We designed and engineered a yeast genetic screen to isolate novel transcriptional machinery components such as elongation factors and chromatin remodelling components. When artificially recruited to a promoter, these components can stimulate gene expression. For example, fusion of known machinery components to the bacterial DNA binding domain (DBD) of LexA results in activation of test genes bearing a binding site for LexA. In a previous study, the coding region of the LexA DBD was fused to random bacterial DNA sequences and, surprisingly, 1% of all random sequences tested were able to activate transcription simply because they were negatively charged. By inserting the LexA site downstream of the transcriptional start site of our reporter genes, we predict that we will exclude these same negatively charged sequences, which do not have a specific role in transcription. The rationale is that at this location classical activators, which are negatively charged, cannot activate transcription. The ability of transcriptional machinery components to activate transcription from a downstream location, however, was demonstrated. In order to isolate transcriptional machinery components, we prepared a yeast genomic DNA library fused to DNA encoding the LexA DBD for transformation into a yeast strain containing reporters bearing a downstream LexA operator site. Using this system, we may isolate novel components of the transcriptional machinery as well as already identified components. This genetic screen should also be able to isolate transcriptional machinery components from higher eukaryotic organisms.

To elucidate mechanisms involved in the regulation of a number of mammalian cell differentiation events, studies were performed to characterize the transcriptional repression functions of the Groucho/TLEs, Hairy and Enhancer of split (Hes), and Runt-related (RUNX) protein families. These proteins have been shown to have overlapping or antagonistic roles in a number of cell differentiation processes, in both Drosophila and mammals. More specifically, the aim was to elucidate mechanisms by which Hes and RUNX-mediated transcriptional control is modulated, with respect to their interactions with each other and the Groucho/TLE proteins. / It is shown here that mammalian Hes1 requires TLEs to repress gene expression, similar to the requirement Drosophila Hes proteins have for Groucho. It is shown further that TLEs also interact with mammalian RUNX proteins and inhibit RUNX's ability to transactivate gene expression. In contrast, Hes1 also physically associates with RUNX and enhances RUNX-mediated transactivation. These results, together with our additional finding that RUNX can inhibit Hes1-mediated transcriptional repression, suggest for the first time that RUNX and Hes1 proteins regulate each other's transcriptional functions. / This thesis also shows that Hes1 can be found in a discreet nuclear subcompartment involved in the regulation of gene expression, termed the nuclear matrix. Nuclear matrix binding is mediated by the same Hes1 C-terminal domain that acts as a binding site for Groucho/TLEs, which also localize to the nuclear matrix and are required for Hes1-mediated transcriptional repression. Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of its C-terminal region, indicating that Groucho/TLEs act as transcriptional corepressors for Hes1. / Together, the material presented in this thesis suggests that Hes1 and RUNX proteins interact with each other to modulate gene expression in the development of a number of tissues.

Protein translation in mammalian cells can be divided into three stages: initiation, elongation, and termination, which require several factors. The peptide elongation factor 1A (eEF1A-1), which was formerly referred to as eEF-1alpha, is a guanosine triphosphate (GTP) binding protein and it is responsible for bringing aminoacyl-tRNA to the ribosomes in the process of protein synthesis. The S1/eEF1A-2 factor also referred to as S1, is an isoform of eEF1A-1. Both proteins are expressed from two distinct genes and share 92% identity in their amino acid sequences. Besides the tissue specific expression S1/eEF1A-2, little is known about the functions of the S1/cEF1A-2 isoform. The objective of this thesis is Thus to investigate the function of the newly discovered peptide elongation factor A-2. The fact that the eEF1A-1 and Sl/eEF1A-2 isoforms' expressions are inversely controlled during development, led me to hypothesise that S1/eEF1A-2 down-regulates eEF1A-1 expression. The goal of the present work was to establish whether S1/eEF1A-2 is responsible for the down-regulation of eEF1A-1 during development in brain, heart and muscle, and how its expression influences cell biology. To address this hypothesis, several cell lines were transduced with an adenovirus expressing S1/eEF1A-2. Ectopic expression of S1/eEF1A-2 in the cardiomyocyte cell line H9c2 led to a down-regulation of eEF1A-1. Similar findings were observed in neuron-differentiated P19 cells, Hela cells, and WI38 cells. Furthermore, S1/eEF1A-2 expression led to a reduced rate of peptide elongation as demonstrated by ribosomal transit time analyses. My data suggest that S1/eEF1A-2 may compete with eEF1A-1 in peptide elongation, leading to a reduced elongation rate, which could be responsible for the relative down-regulation of eEF1A-1. This would imply that terminally differentiated cells, which express high levels of S1/eEF1A-2 (neurones, myocytes, and cardiomyocytes), have a distinct kinetic of peptide elonga

DNA replication initiates at replication origins and involves the ordered assembly of a pre-Replicative Complex (pre-RC), consisting of the ORC1-6, Cdc6, Cdt1 and MCM2-7 proteins. The Ku heterodimer (Ku70/Ku80) was previously implicated in the pre-RC assembly but its mechanism of action is yet unknown. Here, Ku is shown to form a complex with DNA topoisomerase IIβ (topo IIβ) both in vitro and in vivo and target it onto replication origins. The enzymatic activity of topo IIβ creates transient, origin-specific dsDNA breaks thus affecting the DNA topology during pre-RC assembly. Pharmacological inhibition of the topo II activity interferes with the pre-RC assembly at the level of ORC, Cdt1 and Cdc45 recruitment resulting in prolonged G1 phase. Similarly, RNAi-mediated inhibition of the Ku80 expression leads to reduced chromatin loading of pre-RC proteins, decreased origin activation, and activation of a cell cycle checkpoint through a mechanism involving low Cdk2 activity. Overall, the data implicate Ku in the initiation of DNA replication through regulation of the DNA topology and suggest the interplay of the DNA replication and cell-cycle machineries. Pre-RC assembly is followed by the recruitment of the replicative machinery which initiates DNA replication. Study of the chromatin structure of the human lamin B2 and hOrs8 and the monkey Ors8 origin revealed that dynamic changes within the levels of post-translationaly modified histone H3 occur during their activation, which differ between early- and late-firing origins. Late origins have a compact chromatin structure during S phase, which transiently opens during their activation, whereas early origins display histone modifications associated with open chromatin structure, which is only passively affected during their activation by passage of the replication fork. / La réplication de l’ADN commence aux origines de réplication et nécessite l’assemblage méthodique du complexe pré-réplicatif (pre-RC), qui consiste des protéines ORC1-6, Cdc6, Cdt1 et MCM2-7. L’hétérodimer Ku (Ku70/Ku80) a été précédemment impliqué dans l’assemblage du pre-RC, mais le mécanisme de cette action est inconnu jusqu’à présent. Ici, Ku est montré former un complexe avec la topoisomérase IIβ (topo IIβ), tant in vitro qu’in vivo et le conduire aux origines de la réplication. L’activité enzymatique de topo IIβ crèe de brisures transitoires et specifiques aux origines, lesquelles affectent la topologie de l’ADN pendant l’assemblage du pre-RC. L’inhibition pharmacologique de l’activité de topo IIβ tripote avec l’assemblage du pre-RC au niveau du récrutement des protéines ORC, Cdt1 et Cdc45, ayant comme résultat la prolongation de la phase G1. De même, une inhibition de l’expéssion de Ku80 mène à un chargement réduit des proteins du pre-RC sur la chromatine, à une activation réduite des origines de réplication, ainsi qu’ à l’activation d’un contrôle du cycle céllulaire par un mécanisme qui implique une activité réduite de Cdk2. En général, les donnés impliquent Ku dans l’initiation de la réplication de l’ADN par la régulation de la topologie de l’ADN et suggèrent une réciprocité entre les machinéries de la réplication de l’ADN et du cycle cellulaire. L’assemblage du pre-RC est suivi du récrutement de la machinérie réplicative, qui initie la réplication de l’ADN. Une étude de la structure de la chromatine des origines de la réplication humaines lamin B2 et hOrs8, ainsi que celle du singe Ors8, a montré des changements dynamiques aux niveaux des histones H3 modifiés qui ont lieu pendant leur activation et lesquelles diffèrent entre les origines qui sont actives tôt et tard dans la phase S. Les dernières possèdent une structure de chromatine qui est co

Elongation factor-1 alpha (EF-1$ alpha$) is a ubiquitous, highly conserved protein, that functions in peptide elongation during mRNA translation. A cDNA, S1, was isolated in rat; its predicted amino acid sequence shares high similarity (92.6%) with mouse and human EF-1$ alpha$. The principal objective of this thesis is the molecular characterization of S1, a member of the mammalian EF-1$ alpha$ gene family. We have cloned an EF-1$ alpha$ cDNA in rat, and showed that rat S1 and rat EF-1$ alpha$ are two different mRNAs that share extensive sequence similarity in the coding region, but differ considerably in their noncoding regions. The 3$ sp prime$ noncoding regions of S1 and EF-1$ alpha$ were subcloned into the pGEM3z plasmid and cRNA probes were synthesized using an in vitro transcription method. Northern analysis and RNase protection assays have shown that S1 mRNA can only be detected in rat and mouse brain, heart, and muscle, whereas EF-1$ alpha$ mRNA can be detected in all tissues. These results were confirmed by non-radioactive in situ hybridization. A strong S1-positive signal was detected in the hippocampus, cerebral cortex, medulla, and cerebellum of rat brain, whereas liver sections were negative. Certain cells, such as Purkinje neurons in the cerebellum, and motor neurons of the lower brain stem, were S1-positive. On the other hand, EF-1$ alpha$ mRNA was detected in all cell types that we have examined so far. These results prompted us to hypothesize that S1 is a terminal differentiation-specific EF-1$ alpha$ isoform, whose expression is limited to permanently growth-arrested cells. We tested this hypothesis by investigating the expression of S1 and EF-1$ alpha$ during rat development. S1 upregulation correlates with the terminal differentiation process during in vivo neurogenesis and myogenesis. Furthermore, S1 expression is dependent upon the formation of multinucleated myotubes during myogenesis in culture. EF-1$ alpha$ mRNA levels are down-regulated

The KT cell line was developed to study the nature of spontaneous mutations in eukaryotic cells. The plasmid pSV2neoKT, containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene, was used to transfect human AK143tk$ sp-$ cells. A $tk sp+$, G418$ sp{ rm R}$ clone, containing a single integrated copy of the plasmid, was isolated and expanded to form the KT cell line. / Plasmid rescue was designed to recover a 15kb autonomously replicating plasmid containing the integrated pSV2neoKT sequences and the cellular DNA surrounding the integration site. Concurrently, a second method was pursued to clone each junction site separately as two smaller fragments using the cloning vectors pUC119 or pBR322. / Three variations of the polymerase chain reaction (PCR) were also used to amplify the cellular DNA flanking the integration site: anchored PCR, single-specific-primer PCR and inverse PCR.

Aberrant activation of Notch receptors has been implicated in breast cancer. We and other have shown that the expression of a mutant Notch transcript coding mostly for the intracellular domain of Notch1 (Notch1IC) causes transformation of cells in culture and development of tumors in transgenic mice. However, the mechanisms contributing to Notch1IC-induced tumor formation remain elusive and the long latency before the appearance of tumors in Tg mice seems to indicate that Notch requires the collaboration of secondary mutations to induce transformation and tumorigenesis. In the aim of studying direct downstream effects of Notch1IC expression, we generated a Tet-ON inducible expression system for Notch1IC in Hc11 mammary epithelial cells. In the inducible cell lines established, expression of the transgene is only activated upon addition of doxycycline (DOX) to the culture media. The inducible cells were capable of forming colonies in soft agar when subjected to continuous DOX induction and transplantation of the inducible cells in DOX-treated recipient mice caused tumor formation with metastasis to the lungs. We performed a genome-wide microarray-based expression analysis to compare gene expression following 24-hr Notch1IC induction to that of their uninduced counterparts. There were 26 genes identified as being upregulated 2-fold and more upon expression of Notch1IC, while 5 genes were found to be downregulated. Most of these genes identified represent novel candidate Notch1 targets. Of the candidate Notch1IC targets found to be upregulated, expression of the transcript for M-cadherin, also known as CDH15, was most significantly elevated with a change of over 19-fold. M-cadherin is a member of the cadherin family of type I single-pass transmembrane domain proteins that mediate calcium-dependent cell adhesion. M-cadherin, first identified in myogenic mouse cells, is found predominantly in developing skeletal muscles and is highly expressed during secondary myogenesis. In mature skeletal muscle, M-cadherin is mainly detectable in satellite cells. A role for M-cadherin in tumors of epithelial origin has not been previously documented, nor has it been associated with the Notch signaling pathway. We first confirmed that M-cadherin was significantly upregulated by semi-quantitative RT-PCR in the Notch1IC-inducible system. In a time-course experiment, M-cadherin upregulation was observed within 2 hours of Notch1IC induction. In vivo, Notch1IC expression in mammary tumors from Tg mice also correlated with strong expression of M-cadherin, whereas M-cadherin was undetectable in non-Tg mammary glands. We further determined that the transcriptional upregulation of M-cadherin occurs, at least in part, through the canonical CSL-dependent Notch1 pathway. Moreover, using shRNA-mediated depletion of M-cadherin in cell lines derived from mammary tumors from our MMTV/Notch1IC, we were able to investigate the function of M-cadherin in Notch1IC-induced oncogenesis. Through in vitro and in vivo assays, M-cadherin was shown to be required for the transformation of MMTV/Notch1IC cells as well as for their ability to form tumors in mice. In light of these findings, we also examined gene expression of M-cadherin in several human cancer cell lines, including different subtypes of breast cancers. M-cadherin expression was confirmed and appears to also be activated by Notch1IC in a number of breast cancer cell lines. A cursory survey of publicly available human cancer gene expression datasets further corroborated an implication for M-cadherin in human cancers, as well as a correlation between the levels of expression of M-cadherin and Notch1. A better understanding of the mechanisms of action of M-cadherin could shed light on targeted therapeutic approaches for the treatment of Notch1-overexpressing cancers and possibly other human carcinomas. / L'activation aberrante des récepteurs Notch a été impliqué dans le cancer du sein. Notre groupe ainsi que quelques autres ont démontré que l'expression d'un transcrit Notch1 muté, codant principalement pour le domaine intracellulaire de Notch1 (Notch1IC) provoque la transformation des cellules en culture et le développement de tumeurs chez les souris transgéniques. Cependant, les mécanismes contribuant à la tumorigénèse induite par Notch1IC demeurent méconnus et la longue période de latence avant l'apparition de tumeurs chez les souris Tg semble indiquer que Notch nécessite la collaboration des mutations secondaires pour engendrer la transformation cellulaire et la formation de tumeurs. Dans le but d'étudier les effets directs en aval de l'expression de Notch1IC, nous avons généré un système d'expression inductible Tet-ON pour Notch1IC dans les cellules épithéliales mammaires Hc11. Dans les lignées cellulaires inductibles établies, l'expression du transgène n'est activée que lors de l'addition de doxycycline (DOX) au milieu de culture. Les cellules inductibles sont capables de former des colonies en agar lorsqu'elles sont induites à la DOX en continu, et elles forment des tumeurs avec métastases aux poumons lorsque transplantées dans des souris traitées à la DOX. Nous avons effectué une analyse de l'expression du génome entier par micropuce dans le but de comparer l'expression des gènes à la suite de l'induction Notch1IC durant 24 heures, à celle de cellules homologues non-induites. 26 gènes ont été identifiés comme étant régulés à la hausse (2 fois et plus) suite à l'expression de Notch1IC, tandis que 5 gènes ont été identifiés comme étant régulés à la baisse. La plupart des gènes ainsi identifiés représentent de nouvelles cibles candidates de Notch1.Parmi ces cibles candidates, l'expression du transcrit pour M-cadhérine (CDH15) a été le plus significativement élevée (19 fois). M-cadhérine, une molécule d'adhésion cellulaire, a été identifiée dans les cellules myogéniques de souris; la protéine est principalement exprimée durant le développement de muscles squelettiques et au cours de la myogenèse secondaire. Dans le muscle squelettique mature, M-cadhérine est principalement détectable dans les cellules satellites. Fait intéressant, un rôle pour M-cadhérine dans les tumeurs d'origine épithéliale n'a pas été précédemment documenté, d'autant plus que M-cadhérine n'a pas été associée à la voie de signalisation Notch.Nous avons d'abord confirmé la surexpression de M-cadhérine par RT-PCR semi-quantitatif dans les cellules Notch1IC-inductibles. In vivo, l'expression de Notch1IC dans les tumeurs mammaires de souris Tg corrélait également avec une forte expression de M-cadhérine. Nous avons également déterminé que la régulation de la transcription de M-cadhérine se produit, au moins en partie, par la voie de signalisation canonique (CSL-dépendante) de Notch1. Par ailleurs, en utilisant des shRNA pour supprimer l'expression de M-cadhérine dans des lignées cellulaires dérivées de tumeurs mammaires provenant de nos souris MMTV/Notch1IC, nous avons pu étudier la fonction de M-cadhérine dans l'oncogénèse induite par Notch1IC. Par le biais d'essais in vitro et in vivo, nous avons démontré que M-cadhérine était requise pour la transformation des cellules MMTV/Notch1IC ainsi que pour leur capacité à former des tumeurs chez la souris. Par la suite, nous avons également confirmé l'expression de M-cadhérine dans plusieurs lignées cellulaires de cancer du sein. Un bref survol de bases de données d'expression génique dans des cancers humain suggère que M-cadhérine serait impliquée dans plusieurs types de cancers, et qu'il y aurait une corrélation entre les niveaux d'expression de M-cadhérine et de Notch1 dans certains cancers du sein.Une meilleure compréhension des mécanismes d'action de M-cadhérine pourrait mener à de nouvelles approches thérapeutiques ciblées pour le traitement des cancers surexprimant Notch1.

Inherited disorders of calcium homeostasis, familial hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are caused by inactivating mutations, and autosomal dominant hypocalcemia (ADH) is caused by activating mutations in the calcium-sensing receptor (CASR). This receptor belongs to group C of the G-protein-coupled receptor (GPCR) superfamily, one of the largest groups of cell-surface receptors. The CASR is expressed abundantly in the parathyroid gland and in the cells lining the kidney tubule and senses small changes in circulating calcium concentration and once activated it inhibits parathyroid hormone (PTH) secretion and renal tubule calcium reabsorption. The CASR undergoes core (immature) N-linked glycosylation in the endoplasmic reticulum (ER). Once appropriately folded, the core-glycosylated receptor transits through the Golgi apparatus, becomes fully (mature) glycosylated, and is expressed at the cell surface. / The main focus of this Ph.D thesis work was a functional characterization of mutant CASRs. At present over 100 CASR mutations have been published. Our lab has created a database http://www.casrdb.mcgill.ca/, of these mutations which is regularly updated. The CASRdb helps to better understand and analyze the mutations with respect to clinical and biochemical profiles, and aids in receptor structure-function analyses in addition to providing an important communication link between researchers working on this receptor and related areas. / We examined the DNAs from patients with calcium homeostasis disorders and identified mutations and polymorphisms in the coding region of the CASR gene in several of these individuals. Most of the mutations are novel and some are recurring. / Two of the identified mutations (L11S and T14A) are in the signal peptide of the receptor (another mutant, L13P, was previously reported but not analyzed functionally). In the CASR, the N-terminal signal peptide of 19 amino acids likely plays a key role in directing the nascent polypeptide chain into the ER. We demonstrated that indeed the wild-type CASR, and mutant T14A, nascent polypeptides were targeted to the ER in vitro, and underwent core N-glycosylation. In contrast, the L11S and L13P mutants did not translocate into the ER and did not undergo glycosylation. This study is the first to examine the function of the CASR signal sequence and shows that naturally occurring mutations that disrupt the hydrophobic core of this region lead to impaired cotranslational processing causing parathyroid dysfunction. It is the first study to analyze the impairment of a GPCR's signal peptide region resulting in human disease. / The CASR exists at the plasma membrane as a homodimer although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H or R66C inactivating mutations (identified in FHH patients), which cause the receptor to be retained in the ER. Using a biochemical and biophysical analysis we demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER whereas another mutant, N583X, was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface. FHH may present atypically as familial isolated hyperparathyroidism (FIHP). I have performed in vitro analyses of mutations L159P, E250K, V268delfsX273, T445A, and R886P identified in FIHP kindreds. In addition, we have identified two novel inactivating mutations (M74L and L521delfs.X555) in FHH patients. / We have analyzed all mutants by transiently transfecting them in human embryonic kidney (HEK293) cells and comparing them to the wild-type receptor. Western blot analysis demonstrated that the mutant receptors were, in general, as well expressed as the wild-type receptor. However, we observed that the 2 truncation mutants (V268delfsX273 and L521delfsX555) migrated at lower molecular weights. Immunocytochemistry and confocal microscopy studies showed that both of these mutants, as well as some of the others, had impaired cell surface expression. Several mutants had impaired MAPK responses to increasing extracellular calcium concentrations relative to the wild-type receptor. On the other hand, T445A behaved like the wild-type receptor in all the assays, suggesting that this change represented a rare benign polymorphism. In addition, three common CASR polymorphisms (A986S, R990G and Q1011E) were identified in several individuals, and were expressed at similar levels to wild-type on western blot analysis. R886P was the least impaired mutant in all assays. E250K was responsive to some extent in the MAPK assay, but the rest of the mutants had clearly impaired function. Identification and functional analyses of naturally-occurring CASR mutations provides important clinical information to physicians and patients. In addition, assessment of functional deficiencies of particular mutants provides new insight into how the receptor works.

Her2 is amplified in 25-30% of breast cancer cases, but there are no mutations that correspond with this amplification (Her2wt). A naturally occurring splice variant of human Her2 excludes exon 16 (Her2-delta-Ex16) and encodes a constitutively active receptor. It is possible that genomic amplification of her2 could increase the expression of Her2?Ex16 past a threshold level required for carcinogenesis. Her2wt and the alternatively spliced Her2-delta-Ex16 were expressed in a physiologically relevant cell line and characterised. First, it was remarked that the tertiary structure of regions both proximal and distal to the domain encoded by exon 16 differed between Her2wt and Her2-delta-Ex16 protein. Second, Her2-delta-Ex16 appeared to be resistant to metalloproteinase-mediated cleavage. Third, Her2wt was observed to internalise through EEA1-positive endosomes, translocate to the nucleus and be responsive to the drug geldanamycin while Her2-delta-Ex16 was largely retained in the plasma membrane and was only slightly responsive to geldanamycin. / Le gène her2 soit amplifié dans 25-30% des cas de cancer du sein, et aucune mutation ne semble accompagner cette amplification génomique. Il existe un variant naturel d'épissage du gène Her2 présentant une délétion au niveau de son d'exon 16 (Her2?Ex16) qui conduit à l'expression d'un récepteur constitutivement actif. Il est possible que l'amplification génomique d'her2 pourrait augmenter l'expression d'Her2?Ex16 et favoriser ainsi la carcinogenèse. Her2wt et Her2-delta-Ex16 étaient exprimés de manière hétérologue dans des cellules normales de glandes mammaires de souris (NMuMG). Premièrement, la structure tertiaire des régions proximales et distales au domaine codé par l'exon 16 différait entre les protéines Her2wt et Her2-delta-Ex16. Diexièmement, Her2-delta-Ex16 était résistant au fendage du metalloproteinase. Troisièmement, Her2wt internalisait par des endosomes, transférait au noyau et était sensible au geldanamycin tandis que Her2-delta-Ex16 était en grande partie maintenu dans la membrane cellulaire et était légèrement sensible au geldanamycin.

Gliomas are the most common primary brain tumours found in adults. The median survival of patients diagnosed with the most malignant form, glioblastoma multiforme (GBM), is 9-12 months and has changed little over the years despite advances in medical technology. Gene therapy may offer new solutions to treat this resistant disease. Hence, we tested three different gene therapy strategies. In our first study, we tested the efficacy of targeted therapy to correct common aberrations found in gliomas including amplification/mutation of receptor tyrosine kinases (RTK) and loss of PTEN, which result in an overactive PI3K/Akt pathway. Without PTEN, FOXO transcription factors are inactivated, and the cell becomes resistant to apoptosis and cell cycle arrest. By using an adenoviral vector (AdV) expressing an activated FOXO1 mutant (AdFOXO1;AAA), we restored apoptosis and cell cycle arrest, reduced tumour volume and prolonged survival in an intracerebral xenograft model. Secondly, we examined the therapeutic capacity of a novel replicating/non-disseminating AdV expressing the fusion protein of cytosine deaminase and uracil phosphoribosyltransferase (CU). CU can convert the non-toxic pro-drug, 5-fluorocytosine (5-FC) to the tissue diffusible chemotherapeutic drug, 5-fluorouracil (5-FU) to target dividing cells. In vitro, the replicating vectors were superior to the non-replicating vectors, but the fully replicating/disseminating vector did not perform considerably better than the replicating/non-disseminating vector suggesting that dissemination may not be advantageous. In vivo, the replicating/non-disseminating vector administered in conjunction with 5-FC prolonged survival in both an athymic and an immunocompetent mouse model. Moreover, an immune bystander effect in vivo was mediated by macrophages and T cells. Lastly, we investigated a method to harness a tool of the immune system, IFN-ß; this cytokine is known to have anti-angiogenic, anti-proliferative, and immunomo / Les gliomes sont des tumeurs primaires de cerveau les plus communes retrouvées dans les adultes. La survie médiane des patients diagnostiqués avec la forme la plus maligne, le glioblastome multiforme (GBM), est de 9 à 12 mois et a peu changé au cours des années en dépit des avances en technologie médicale. La thérapie génique peut offrir de nouvelles solutions pour traiter cette maladie résistante. Durant nos travaux, nous avons examiné trois stratégies différentes de thérapie génique Dans notre première étude, nous avons examiné l'efficacité de la thérapie visée à corriger des anomalies communes retrouvées dans les gliomes, comprenant l'amplification/mutation de récepteurs de type tyrosine kinase (RTK) et la perte de PTEN, qui mènent en conséquence à une voie activée de PI3K/Akt. Sans PTEN, les facteurs de transcription FOXO sont inactivés, et la cellule devient résistante à l'arrêt du cycle cellulaire et à l'apoptose. En utilisant un vecteur adénoviral (AdV) exprimant une protéine activée du mutant FOXO1 (AdFOXO1;AAA.), nous avons reconstitué les signaux pour l'arrêt du cycle cellulaire et l'apoptose in vitro ainsi que in vivo. Deuxièmement, nous avons examiné la capacité thérapeutique d'un nouveau vecteur adénovirale qui a la capacité de se répliquer sans provoquer de lyse cellulaire et qui exprime en plus la protéine de fusion uracile phosphoribosyltransférase/cytosine déaminase (CU). La protéine CU peut convertir le promédicament non-toxique, le 5-fluorocytosine (5-FC) à la drogue chimiothérapeutique diffusible, le 5-fluorouracile (5-FU) qui a comme cible des cellules en division cellulaire. In vitro, les vecteurs à capacité de répliquation étaient meilleurs que ceux qui ne pouvaient pas se répliquer. In vivo, le vecteur en présence du 5-FC a prolongé la survie de deux modès animaux (avec et sans sytèmes immunitaires). Dans un dernier temps, nous avons étudié une méthode pour exprimer l'IF