Retinoic acid (RA), the major biologically active form of vitamin A, plays important roles in regulating a broad range of biological processes. / Progressive loss of RARs is associated with skin carcinogenesis both in human and animals. Despite such observations, the biological significance of RAR loss in skin carcinogenesis has not yet been clarified. To this end, we established keratinocyte cell lines deficient in RARalpha, RARgamma, or both and employed a well-established tumorigenesis model to investigate whether loss of RARs is causally related to skin tumorigenesis. We found that RARgamma is the major RAR subtype mediating the growth and AP-1 inhibitory effects of RA on keratinocytes in vitro. Consistent with this observation, loss of RARgamma, but not RARalpha, predisposed keratinocytes to tumor formation, suggesting that RARgamma may act as a tumor suppressor. Reconstitution of RARs in the RARalphagamma-/- keratinocytes inhibited their tumorigenic potential, further proving that RARs have tumor suppressive effects. / As expected, expression of dnRARalpha resulted in profound epidermal defects. Intriguingly, dnRAalphaDBD caused a virtually identical skin phenotype, suggesting that dnRARalpha acts to affect epidermal development via a DNA-binding-independent mechanism. The epidermal phenotype of these transgenic mice is reminiscent of that seen in the p63-/- mice, and p63 expression was indeed significantly reduced in the epidermis expressing dnRARalpha or dnRARalphaDBD, suggesting that downregulation of p63 by dnRARalpha may be attributable to the epidermal phenotypes associated with the transgenic mice. These observations also suggest that DNA-binding is not required for dnRARalpha to attenuate p63 expression in the epidermis. Consistent with these observations, I also found that p63 is indeed not a RAR-target, as no overt changes in p63 expression were observed in the RARalphagamma-/- epidermis, which appeared normal. (Abstract shortened by UMI.)

Acute Promyelocytic Leukemia (APL) results from an accumulation of undifferentiated promyelocyte progenitors. Complete clinical remission of APL can be achieved through therapy with retinoic acid (RA). However, patients treated with RA alone almost invariably develop resistance to RA. We have found that tumor necrosis factor (TNF) and RA can synergize to induce differentiation of RA sensitive APL cells in a much shorter period of time than RA alone. In addition, this combination can also overcome the maturation block of RA resistant APL cells and the non-APL leukemia cell line U937 leading to differentiation. Correlating with this, we found synergistic induction of several genes at early and late time points in both the RA sensitive and resistant cell lines. Through use of neutralization antibodies, we found the protein product of one of these genes, the receptor for the macrophage colony stimulating factor, was important in mediating the differentiation effects of TNF and RA. / To better understand transcriptional activation with RA and TNF we used the promoter of a gene synergistically induced by RA and TNF, Dif-2, as a model to investigate the mechanism whereby the two agents interact on the level of transcription at early time periods. We used ChIP analysis to study the accessibility of the promoter to binding by various transcription factors and the recruitment of cofactors in response to RA and TNF. We found that upon RA treatment, there was a release of corepressors and a decrease in histone methylation. This was accompanied by a subsequent increase in binding of the transcription factor PU.1, a recruitment of coactivators, as well as Snf5 (a component of the Swi/Snf complex), and an increase in histone acetylation. Interestingly, TNF could only stimulate NF-kappaB (downstream effectors of TNF) binding to the Dif-2 promoter when cells were cotreated with RA. Furthermore, TNF and RA lead to a heightened level of active, phosphorylated RNA Pol II at the Dif-2 promoter. Correlating with this was heightened recruitment of TFIIH, a known Pol II kinase. This may represent a mechanism whereby RA and TNF act in synergy to activate Pol II. These data suggest a novel mechanism for synergy between signaling pathways where RA can trigger a conformation change in a target promoter/enhancer region resulting in a more open conformation that is conducive to transcription factor binding in response to other stimuli.

Telomerase is an unusual reverse transcriptase (RT) that catalyzes the de novo addition of telomeric DNA repeats to telomeres. Telomerase activity counteracts the progressive loss of telomeric DNA over successive rounds of DNA replication, and is important for the immortality of most eukaryotic cells. Telomerase is distinct from other RTs in that its catalytic subunit (TERT: telomerase reverse transcriptase) stably associates with a telomerase RNA (TR) component that contains a short template used to direct synthesis of telomeric repeats. Telomerase also exhibits a unique, repeat addition form of processivity that permits the addition of multiple telomeric repeats to a single substrate by repetitive reverse transcription of the template. The TERT proteins consist of a central region of RT-conserved motifs, which is flanked by extensive telomerase-specific N- and C-terminal sequences. The N terminus constitutes nearly half of the TERT protein, and is an excellent candidate site for telomerase-specific catalytic functions. Using a mutagenic approach, we investigated the contributions of human TERT N-terminal sequences to telomerase catalytic function and nucleic acid interactions. We found that the hTERT N terminus contains two RNA interaction domains, RID1 and RID2. RID1 was functionally and physically separable from the remainder of hTERT, and may constitute an hTERT polymerase accessory domain. We investigated the catalytic function of two RID1- and RID2-interacting regions in the human TR, the pseudoknot/template domain and the P6.1 helix. RID1-pseudoknot/template domain interactions were essential for repeat addition processivity, and RID2-P6.1 interactions mediated telomerase assembly and were required for basic polymerase function. Repeat addition processivity is thought to be partly dependent on an anchor site(s) in TERT that stabilizes telomerase-DNA interactions. We found that RID1 mutations also reduced the affinity of human telo

Liver transplantation continues to be the only remedy for end-stage liver disease. Moreover, the number of recipients far exceeds the number of donors and patients die on waiting lists. Unfortunately, not all grafts survive the process of transplantation and marginal livers are discarded, as they would not tolerate the stresses of ischemia-reperfusion and therefore would not survive the process of transplantation. If we are to resolve these problems, and decrease the chasm between the donor and recipient numbers, we need to characterize how a normal liver survives the process of transplantation. / We have developed a protocol that allows us to characterize the normal liver's response to transplantation. Indeed, a liver that endures the process of transplantation must be able to limit the amount of damage caused by the various stresses related to cold ischemia and warm oxygenated reperfusion. Unfortunately, the process of transplantation is not easily amenable to the reduction much less the elimination of these stresses. Therefore, a means of investigating what happens to the liver during the process of transplantation was needed that would take into account the global effect of these variables on the graft's survival. / The underlying hypothesis of my thesis is that the surviving liver invokes protective mechanisms to moderate the damage that could occur as a result of transplantation in part by regulating the level and type of expressed genes. Using microarray technology, we determined the identity of the mRNAs that revealed a degree of regulation, either up- or down-regulation during the reperfusion phase of transplantation. Furthermore, because ischemia precedes reperfusion, the process of reperfusion per se includes all of the stresses associated with ischemia, e.g. all reperfused livers were ischemia preconditioned. Thus, to conduct our analysis biopsy specimens were taken at three time-points during the peri-reperfusion phase of the operation. Our methodology not only permitted us to identify regulated genes, it also allowed us to control for recipient blood-borne contribution of messenger RNA. Because the last biopsy specimen was taken 1h post-reperfusion, our list was comprised of immediate early genes. / Of the other immediate-early genes that were on our list, we found an up-regulated gene that coded for map kinase phosphatase-1 (MKP-1). Immunohistochemistry preformed on frozen human liver sections revealed expression of MKP-1 in hepatocytes. MKP-1 is a phosphatase is best known for JNK-1, p38MAPK and ERK1/2/5 dephosphorylation. Using transplantation relevant stresses in vitro, and HepG2 as a cellular model for hepatocytes, we characterized mkp-1 mRNA regulation. Furthermore, using the same protocol and MKP-1 shRNA expressed in HepG2, we found that a lack of MKP-1 protein expression increased apoptosis. The second gene, als2, we investigated was slightly down-regulated and coded for a protein called alsin. The latter is a RhoGEF, a guanine nucleotide exchange factor that activates Rac1, Rab5 and Ran. We proceeded to characterize alsin mRNA regulation in vitro using ischemia-reperfusion relevant stresses, as we did for MKP-1. Finally, we are seeking to determine if or how the Rho proteins (Rac1, Rab5 or Ran) are implicated during the "reperfusion" phase of the operation. All in all, our results indicate a hepatic coping mechanism invoked for the purpose of reducing the damage caused by the trauma of ischemia and reperfusion. / La transplantation du foie demeure le seul remède pour les maladies hépatiques mortelles. Malheureusement le nombre de donneurs d'organe est inférieur au nombre de patients. Cette situation s'aggrave lorsque l'on considère qu'un certain nombre de foies transplantés ne survivent pas et que ce ne sont pas tous les foies qui s'avèrent utilisables pour la transplantation. En effet, ces foies de « qualité inférieure » ne survivraient pas au traumatismes de l'ischémie et de la réperfusion donc ils sont refusés. Actuellement il n'existe pas de solution pour pallier ce nombre de foies à transplanter. fr / Nous avons développé une approche permettant de caractériser les mécanismes par lesquels un foie normal survit au processus de la transplantation. Un tel foie doit être en mesure de diminuer ou du moins de contrôler les effets toxiques associés aux stress d'ischémie froide et de la réperfusion tiède oxygénée. Malheureusement, le processus de transplantation ne peut être manipulé facilement de façon à réduire ou à éliminer les variables qui lui sont associées. Compte tenu ces observations, une analyse détaillée de ce phénomène doit tenir compte de l'effet global de ces variables sur la survie de la greffe. / Une hypothèse fondamentale de mon travail de thèse présuppose qu'un foie qui survit au processus de la transplantation doit être en mesure de contrôler les effets toxiques des stress associés à ce processus. Cette régulation peut être accomplie en partie par des mécanismes de contrôle de l'expression des gènes. En utilisant des biopuces à ADN, nous avons déterminé l'identité des ARN messagers régulés positivement ou négativement au cours de la phase de réperfusion de la transplantation. Cette phase de la transplantation est ciblée pour notre étude, puisque que le processus d'ischémie précède la réperfusion, le processus de réperfusion englobe tous les stress associés à l'ischémie. Afin d'accomplir notre analyse, des biopsies hépatiques ont été prélevées à trois intervalles de temps différents. Notre protocole expérimental nous a permis non seulement d'établir une liste de gènes qui étaient régulés à la hausse ou à la baisse, une heure après le ré-établissement du flot sanguin, mais aussi nous a permis d'éliminer les ARN messagers provenant du sang du receveur. Étant donné que la dernière biopsie prélevée était à une heure après la réperfusion du foie par la veine porte hépatique, notre liste de gènes fut composé de gènes immédiats précoces. / Parmi les gènes identifiés, le messager codant pour la map kinase phosphatase-1 (MKP-1), était régulé à la hausse. En utilisant des coupes de foie humain, l'immunohistochimie a révélé l'expression hépatocytaire de la protéine MKP-1. Cette protéine déphosphoryle les map kinases JNK-1, p38MAPK et ERK1/2/5. En simulant les stress d'ischémie et de réperfusion sur des cellules d'origine hépatomique, HepG2, nous avons caractérisé la régulation de l'ARN messager de MKP-1. De plus, en utilisant le même protocole nous avons déterminé les conséquences d'une pénurie de la protéine MKP-1 pour les cellules HepG2. Pour ce, nous avons exprimé du « shRNA » dirigé contre l'ARN de MKP-1, dans ces cellules, afin de diminuer l'expression de MKP-1. Ceci a causé une augmentation d'apoptose. Le second gène à l'étude était celui d'als2 qui code pour la protéine alsine, dont l'ARN messager était légèrement diminué en quantité lors de son évaluation par biopuces. L'alsine est une « RhoGEF », une protéine échangeuse de nucléotides, qui active les RhoGTPases Rac1, Rab5 et Ran. Nous avons procédé à la caractérisation de ce gène, en utilisant les conditions expérimentales préétablies avec MKP-1. La dernière étape se résume à déceler si ou comment les protéines RhoGTPases soient Rac1, Rab5 ou Ran, sont impliquées dans la phase de réperfusion du foie. En conclusion, nos résultats indiquent que le foie transplanté utilise différents mécanismes pour contrecarrer les effets des stress d'ischémie et de réperfusion.

The aim of this study was to create a pancreatic ductal cell line model to study Notch signaling and its effect on endocrine differentiation. / Briefly, Notch2 was silenced in a pancreatic adenocarcinoma cell line, Panc 02.03, by transfecting cells with human Notch2 siRNA. Our results unexpectedly show that despite silencing Notch2, there is evidence of increased Notch pathway activation. First, there is a significant upregulation of Notch ligand Jag1 and Notch target Hes1. Hes1 in turn represses Ngn3, thus we see a significant downregulation of Ngn3. When Ngn3 is repressed, an endocrine fate is inhibited. This notion is supported by a downregulation of pro-endocrine transcription factors NeuroD1, Pax6, and IA1, and also a downregulation of endocrine genes such as insulin, glucagon, and pancreatic polypeptide. Lastly, our data show a significant downregulation of stemness marker Nestin, which further suggests that Panc 02.03 are in a more dedifferentiated state through an increased Notch pathway activation. / We suggest that there may be a compensatory mechanism to maintain Notch activation in Type 2 Diabetes Mellitus (T2DM) as well as in pancreatic adenocarcinoma despite Notch2 gene silencing. Our study thus poses a new question; is an aberrantly active Notch pathway under the master control of an alternate signaling mechanism in T2DM? Elucidating this question will be important in order to develop better therapeutic targets for the treatment of T2DM. / Le but de cette étude était de créer un modèle de cellules canalaires du pancréas pour étudier la signalisation du gène Notch et son effet sur la différenciation des cellules endocrines. / Brièvement, le gène Notch2 a été rendu silencieux dans une lignée de cellules pancréatiques cancéreuses, Panc 02.03, par transfection avec des siARN du gène Notch2. Nos résultats montrent que de manière inattendue, malgré que le gène Notch2 soit rendu silencieux, il y des evidences d'une augmentation de l'activation de la voie Notch. Premièrement, il y a une importante régulation positive du ligand du gène Notch, Jag1 et cible directe Hes1. À son tour, Hes1 réprime Ngn3, ce qui se traduit donc par une régulation négative significative de Ngn3. Ainsi, lorsque le gene Ngn3 est réprimé, la fonction endocrine est inhibée. Cette notion est soutenue par une régulation négative pro-endocrine de facteurs de transcription NeuroD1, Pax6 et IA1, et aussi une régulation négative des gènes endocriniens, tels l'insuline, le glucagon et le polypeptide pancréatique. Enfin, nos données montrent une régulation négative de marqueur des cellules souches, Nestin, qui suggère en outre les cellules Panc 02.03 sont dans un état plus dédifférencié par le biais d'une augmentation de l'activation du passage du gène Notch. / Nous suggérons qu'il y a peut-être des mécanismes de compensation pour maintenir l'activation de Notch dans le T2DM ainsi que dans l'adénocarcinome pancréatique malgré que le gène Notch2 reste silencieux. Notre étude pose donc une nouvelle question: Y a-t-il une activation abberante de la voie Notch dans le T2DM qui serait alors contrôlée par un autre mechanism alternative clé? Élucider cette question sera important afin de développer de meilleures cibles thérapeutiques pour le traitement du diabète de type 2 Mellitus.

Many of the underlying genetic causes of Uveal Melanoma (UM) metastatic disease still remain to be elucidated. This particular cancer is the most common primary malignant intraocular tumor in adults, comprising approximately 5% of all diagnosed melanoma cases. Despite its rarity, approximately 50% of patients diagnosed with UM will develop metastatic disease. Due to the poor understanding of the genetic mechanisms that cause UM metastatic disease, further investigations into elucidating these mechanisms are necessary. Fresh biopsied ocular tumor tissues are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology now allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue to run genomic cancer arrays by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between two UM clinical-histopathological parameters (Metastasis, Cell Type). ANOVA testing identified 106 genes with a significant change in transcript abundance (p < 0.05, Welch T-test) between tumors from patients that died of metastasis to those that did not. Similarly, 64 genes significantly differed in transcript abundance when the spindle and epithelioid cell type parameters were compared. As a predictor of metastasis-dependant death, 12 genes were shown to correctly predict this parameter in 27/37 tumors (73%). As a predictor of UM cell type we successfully predicted the classification of 27/31 (87.1%) non-mixed tumors with a significant difference in transcript abundance between 10 genes.We sought to validate these transcriptional findings at the DNA and protein level using comparative genomic hybridization arrays (aCGH) and immunohistochemistry. LIG4 (predictor of metastasis) and ErbB3 (predictor of UM cell type) correlated well at both levels in independent patient sample populations.In addition, DASL analysis revealed an up-regulation of PAI-1 serum bio-marker expression in patients that developed metastatic disease. ELISA analysis of UM patient plasma revealed a positive correlation of increased PAI-1 plasma protein expression with increasing tumor height (n = 11, r = 0.6525, p = 0.0295). To the best of our knowledge, this is the first study to have performed and validated a retrospective transcriptional analysis using RNA extracted from FFPE primary UM. / À ce jour, l'étude des facteurs génétiques reliés au mélanome uvéal (MU) est en pleine croissance. Ce type de cancer représente la principale forme de tumeur intraoculaire maligne au sein de la population adulte, comprenant approximativement 5% de tous les cas de mélanome diagnostiqués. Plus de 50 % des individus chez qui on a diagnostiqué la présence d'un mélanome uvéal développeront des métastases dans divers organes, principalement au foie. On connaît mal le mécanisme biologique de cette maladie et c'est pour cette raison que l'étiologie du mélanome uvéal est nécessaire.Il est difficile d'obtenir des échantillons de biopsie de tissus cancéreu oculaires pour extraire les acides nucléiques. A ce jour, la technologie nous permet d'extraire l'ARN de tissus fixés dans la formaldehyde et enrobés de paraffine (FFPE « Formaldehyde Fixed and Paraffin embedded »), en totalité pour ensuite analyser les génomes par la méthode cDNA mediated Annealing Sectioning and Ligation (DASL). Notre but est d'établir la corréler les differences des gènes transcrit entre deux paramètres clinico-hispopathologiques MU (Métastase, Type de cellule). A partir des tests ANOVA, nous avons identifié 106 gènes possédant un changement de transcription significatif (p < 0.05, Welch T-test) entre les tumeurs des patients qui sont décédés de métastases et ceux qui ont survecu. De plus, 64 génes possédant un changement de transcription significatif lorsque les paramètres des cellules fusiformes et des cellules épithélioïdes ont été comparés. 12 gènes ont correctement prédit la mort dépendant des métastases dans 27 des 37 tumeurs testés. Grace à cette méthode, notre groupe a réussi à prédire la classification de 27/31 tumeurs non-mixtes entre 10 gènes pour des cellules de type MU.Nous avons validé nos découvertes au niveau de l'ADN et des protéines en utilisant des tests d'hybridization comparative génomique (aCGH) et de l'immunohistochimie. Une bonne corrélation de LIG4 (métastase) et ErbB3 (cellules de type MU) est obtenue dans nos échantillons de population de patients indépendants.De plus, les analyses DASL ont révélé une expression de haute-régulation de sérum PAI-1 bio marqueur dans les échantillons des patients qui ont développé de métastases. D'après les analyses ELISA du plasma des patients avec du MU, une corrélation positive entre l'augmentation d'expression de protéines de plasma PAI-1 avec une augmentation de l'hauteur des tumeurs (n = 11, r = 0.6525, p = 0.0295).D'après nos connaissances, ce travail est la première étude qui ce validé l'analyse rétrospective transcription avec de l'ARN extrait d'échantillons FFPE MU primaire.

Oncolytic virus therapy is a new form of cancer treatment that uses viruses that preferentially infect and lyse cancer cells. Vesicular stomatitis virus (VSV) is a strong oncolytic virus candidate that infects multiple tumor cells, produces rapid viral replication in malignant cells and spreads quickly in the tumor. Defects in the interferon (IFN) antiviral pathway are common in tumor cells and such defects are accountable for the sensitivity to VSV infection and replication in several malignant cells. The intrinsic mitochondrial apoptotic pathway plays a crucial role in VSV-induced apoptosis and disturbance of this pathway is responsible for resistance to VSV oncolysis of cancer cells. The antiapoptotic protein B-cell lymphoma 2 (BCL-2) is commonly overexpressed in tumor cells, especially in hematological malignancies, and is strongly related to resistance to cancer therapy. Chronic lymphocytic leukemia (CLL) is an accumulative disease of mature-looking CD5+/CD19+ lymphocytes, caused by defects in apoptosis rather than increase in proliferation. CLL patients express high levels of the BCL-2 protein which correlates with poor treatment outcome. Small-molecule BCL-2 inhibitors showed promising anticancer properties in preclinical models. A phase I clinical trial demonstrated modest activity against CLL. Based on our observation that CLL cells are resistant to VSV-induced cell death due to overexpression of BCL-2, we hypothesized that inhibition of BCL-2 could restore VSV oncolytic potential in primary CLL cells. In fact, BCL-2 inhibitors EM20-25IVand obatoclax sensitized primary CLL cells to VSV-induced cell death. Mechanistically, while VSV infection triggered Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) upregulation, obatoclax blocked the ability of BCL-2 to dimerize with the proapoptotic BCL2-associated X protein (BAX). Together, NOXA expression and BAX release were able to efficiently induce apoptosis. Moreover, our data demonstrated a direct interaction between NOXA and BAX. Together, these data indicate that the use of BCL-2 inhibitors may improve VSV oncolysis in apoptosis-resistant hematological malignancies characterized by overexpression of anti-apoptotic proteins such as BCL-2. / Oncolytic virus therapy is a new form of cancer treatment that uses viruses that preferentially infect and lyse cancer cells. Vesicular stomatitis virus (VSV) is a strong oncolytic virus candidate that infects multiple tumor cells, produces rapid viral replication in malignant cells and spreads quickly in the tumor. Defects in the interferon (IFN) antiviral pathway are common in tumor cells and such defects are accountable for the sensitivity to VSV infection and replication in several malignant cells. The intrinsic mitochondrial apoptotic pathway plays a crucial role in VSV-induced apoptosis and disturbance of this pathway is responsible for resistance to VSV oncolysis of cancer cells. The antiapoptotic protein B-cell lymphoma 2 (BCL-2) is commonly overexpressed in tumor cells, especially in hematological malignancies, and is strongly related to resistance to cancer therapy. Chronic lymphocytic leukemia (CLL) is an accumulative disease of mature-looking CD5+/CD19+ lymphocytes, caused by defects in apoptosis rather than increase in proliferation. CLL patients express high levels of the BCL-2 protein which correlates with poor treatment outcome. Small-molecule BCL-2 inhibitors showed promising anticancer properties in preclinical models. A phase I clinical trial demonstrated modest activity against CLL. Based on our observation that CLL cells are resistant to VSV-induced cell death due to overexpression of BCL-2, we hypothesized that inhibition of BCL-2 could restore VSV oncolytic potential in primary CLL cells. In fact, BCL-2 inhibitors EM20-25IVand obatoclax sensitized primary CLL cells to VSV-induced cell death. Mechanistically, while VSV infection triggered Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) upregulation, obatoclax blocked the ability of BCL-2 to dimerize with the proapoptotic BCL2-associated X protein (BAX). Together, NOXA expression and BAX release were able to efficiently induce apoptosis. Moreover, our data demonstrated a direct interaction between NOXA and BAX. Together, these data indicate that the use of BCL-2 inhibitors may improve VSV oncolysis in apoptosis-resistant hematological malignancies characterized by overexpression of anti-apoptotic proteins such as BCL-2.

Pancreatic and duodenal homeobox 1 (Pdx1) was one of the first pancreas specific genes isolated. It is expressed in early pancreatic buds, throughout the duodenum and localized to insulin producing cells in the adult. Pdx1 plays a fundamental role in pancreas development as the loss-of-function of Pdx1 in mice and frogs result in absence of pancreatic tissue. In humans, Pdx1 homozygous mutations lead to pancreas agenesis, while heterozygous mutations result in type 2 diabetes. Our laboratory studies the role of Pdx1 in promoting ectopic pancreatic cell fates. Using Xenopus laevis as a model, we previously showed that the overexpression of a modified form of Pdx1, Pdx1-VP16, is sufficient to convert liver to pancreas. Whether Pdx1 is able to promote ectopic pancreas in naïve endoderm has yet to be determined. To achieve this, Pdx1 mRNA was overexpressed in the anterior endoderm. The overexpression resulted in ectopic tissue with reduced expression of exocrine and endocrine differentiation markers. In addition, stomach, duodenum and liver organogenesis was severely perturbed. To ascertain the identity of this ectopic tissue, microarray analysis was performed which confirmed the reduction in pancreatic endocrine and exocrine cells as well as the reduction in stomach, duodenum and hepatic tissue. Moreover, the genes highly upregulated suggest a pancreatic stellate cell phenotype. The information obtained from the gain-of-function analysis will help explain the role of this transcription factor in regulating the initial stages of pancreatic cell fate specification. / Pancreatic and duodenal homeobox 1 (Pdx1) a été l'un des premiers gènes spécifiques du pancréas à être isolé. Pdx1 est exprimé tôt dans les bourgeons pancréatiques, à travers le duodénum et, à l'âge adulte, dans les cellules productrices d'insuline. Pdx1 joue un rôle fondamental quant au développement du pancréas car l'absence de ce gène chez les souris et les grenouilles empêche la formation et l'élaboration du tissue pancréatique. Chez les humains, les mutations homozygotes du gène Pdx1 engendrent l'agénésie du pancréas, tandis que les mutations hétérozygotes engendrent le diabète du type 2. Notre laboratoire étudie le rôle de Pdx1 et sa capacité à promouvoir le sort des cellules pancréatiques ectopiques. En utilisant Xenopus laevis comme modèle, nous avons précédemment démontré qu'une surexpression d'une forme modifié de Pdx1, Pdx1-VP16, est suffisante pour convertir le foie en pancréas. Il reste encore à savoir si Pdx1 est capable d'inciter la formation d'un pancréas ectopique parmi un endoderme naïf. Nous avons donc surexprimé l'ARNm Pdx1 parmi l'endoderme antérieur. La surexpression a conduit à du tissu ectopique avec une expression réduite des marqueurs de différenciations exocrines et endocrines. En outre, l'organogenèse de l'estomac, du duodénum et du foie a été gravement perturbée. Pour vérifier l'identité de ce tissue ectopique, une analyse du profil d'expression génétique par puce à ADN a été réalisée et a confirmée la réduction de cellules pancréatiques endocrines et exocrines, ainsi que la réduction des tissues de l'estomac, du duodénum et du foie. De plus, les gènes fortement induits suggèrent des cellules pancréatiques de phénotype stellaire. Les informations obtenues par l'analyse de gain de fonction permettront d'expliquer le rôle de ce facteur de transcription dans la régulation de la phase initiale de la spécification du sort des cellules du pancréas.

Invasion and metastasis are crucial steps in breast cancer for patient's survival. Recent studies suggest the involvement of loss of DNA methylation resulting in the activation of prometastatic genes, such as uPA and heparanase, during metastasis progression. Also, global DNA hypomethylation and regional DNA hypermethylation are associated with tumorigenesis. Methylated CpG binding protein 2 (MBD2) binds to methylated DNA and demethylates it. Early growth response 1 (EGR1) is also implicated in metastasis and DNA demethylation. We show in this study that MBD2 expression in breast cancer cell lines correlates with uPA expression. Moreover, MBD2 overexpression leads to a global DNA hypomethylation. Furthermore, MBD2 and EGR1 expression induces uPA promoter activity alone or in combination. We also demonstrate a physical interaction between EGR1 and uPA promoter as well as EGR1 and MBD2. Taken together, these data demonstrate that MBD2 in association with EGR1 regulates uPA gene expression through DNA demethylation, and involvement of MBD2 in regulation of metastasis progression.

Eukaryotic cells are divided into subcellular organelles that generate appropriate molecular environments for the functions they harbour. As such, subcellular localization is a key characteristic that provides valuable clues regarding protein function and, when studied globally, a better understanding of cellular processes. The organelles of the secretory pathway are responsible for the processing of all proteins destined for secretion, the plasma membrane as well as their own resident proteins. This group of organelles is difficult to study experimentally because they are difficult to purify to homogeneity. / To facilitate the investigation of the endoplasmic reticulum (ER) and more generally, the secretory pathway, we have created Hera, a publicly accessible protein localization database. Originally designed to house characteristics of ER proteins, it currently contains tens of thousands of proteins from different organisms and subcellular compartments. Hera was originally used to investigate features of ER proteins, providing insight into the extent of usage of various localization mechanisms, including both well-studied but also non-classical and novel mechanisms. / Hera was subsequently used to create Bayesian network type localization predictors. By considering the combinatorial presence of motifs, domains, targeting signals and using in some cases, protein interaction information, our predictors achieve high accuracy and coverage. When our predictions are compared with localization annotations from high-throughput studies in both human and yeast, we find that disagreements mainly involve proteins in the secretory pathway. Our predictors can be used to independently validate these large-scale studies. We further refined the localization prediction of the whole yeast proteome by distinguishing proteins localized to the lumen or membrane of various organelles from cytosolic proteins peripherally associated with these organelles. / Hera was also used to investigate efficient and informative approaches to interrogate interaction networks in order to gain insight into the relationship between proteins/genes of interest. By combining interaction and refined localization information, we constructed localizome-interactome networks of whole organelles. Such models provide insight into global organellar characteristics and inter-organellar mechanisms of communication. / The research presented in this thesis demonstrates that the integration, in an appropriate framework such as Bayesian networks, of widely available information such as localization and interaction data allows to gain deep insights into cellular processes.