré que la traduction de l'allèle trouvé dans une proportion plus élevée dans la fraction de polysomes lourds produit plus de protéines par rapport à l'autre allèle dans les même conditions.Conclusion: Les deux SNPs (le rs1131017 qui est fréquent et le rs17118262, de moindre fréquence) exercent un effet significatif sur la quantité de protéine RPS26 produite. Combiné à l'effet déjà connu des SNPs sur les niveaux de transcription, leur effet sur la variation du niveau de traduction est peut-être aussi important dans la détermination du niveau total de protéines et par le fait même contribuer au risque de maladie de manière significative. RPS26 au niveau du diabète de type I en est un parfait exemple.

Salmonella infections remain a global health concern exacerbated by the emergence of multidrug resistant strains. Host genetics have been demonstrated to influence the immune response to Salmonella infections. Adopting the susceptible wild-derived MOLF/Ei mice as a model of systemic Salmonella Typhimurium infection has previously identified a resistance locus Ity2 and a susceptibility locus Ity3. / Both the Ity2 and Ity3 loci were validated through the creation of congenic mice and through linkage confirmation in a new segregating cross. Refinement of the critical intervals was achieved using subcongenic mice. Transcriptional profiling of the congenic mice identified differentially regulated candidate genes within the two loci that can be examined for potential relevance to Salmonella infection in MOLF/Ei mice. / Previous studies proposed Tlr5 as a candidate for Ity3 based on the sequence variants and reduced expression in MOLF/Ei liver. Tlr5 function was characterized in vitro and in vivo using the Ity3 congenic strains. Both analyses pointed towards the exclusion of this gene as a candidate for Ity3 due to discordant functional responses between MOLF/Ei and the Ity3 congenics. / Ncf2 was pursued as a candidate for the Ity3 locus based on its map position and its antimicrobial role in Salmonella infection. Sequencing of Ncf2 led to the identification of one non-conservative amino acid in a conserved domain of the protein. Functional analysis indicated a reduced response attributed to the MOLF/Ei allele, suggesting a potential involvement of Ncf2 in Salmonella pathogenesis of Ity3 congenic mice. / Taken together, we have been able to narrow down the critical intervals and confirm the contribution of Ity2 and Ity3 to Salmonella infection. We have eliminated Tlr5 but highlighted the potential involvement of Ncf2 for the Ity3 locus. The genetic dissection of the host response in MOLF/Ei mice has captured the inherent complexity of the immune response towards Salmonella infection. Our findings have also revealed the genetic and phenotypic diversity of the wild-derived MOLF/Ei mice. Furthermore, a complete genome scan of the additional informative cross as well as the validation of candidates identified here, should resolve of the intricacies of the susceptibility of MOLF/Ei mice and contribute to our understanding of Salmonella pathogenesis.

Since the declaration of “War on cancer” in 1971, and with the insight provided by recent advances in human genetics and in molecular technology , the field of cancer biology has been expanded rapidly providing hope that a cure for this lethal disease will be found. One of the major contributions of the field of cell biology to the understanding of malignant diseases has been the identification of growth factors and their receptors as major promoters of transformation and malignant progression. In particular, the appreciation of the central role that receptor tyrosine kinases (RTK) play in different cancers has led to development of effective therapeutic reagents. One of the RTK implicated in malignant progression is the receptor for the type 1 insulin like growth factor (IGF-IR) that has been identified as a target for anti-cancer treatments. Cancer gene therapy is a rapidly developing modality for cancer therapy. Many gene therapy strategies have been developed generally targeting the genes and proteins involved in cancer initiation and progression. To design a successful gene therapy strategy requires an understanding of the molecular basis of cancer progression and knowledge of human and animal genetics and physiology. In the present work, I have introduced two different strategies to inhibit liver metastases formation using established human and murine cancer cell lines. The first strategy is based on targeting the IGF-IR in tumor cells using an antisense technology (chapter 2). This strategy was also shown to be applicable in cancer gene therapy of glioblastoma growing in the brain (chapter 3). Very interestingly and for the first time, we showed that reduction of IGF-IR expression levels in glioma cells can induce a state of dormancy, providing a unique model to study this clinically important phenomenon. As the second strategy, I designed a novel soluble IGF-IR molecule. I showed that expression of this molecule in tumor cells caused an inhibitio / Depuis la “déclaration de la guerre contre le cancer” en 1971 et grâce aux avancées en génétique humaine et technologie moléculaire, le domaine de la biologie du cancer s’est rapidement étendu, fournissant l’espoir qu’un traitement pour cette maladie léthale sera trouvé. Une des contributions majeures du domaine de la biologie cellulaire pour la compréhension des maladies malignes a été la mise en évidence du rôle des facteurs de croissance et de leurs récepteurs dans la transformation et la progression maligne. En particulier, le rôle central que jouent les récepteurs à tyrosine kinase (RTK) dans différents cancers a abouti au développement d’agents thérapeutiques efficaces. Un des RTK impliqué dans la progression maligne est IGF-IR, le récepteur de type 1 du facteur de croissance de l’insuline qui a été identifié comme étant une cible pour des traitements anti-cancéreux. La thérapie génique est en pleine voie de développement pour la thérapie du cancer. De manière générale, de nombreuses stratégies de thérapie ont été développées en ciblant les gènes et protéines impliqués dans l’initiation et la progression du cancer. L’élaboration d’une stratégie de thérapie génique efficace nécessite la compréhension des bases moléculaires de la progression du cancer ainsi que la connaissance de la génétique et la physiologie humaine et animale Dans ce travail, j’ai introduis deux stratégies différentes pour inhiber la formation de métastases du foie en utilisant des lignées cellulaires cancéreuses humaines et murines. La première stratégie est basée sur le ciblage de IGF-IR dans des cellules tumorales en utilisant une stratégie antisens (chapitre 2). Cette stratégie s’est également avérée être appliquable à la thérapie génique du cancer de glioblastome dans le cerveau (chapitre 3). De facon intéressante et ce, pour la première fois, nous avons montré que la diminution de l’exp

Skeletal development and homeostasis is comprised of complex events characterized by the presence of cell specific regulators and markers. Aberrant expression of any of the regulating factors usually results in metabolic bone disease. Therefore understanding the mechanisms of skeletal homeostasis may disclose new therapies for treating these disorders. While the complete pathway that defines normal osteoblast differentiation remains indeterminate, several regulatory factors have already been identified and characterized. Using gene targeting technology, the importance of parathyroid hormone-related peptide (PTHrP) in skeletal biology has been well established, as PTHrP^-/- and PTHrP^+/- mice demonstrate skeletal abnormalities. Several lines of evidence from in vivo and in vitro studies suggest that PTHrP has unique roles in osteoblast biology, independent from its well-established roles in chondrocytes. The presence of an intronic variable number tandem repeat sequence (VNTR) in the human PTHrP gene has been proposed to provide a novel mechanism of transcriptional gene regulation in osteoblasts. A preliminary survey indicated that osteoporotic patients with low bone mineral density have the shortest allele of the VNTR. In this work, we set out to investigate the role of the minisatellite in PTHrP expression and as a potential predictor of decreased bone mineral density. However, in a large sample of osteoporotic patients, a correlation between bone mineral density and VNTR length was not apparent. Also, the presence of the VNTR, or its length, seemed not to affect PTHrP promoter activity in osteoblast-like cells. EMSA experiments demonstrated the ability of the VNTR to bind proteins whose natures remain unclear. We also studied the effect of the newly identified zinc-finger transcription factor, Osterix, on the GC-rich PTHrP promoter. Osx did not bind the promoter or affect its transcriptional activity. However, unexpectedly, we observed that the transcription factor Cbfal, the master regulator of osteoblast differentiation, decreases PTHrP gene expression, likely through a molecular intermediate. Our investigations have shed some light on the role of PTHrP in osteoblast differentiation, and its regulation in this process by polymorphic elements and transcription factors specific to osteoblasts. The potential role of these parameters in the homeostatic regulation of the skeleton and the development of metabolic bone diseases such as osteoporosis warrants further investigation.

Wilms' tumor is an embryonal malignancy of the kidney that affects approximately 1 in 10,000 children under the age of 5 years. These tumors arise from the metanephric blastemal cells that do not follow the normal course of cellular differentiation, but instead continue along the path of proliferation. A Wilms' tumor predisposing gene, WT1, situated at 11p13 has been cloned and spans 50 kb of genomic DNA. It encodes a putative transcription factor, with an NH$ sb2$-terminus rich in proline and glutamine residues and a COOH-terminus composed of four zinc fingers of the Cys$ sb2$-His$ sb2$ type. The WT1 gene is alternatively spliced, giving rise to four mRNA and protein isoforms, encoding polypeptides ranging in size from 45-49 kDa. Although some Wilms' tumors occur in familial setting (1-2%), most cases occur as a sporadic form. Of the sporadic tumors analyzed to date for WT1 mutations, only the zinc finger domains have been evaluated.We have investigated the status of the complete WT1 gene in 101 tumors to document the frequency with which WT1 is mutated in sporadic and hereditary Wilms' tumors. We have identified homozygous, hemizygous and heterozygous mutations in 10 tumors, of which 7 were found to occur within the zinc finger domain. Our results indicate that the frequency of mutations (insertions, deletions, base substitutions) in sporadic tumors is $ sim$6%. We have been able to identify novel mutations in addition to already documented ones.

Many organisms can execute a dormant state or diapause to survive harsh environmental conditions for extended durations. When Caenorhabditis elegans larvae enter the dauer diapause, they completely arrest development and feeding, but remain active and motile, yet become stress-resistant and extremely long-lived. Entry into dauer is associated with a reduction in insulin-like signalling, the establishment of a generalized cell cycle arrest, the accumulation of nutritive resources and a concomitant global change in metabolism. The precise molecular and physiological processes that induce cell cycle quiescence and enable long-term survival in the absence of caloric intake however remain largely unknown. I show here that the C. elegans orthologs of PTEN, STRAD, LKB1 and AMPK (α1, α2, β1, β2 subunits) cooperate to establish quiescence in the germline stem cell population during dauer development. Interestingly, germline mutations in LKB1 cause predisposition to cancer in humans, while mutations in STRAD or AMPK subunits do not seem to cause cancer. In C. elegans, LKB1 also regulates embryonic polarity, while STRAD and AMPK are dispensable for this process. Thus, my data suggest that LKB1/STRAD regulate cell growth/proliferation through AMPK, while LKB1 also acts independently to regulate polarity, and that this function may be critical for tumor suppression in human. In addition, I show that C. elegans larvae that lack LKB1/AMPK signalling rapidly consume their stored energy and prematurely expire following vital organ failure. This signalling pathway acts in adipose-like tissues to downregulate triglyceride hydrolysis so that these fat reserves are rationed to last the / Plusieurs organismes peuvent entrer en dormance, ou diapause, pour survivre à des conditions environnementales précaires pour une durée prolongée. Lorsque des larves de Caenorhabditis elegans entrent en diapause dauer, elles cessent complètement de se développer ainsi que de se nourrir, cependant elles demeurent actives et mobiles, tout en acquérant une résistance au stress et une longévité extrême. L'entrée en stade dauer est accompagnée d'une réduction de signalisation par l'insuline, de l'établissement d'un arrêt généralisé du cycle cellulaire, de l'accumulation de ressources nutritives et d'un changement global au niveau du métabolisme. Les processus physiologiques et moléculaires précis qui induisent la quiescence cellulaire et permettent la survie prolongée en l'absence de tout apport calorique, demeurent toutefois essentiellement inconnus. Je montre ici que les orthologues de PTEN, STRAD, LKB1 et de AMPK (sous-unités α1, α2, β1, β2) chez C. elegans coopèrent dans l'optique d'établir la quiescence cellulaire dans la population de cellules germinales souches durant le développement de la larve dauer. Il est intéressant de préciser que chez l'humain, une mutation de LKB1 dans la lignée germinale provoque une prédisposition au cancer, tandis qu'une mutation dans une sous-unité de STRAD ou d'AMPK ne semble pas causer de cancer. Chez C. elegans, LKB1 régule aussi la polarité embryonnaire, tandis que STRAD et AMPK sont dispensables pour ce processus. Donc, mes données suggèrent que LKB1/STRAD régulent la croissance et la prolifération des cellules à travers AMPK, tandis que LKB1 fonctionne aussi indépendamment pour contrôler

Through this investigation I hoped to illuminate and characterize any combinatorial relationships that might exist amongst the regulatory sequences that drive expression of the myelin basic protein gene. With such insight, I expected to learn more of the mechanism/s regulating myelin sheath formation, maintenance and repair. In the search for regulatory regions within the mbp gene, four highly conserved non-protein-coding modules were found in its 5' flanking sequence. In the context of reporter constructs in transgenic mice, these regulatory modules confer distinct cell specificity and developmental expression programs. M1 and M3 drive expression in oligodendrocytes while M4 drives Schwann cell expression. However, the expression programs realized by these reporter constructs also exposed higher levels of regulatory organization; e.g., when M3 is dissociated from neighboring flanking sequences it acquires the ability to drive transient expression in Schwann cells. To further understand how the four known mbp regulatory modules orchestrate the expression of the mbp gene, I first generated reporter constructs designed to contain most module combinations. These were inserted in single copy in a common orientation at a common site 5' of the HPRT locus and their qualitative and quantitative regulatory potential was analyzed. Based on the expression programming of reporter genes, M3 is critical for achieving high levels of mbp expression in myelinating oligodendrocytes. To determine if the expression phenotypes of reporter genes accurately reflected the activity of enhancers in the context of the endogenous locus, I used gene targeting to delete M3 in the endogenous mbp loc / Au cours de ma recherche j'ai voulu mettre en évidence les combinatoires existantes parmi les séquences régulatrices qui gouvernent l'expression du gène de la Protéine Basique de la Myéline (MBP). Ces connaissances devraient pouvoir me permettre de mieux comprendre les mécanismes régulant la formation, la maintenance et la réparation de la gaine de myéline. Lors de l'étude de la régulation du gène de la MBP, quatre modules non codant extrêmement conservés ont été trouvés en amont du gène de la MBP. Dans des souris transgéniques, ces modules confèrent des propriétés d'expression temporelle et tissu spécifique aux constructions. Les modules M1 et M3 gouvernent l'expression dans les oligodendrocytes alors que le module M4 gouverne l'expression dans les cellules de Schwann. Cependant, les programmes d'expression de ces constructions révèlent une organisation de plus haut niveau : Quand M3 est dissocié des séquences flanquantes du gène de la MBP il acquière la capacité de gouverner une expression transitoire dans les cellules de Schwann. Pour comprendre comment les quatre modules régulateurs de la MBP orchestrent l'expression du gène, j'ai d'abord généré des constructions présentant la plupart des combinaisons de modules. Ces constructions ont été insérées en copie unique de même orientation et dans un locus commun en 5' du gène HPRT afin de pouvoir comparer qualitativement et quantitativement leur expression. L'analyse de ces résultats montrent que le module M3 est critique pour obtenir un haut niveau d'expression durant la myélination des oligodendrocytes. Pour déterminer si l'expression des constructions reflète correctement

Eukaryotic genomes possess significant amounts of highly tandemly-arrayed repetitive DNA sequences in their constitutive heterochromatin. Heterochromatin has been involved in processes, such as position-effect variegation (PEV), that disrupt normal expression of euchromatic genes. The role of highly repetitive DNA sequences, also termed satellite DNAs, in gene silencing has therefore aroused a great deal of interest. Previous plasmid transfection studies into tk-/neo- human cells had shown that the location (in cis) of a 1.797 kb human satellite II DNA sequence next to the 5' end of the neo gene, exerted a severe negative effect on the recovery of stable tk+/neo + transfectants, in the presence of HAT and G418 drugs. As shown in this thesis, positioning of the same satellite II DNA sequence at another location, between the 3' ends of the tk and neo genes, led to the loss of this negative effect. To identify a possible size limit to the silencing effect exerted by the satellite II DNA, fragments of 813 and 983 by of the 1.797 kb DNA sequence were also inserted near the 5' end of the neo gene. These sequences were still able to exert a drastic negative effect on the recovery of stable tk+/neo + transfectants. The effect was found to be dependent on the orientation of the satellite II DNA fragments relative to the marker genes. Further reduction in the size of the satellite II DNA sequence, to fragments of 354 to 620 bp, revealed a significant reduction in the negative effect. Selection of the transfected cells in media containing single drugs (i.e. HAT or G418) showed that the neo gene in the plasmids was more affected than the tk gene. Analysis of the integration pattern of the plasmid vector and a plasmid containing the 1.797 kb satellite II DNA indicated that the satellite II DNA did not influence the plasmid site of integration.

Mouse genome mapping is an essential integrating part of the human genome project because mouse has long been a valuable animal model for the investigate human genetic diseases. However, application of fluorescence in situ hybridization (FISH) in mouse has been hindered by technical difficulties related to the characteristics of mouse cytogenetics. To implement FISH as a tool for the mouse genome project, we developed high-resolution FISH and applied it to mouse and comparative genetics. / Using an original technique, we obtained high-resolution replication R-banded mouse chromosomes (over 500 bands per haploid). We constructed a new high-resolution R-banding idiogram, for an easier identification of mouse chromosomes allowing mouse gene mapping with greater precision. / Two mouse renal Na-Pi cotransporter genes (Npt1 and Npt2) were assigned to mouse chromosome region 13A3-A4 and 13B respectively. The autosomal localization excluded Npt1 and Npt2 as direct disease genes for X-linked hypophosphatemia and implicated them as candidate genes for autosomally inherited hypophosphatemia, hereditary hypophosphatemic rickets with hypercalciuria and hypophosphatemic bone disease. This study demonstrated that gene mapping using FISH is a direct approach in linking genetic diseases and relevant function genes. / In order to directly generate information transferable from mouse to human, we conducted comparative gene mapping study by FISH. Human HLCS, mapped to human chromosome 21q22.1, was assigned to mouse chromosome region 16C4 with a human probe. Conversely, mouse Mborg-1 gene, localized to mouse chromosomes 2H1, was mapped to human chromosomes 20q13 with the murine probe. Moreover, two clusters of mouse gene/DNA marker in the region of Bcg and Lp locus at mouse chromosome 1C4, 1H1 respectively were cross-mapped to human chromosomes 2q35 and 2q23 respectively. The results yields higher resolution than linkage studies and thus allowed us to narrow down the chromosomal region containing the human homologues, as a first step for the cloning of the human genes. / In an attempt to positionally clone the Lp gene, we established for the first time a linear relationship between optical distance in interphase nuclei and physical distance between DNA sequences, ranging from 130kb to 1500kb, using two-color FISH. The relative position and genomic distance between four markers flanking Lp locus was effectively estimated.

Considerable evidence supports a role for vasopressin (VP) as a local regulator of steroidogenesis in rat testes. I show that rat testes contain three distinct VP gene-related transcripts differing in size from the hypothalamic VP mRNA. Two of these transcripts contain exons B and C, but not the exon that encodes the nonapeptide VP. Hence, these transcripts are incapable of generating testicular irVP. The third transcript possesses exon A-like sequences, probably originating from expression of a VP-related gene. This transcript may encode the VP-like peptide that has been detected in testicular extracts. / The major biological effects of oxytocin (OT) are related to reproduction. I show that the tissues comprising the maternal-placental-fetal unit of the rat, i.e. the fetal membranes (placenta and amnion/chorion) and the uterus, possess a single species of OT gene transcript differing in size from the hypothalamic OT transcript as a result of differences in poly(A) tail lengths. The uterus of a term pregnant rat contains about 70 times more OT mRNA than the hypothalamus, identifying the uterus as the major site of OT gene expression at term pregnancy. All three peripheral tissues possess authentic OT and a noncovalent complex between OT and another molecule, probably Neurophysin I (NpI). OT mRNA and OT immunoreactivity are localizcd to several cell types in the placenta and to the endometrial epithelial cells of the pregnant uterus. OT binding studies reveal high numbers of OT receptors in rat uterine tissues (myometrium and endometrium), but no OT binding, sites were found in the fetal membranes. These findings suggest that OT may act in a paracrine fashion in the fetal membranes while uterine OT may act both as an autocrine and paracrine mediator of uterine functions during pregnancy. / Northern blot analysis shows that the OT gene is expressed in the nonpregnant rat uterus and that expression is regulated in a cycle stage-dependent manner. Also, ovarian steroids are involved in regulating the expression of the OT gene in the uterus. Interestingly, prostaglandins may also be involved in the mechanisms whereby the uterine OT gene responds to steroidal stimulation. (Abstract shortened by UMI.)