Les BMPs (Boue Morphogenetic Proteins) sont des morphogènes membres de la superfamille du TGF-[bêta] qui interagissent avec des récepteurs de surface cellulaire à activité sérine/thréonine kinase. L'interaction des BMPs avec ces récepteurs entraîne l'activation d'une cascade de signalisation cellulaire impliquant les facteurs de types R-Smads (Smad1, 5 et 8). La voie de signalisation des BMPs joue des rôles cruciaux dans des processus biologiques tels que l'embryogenèse et l'organogenèse des tissus ainsi que des processus cellulaires tels que la prolifération, la migration et la différenciation cellulaire. De plus, ces derniers semblent aussi impliqués dans les processus de mort cellulaire et dans la tumorigenèse. Malgré un intérêt grandissant pour la signalisation des BMPs, il existe très peu d'études sur les rôles spécifiques joués individuellement par les différents Smads dans la morphogenèse de l'intestin in vivo . Ceci est principalement dû au fait que la délétion classique des différents effecteurs de la voie des BMPs entraîne la mort in utero à cause de multiples défauts dans l'embryogenèse. Le système Cre/loxP, sous le contrôle d'un promoteur tissu spécifique, a été utilisé dans notre laboratoire, dans le but de générer une lignée murine possédant une délétion conditionnelle de l'effecteur Smad5 strictement au niveau de l'épithélium intestinal. Une analyse comparative à l'aide d'un modèle de délétion conditionnelle du récepteur BmpR1a au niveau de l'épithélium intestinal a été effectuée afin de décortiquer spécifiquement le rôle de l'effecteur Smad5 dans le développement de cet organe. Afin de valider les résultats obtenus in vivo nous avons généré un modèle cellulaire nous permettant de mimer l'effet de la délétion de l'effecteur Smad5 à l'aide de la technologie du shRNA.Les résultats obtenus dans les deux modèles suggèrent que Smad5 serait un facteur clé impliqué dans la régulation de la migration cellulaire des entérocytes. En effet, l'invalidation de la voie des BMPs et plus particulièrement de l'effecteur Smad5 dans les souris entraîne une augmentation de la vitesse de migration des cellules le long de l'axe crypte villosité. Ces résultats sont corroborés dans un modèle cellulaire dans lequel l'expression de Smad5 a été inhibée par interférence d'ARN. Dans ce modèle, la migration se fait de façon beaucoup plus compacte en comparaison aux cellules contrôles. L'augmentation de la vitesse de migration cellulaire pourrait être due à un phénomène de relocalisation des protéines de jonctions adhérentes ainsi qu'à une modulation de l'actine filamenteuse. Ce phénomène pourrait faire intervenir les petites protéines G Rho/Rac ainsi que les kinases Src. En plus de l'actine filamenteuse, différentes protéines impliquées dans la formation des complexes de jonctions adhérentes semblent relocalisées dans nos deux modèles (E-cadhérine/[bêta]-caténine). En conclusion, les différents résultats recueillis au cours de mes travaux de maîtrise dans le laboratoire du Dr. Nathalie Perreault nous suggèrent que l'effecteur Smad5 de la voie des BMPs serait un facteur impliqué dans la stabilité des complexes de jonctions adhérentes, régulant ainsi la migration des cellules le long de l'axe crypte villosité.

PTEN est une protéine dotée d'une activité phosphatase qui déphosphoryle les phosphatidylinositols issus de l'activation de la phosphatidylinositol 3-kinase (PI3K). Des mutations germinales du gène PTEN ont été mises en évidence dans le syndrome de Cowden, une maladie caractérisée par le développement de polypes le long du tube digestif et associée à un risque accru de cancer. De plus, la perte d'un allèle de Pten chez la souris conduit à la formation d'hyperplasie et de dysplasie du tractus gastro-intestinal ainsi qu'à des tumeurs notamment au niveau du côlon. Ces observations suggèrent que PTEN joue un rôle important dans le tube digestif. Cependant, ses mécanismes d'action dans les cellules épithéliales intestinales sont peu connus. Nos travaux nous ont permis de mieux caractériser les rôles de PTEN dans ces cellules. Pour ce faire, nous avons d'abord analysé l'effet d'un shRNA inhibant spécifiquement l'expression de PTEN dans les cellules Caco-2/15, une lignée cancéreuse colorectale qui a la particularité de se différencier suite à l'atteinte de la confluence en adoptant un phénotype semblable aux cellules absorbantes de l'intestin grêle. La perte d'expression de PTEN stimule la prolifération de ces cellules. Cette augmentation de la prolifération semble résulter d'une diminution de l'expression de p21 et de p27 ainsi que d'une hausse des cyclines D2 et E. De plus, le shRNA contre PTEN inhibe la différenciation fonctionnelle et morphologique des Caco-2/15. Cela découle partiellement de l'inhibition de l'expression des facteurs de transcription CDX2, HNF-1? et HNF-4?. Les jonctions serrées sont également altérées dans ces cellules. En effet, une réduction importante de l'expression des claudines et une augmentation de la perméabilité transépithéliale a été observée. Une augmentation de la synthèse protéique a aussi été remarquée. De plus, nos résultats laissent également croire que PTEN pourraient jouer un rôle dans la carcinogenèse colorectale. Nous avons effectivement constaté une augmentation du potentiel tumorigénique des Caco-2/l5 exprimant le shRNA contre PTEN suite à l'injection des cellules dans des souris nues. De plus, ces cellules ont des capacités de migration et d'invasion accrues. Nous avons également constaté que les niveaux de PTEN sont plus faibles dans plusieurs lignées cancéreuses colorectales comparativement aux cellules épithéliales intestinales normales. Nous avons aussi analysé le phénotype d'une lignée de souris possédant une délétion du gène Pten exclusivement au niveau de l'épithélium intestinal, générée à l'aide du système Cre/loxP. Macroscopiquement, une organomégalie de l'intestin grêle et du côlon a été observé chez les souris déficientes pour Pten. Histologiquement, nous avons constaté une désorganisation de l'architecture épithéliale intestinale caractérisée par un allongement des villosités et par la présence d'embranchements villositaires. De plus, un épaississement important des couches musculaires a été remarqué. Il y a également une augmentation du nombre de cellules prolifératives au niveau des cryptes intestinales corrélant avec une augmentation des niveaux de ?-caténine et des cyclines D. Finalement, une augmentation du contenu protéique par cellule a également été observée ainsi qu'une activation de la voie mTOR. En conclusion, nos résultats montrent que la phosphatase PTEN est impliquée dans l'établissement de l'architecture générale de l'intestin et qu'elle contrôle la synthèse protéique, la migration, le cycle cellulaire ainsi que la différenciation des cellules épithéliales intestinales. De plus, nos résultats indiquent que la perte d'expression de PTEN pourrait influencer la progression des cancers colorectaux. [Symboles non conformes]

Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Glutamic Acid Decarboxylase (GAD) is a rate-limiting enzyme responsible for synthesis of the inhibitory neurotransmitter GABA. Dopaminergic denervation in rodents by unilateral injections of 6-OHDA or MPTP causes an increase in Gad67 mRNA in the striatum, which is further exacerbated by administration of L-Dopa (Horvath et al., 2011; Katz et al., 2005 Bacci et al., 2002). Denervation of nigrostriatal neurons is the key pathological hallmark of Parkinson's disease, which results in hypokinetic movement and rigidity. Medium spiny projection neurons of the striatum comprise 95% of the neuronal population and utilize Gad67 (encoded by the Gad1 gene) for the synthesis of basal levels of GABA. The contribution of Gad67 to GABA signaling in medium spiny projection neurons in the striatum has not been thoroughly understood in normal or Parkinsonian states. Mice with a deletion in Gad67 in Gpr88 expressing neurons were generated by crossing mice with a floxed exon 2 of Gad1 with mice expressing Cre recombinase under the control of the Gpr88 promoter. The aim of this study was first, to characterize mice with a deletion in striatal Gad67 by immunohistochmical, electriophysiological and behavioral examination to determine whether Gad67 expression contributes to sensorimotor and learning tasks. And next, to investigate whether a downregulation in striatal Gad67 would decrease dyskinesia and affect the impaired motor symptoms following dopaminergic denervation with a unilateral 6-OHDA lesion and subsequent treatment with L-Dopa. In this study, neuronal Gpr88 expression was indicated by GFP reporter expression, which resulted from Cre-mediated excision of exon 2 of the Gad1 gene. Gpr88 expression was confirmed in the striatum, olfactory tubercle, cortex and brain stem. Furthermore, Gpr88 was confined to striatonigral and striatopallidal MSNs in the striatum. Additionally, Cre-mediated GFP reporter expression indicated that Gpr88 expression occurs throughout various brain regions, including the motor and visual areas of the cortex, amygdala, hippocampus and cerebellum during development. The developmental expression of Gpr88 seems to be a highly regulated process that occurs throughout the brain. In the conditional knockout mouse, deleting striatal Gad67 resulted in an upregualtion of Gad67 in the globus pallidus and downregulation in the substantia nigra. The changes in Gad67 expression indicate the effects of inactivating GABAergic signaling in striatonigral and striatopallidal MSNs in the direct and indirect pathways. Mice with a deletion in striatal Gad67 demonstrated compromised performance in spatial learning in the Morris water maze, suggesting that GABAergic striatal signaling in the direct and indirect pathways accounts for cue-based learning and spatial memory. However, inactivation of GABAergic signaling in striatonigral and striatopallidal MSNs does not account for motor deficits such as bradykinesia, akinesia or hypokinesia in intact mice; instead it perpetuates hyperkinetic motor activity. In the second experiment of this study, dopaminergic denervation by a unilateral 6-OHDA lesion induced bradykinesia and hypokinetic motor behavior, as demonstrated by impaired performance in the rota-rod and pole test. Additionally, L-Dopa administration to 6-OHDA lesioned mice evoked abnormal involuntary movements (AIMs) to the same degree in all dyskinetic mice. A deletion in striatal Gad67 did not decrease symptoms of dyskinesia, nor cause a lessening of motor impairment caused by dopaminergic denervation. Complete inactivation of the indirect pathway is believed to limit the inhibition of unwanted actions and may perpetuate dyskinesia, even when striatonigral MSNs of the direct pathway are inactive.

Understanding the recruitment of multipotent skeletal progenitor cells and the factors that influence their differentiation would be helpful in providing a means for harnessing the regenerative capacity of skeletal progenitor cells in bone tissue engineering. In order to track the recruitment of skeletal stem cells in fracture healing, transgenic mice containing a Tamoxifen-inducible Cre recombinase that had been placed under the control of a 2.4 kb Prx1 promotor were used to induce conditional expression in periosteal skeletal stem cells that express the Prx1 gene. In order to initially see the cells expressing Prx1, a green fluorescent protein gene (GFP) had also been put downstream to the Prx1 promotor. We then crossed these Prx1CreER-GFP transgenic mice with a second strain containing the Beta-galactosidase gene that becomes constitutively expressed after recombination by the Cre recombinase. The enzymatic activity of Beta-galactosidase was then used to generate a colormetric staining reaction that was used to visualize the cells in which recombination had occurred based on a blue staining product. The recombination activity should only be present in Prx1 expressing cells and their progeny. The goal of the present study was to assess several different approaches to optimize the Beta-galactosidase enzymatic staining protocol and to visualize the Prx1-expressing cells during fracture healing. These studies further examined those populations of cells in the fracture calluses that became labeled and arose from the stem cell populations that had expressed Prx1 at post-operative day 7 and 14. The optimization of a staining method for histology will allow this study to track Prx1 cell fates in a fracture model both in response to specific drug treatments, mechanical loading of the fracture during healing and under pathological conditions that effect healing.

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

The Cre sequence (ACGT) is a site responsible for the binding of specific transcription factors that determine the activation of genes. Due to its major role in gene transcription, it has become a subject of immense research. The binding of transcription factors to the Cre binding site has been determined to be dependent on DNA conformation. In this study, the effects of flanking sequence around the Cre binding site on the conformation and the dynamics of DNA were investigated. The Cre binding site was studied in its native form with differing flanking sequences to determine the BI/BII profile (conformation) and the magnitude of the energy transition barrier (dynamics) between the BI and BII conformations of each phosphate step of the following three dodecamer sequences: CreACAG, CreGGAG, and CreTATA. In order to obtain the BI/BII profile of each phosphate step, 2D 31P-NMR NOESY and HSQC experiments at various temperatures were utilized. Based of the basic principles of kinetics, the lower the energy barrier between the two conformations, the easier the transition between the BI and BII conformation. Therefore, it was hypothesized that low and high %BII character lead to a large energy barrier (high ∆G‡ values), whereas average %BII character leads to a small energy barrier (low ∆G‡ values). The results of the 2D 31P-NMR experiments of the three dodecamer sequences confirmed this relationship between the %BII character and the magnitude of the energy barrier (∆G‡). However, further conformation and dynamics studies must be conducted to further understand the correlation.

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.