The immunoglobulin superfamily (IgSF) is a large protein superfamily of membrane and soluble proteins that influence recognition, binding, and adhesion. Among members of this family are cell adhesion molecules (CAMs), which form cell-cell contact points that play key roles in development, cell polarization, and cellular fate. Cadherins (CADs) are calcium-dependent proteins of the adherens junction (AJ), and polarize epithelium and endothelium. The tight junction (TJ) is a multiprotein junctional complex whose function is to control the permeability of the paracellular pathway. At the membrane level, TJs are composed of three types of proteins: claudins (CLDNs), occludin (OCLN) and junctional adhesion molecules (JAMs). JAMs are members of the IgSF while CLDN and OCLN are 4-α-helix membrane proteins. Although JAMs are part of the TJ and reside in the same ultrastructure, they are similar to CADs in their secondary, tertiary, and quaternary protein structure. Crystallographic studies of CADs in the presence of calcium yielded trans interactions that resulted in cell-cell contacts. In the absence of calcium, CADs form cis interactions that do not form cell-cell interactions. The crystal structure of JAM-A, has a quaternary organization of a cis dimer. In spite of the many similarities, a link between CADs and JAMs remains unclear. Beyond this point, the association between JAMs, CLDNs, and OCLN in the TJ is vaguely understood. The JAM family (JAM-A, -B, -C and 4) and their tissue-specific distribution indicate that they are key to understanding the TJ’s function and the interplay with the AJ. JAM-A has been used as a prototype for the other three members of the family, but based on current evidence we hypothesized that these proteins may display unique properties to support TJ’s function in a given tissue. Are JAMs affected by calcium just as CADs? Do CLDNs and OCLN make direct contact with JAMs? Do JAMs coordinate the interplay between TJ and AJ? We designed a strategy based on recombinant proteins and biophysical methods to answer these questions. First, we fused the extracellular domain of each JAM to maltose-binding protein (MBP). Our results indicate that JAM proteins have similar secondary structures, but unique tertiary structures. Surface Plasmon Resonance experiments showed that JAM proteins favored heterotypic compared to homotypic interactions. Second, we addressed the effects of cations (Ca2+, Mg2+, Cu2+, Fe2+, Fe3+, and Zn2+) on JAM-A. The exposure of JAM-A to the resulted in changes in its secondary, tertiary structure, and homotypic binding affinity. Finally, we addressed whether cations had an effect on the other TJ components and if there is an interplay with E-CAD. We determined that in the assembly of a simple TJ and AJ, JAM-A and E-CAD are calcium-dependent, while CLDN1 and OCLN are calcium independent. We conclude that TJ components such as CLDN1 and OCLN may work as anchors to maintain cell-cell interactions while JAM-A and E-CAD would be regulated by cations in order to accommodate other homeostatic functions.

La protéine kinase D1, PKD1, est une sérine/thréonine kinase activée par de nombreux facteurs mitogènes. Les études, menées jusqu’à présent sur les fonctions de PKD1, ont montré qu’elle semble jouer un rôle dans la régulation de plusieurs processus biologiques fondamentaux impliqués dans le développement des tumeurs. Cependant, le rôle précis et les cibles de PKD1 restent largement méconnus. Au cours de ce travail, nous avons tout d’abord démontré que l’inhibition de PKD1 dans les cellules de mélanome inhibe la croissance et la motilité cellulaire, induit l’expression de la E-cadhérine et une diminution de la N-cadhérine. D’autre part, nous nous sommes intéressés au rôle des perturbateurs endocriniens dans les cellules tumorales mammaires et avons démontré que PKD1 est une cible des perturbateurs endocriniens (PE). Les PE, tels que le bisphénol A (BPA) et les phtalates, sont des produits chimiques ubiquitaires de notre environnement. Leur rôle dans la croissance tumorale mammaire est bien documenté. Néanmoins, les mécanismes moléculaires précis par lesquels ces molécules agissent demeurent encore inconnus. Au cours de notre travail, nous avons démontré que ces PE induisent de façon dose-dépendante la prolifération des cellules MCF-7 (cellules d’adénocarcinome mammaire) et que ce processus biologique est dépendant de l’expression de PKD1. Ainsi, l’ensemble de ce travail fait apparaître, d’une part, que PKD1 pourrait être une nouvelle cible thérapeutique anti-tumorale potentielle dans le mélanome et que, d'autre part, PKD1est une cible moléculaire de certains PE dans le cancer du sein. / Protein kinase D1, PKD1, is a serine/threonine kinase which can be activated by mitogens and that regulates various functions involved in the development of tumors. However, its precise role and targets are still unclear. Our study demonstrates that PKD1 inhibition in melanoma cells decreased cell growth and motility, and reversed the E- to N-cadherin switch. On the other hand, we examined the role of endocrine disruptors (EDs) in breast cancer cells and identified PKD1 as a target of these compounds. EDs, such as bisphenol A (BPA) and phthalates, have been found molecular mechanisms are still ubiquitously throughout our environment. Their role in breast tumor growth has been well documented. However, their precisemolecular mechanisms are still unknown. Our study demonstrates that EDs induce dose-dependently MCF-7 cell growth and that this biological process is dependent upon PKD1expression. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target in melanoma and identifies PKD1 as a new molecular target of some EDs in breast cancer cells.

One of the numerous complications of obesity is infertility. Leptin has been shown to reverse infertility; however, exact mechanism is poorly understood. Recent evidence indicates Ecadherin/ β-catenin complex, which is a structural constituent of adherens junction, is expressed in the rat ovary during folliculogenesis. We hypothesized that systemic leptin modulates the expression of E-cadherin and β-catenin in dietary-induced obese infertile rats to reverse infertility. Female Sprague-Dawley rats were fed either regular chow diet (RCD) (n=6) or high fat diet (HFD) (n=14). Oestrus cycles were monitored daily until their cycles became irregular. 100 ug/ml of leptin was given intraperitoneally to HFD-fed rats (n=5) with irregular cycles. The control rats HFD (n=9) and RCD received saline. Leptin treatment restored regular estrous cycle and increased the expression of E-cadherin and β-catenin in all the 5 rats (HFD+Leptin). This could represent the mechanism by which leptin reverses infertility in obese infertile rats.

The migration of the cranial neural crest is an essential part of cranio-facial development in every vertebrate embryo. The cranial neural crest (CNC) is a transient population of cells that forms the lateral border of the anterior neural plate. In the tailbud stage Xenopus embryo, the neural crest cells delaminate from the neural tube, and undergo a large-scale migration from the dorsal to ventral region of the embryo. The CNC travels along distinct pathways, and populates specific regions of the embryos face. Once the CNC ceases migrating, it differentiates into a variety of tissues that are essential for cranio-facial structure and function. Some of these tissues include bones, muscle, cartilage, and ganglia. The CNC receives a concert of signals from neighboring tissues during and after CNC migration as well as signals transmitted among CNC cells, which act together to determine the fate of each CNC cell. Therefore, the proper migration of the CNC is an essential part of cranio-facial development. What molecules are important for the process of CNC migration? As one might imagine, a milieu of different molecules and interactions are essential for this complicated embryological process to occur. The work presented in this dissertation will focus on the role of a cell adhesion molecule that is important for Xenopus CNC migration. Typically, the amount of cell adhesion decreases within tissues undergoing migration. This behavior is essential to allow fluidity within the tissue as it moves. However, cell adhesions are fundamental for cell migration to occur because the moving cells need a platform on which to mechanically propel themselves. These interactions can occur between the migrating cell and extracellular matrix molecules (ECM), or can happen between cells. The cranial neural crest utilizes both cell-ECM and cell-cell interactions during the process of migration. The amount of cell adhesion mediated by either of these mechanisms will depend on where the cell is located within the CNC. Cells located at the periphery of the CNC tissue, which is surrounded by a matrix of ECM, will have more cell-ECM interactions. Cells located deeper in the CNC tissue, where there is little ECM, will rely more on cell-cell interactions. The work presented in this thesis focuses on a cell-cell adhesion molecule that is part of the cadherin superfamily of molecules. With this in mind, these studies should be descriptive of the environment within the CNC, and to a less degree the environment between the CNC and the surrounding tissues. The work presented in this dissertation will focus on cadherin-11, which is a classical cadherin that is specifically expressed in the cranial neural crest during its migration. How does cadherin-11 function in the CNC during this process? The work presented here suggests that the main role of cadherin-11 in the CNC is to perform as a cell adhesion molecule. However, too much cell adhesion is inhibitory to migration. In this respect, many of the studies described in this work indicate that cadherin-11 mediated cell adhesion is tightly regulated during CNC migration. Here I show that cadherin-11 is extracellularly processed by ADAM metalloproteases, ADAM9 and ADAM13, which removes the adhesive domain of cadherin-11. This extracellular cleavage event occurs throughout CNC migration, and is likely the main mechanism that regulates cadherin-11 mediated cell adhesion. Cleavage of cadherin-11 by ADAMs does not seem to affect its ability to interact with cytoplasmic binding partners, â-catenin and p120-catenin. This observation supports the idea that the “purpose” of cadherin-11 cleavage is to regulate cell adhesion, and not to induce (cell autonomous) signaling events. Additionally, the secreted extracellular domain of cadherin-11 (EC1-3) retains biological activity. This fragment can bind to a number of cell surface molecules in tissue culture including full-length cadherin-11 and specific members of the ADAM family. This observation suggests that EC1-3 may interact with full-length cadherin-11 molecules in vivo, and inhibit cadherin-11 mediated cell adhesion during CNC migration. EC1-3 can rescue CNC migration in embryos that overexpress cadherin-11, further supporting this hypothesis. Many of the above observations have been published in my first-author paper entitled “Extracellular processing of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest migration” published in the journal Molecular Biology of the Cell in 2009. Some of the unpublished work in this dissertation further focuses on how EC1-3 effects CNC migration in an ex vivo environment. During these studies, the observation was made that overexpression of EC1-3 in a cranial neural crest explant produces abnormal directional movement. In these experiments, it appeared as though certain regions of the CNC explant were “attracting” other regions of the explant. The preliminary studies described in chapter IV are aimed at answering the question; does EC1-3 attract migrating CNC cells? Here, we generated a Matlab program in order to effectively quantify the amount of directional movement of CNC explants presented with a source of EC1-3. In addition to quantifying cell directionality, this program can also decipher between cells moving with random or directed motion, and measure the velocity of cell migration within certain coordinates. Therefore, this program should be useful other ex vivo studies that require the observation of these features. To conclude, the work presented in this dissertation suggests that the role of cadherin-11 during cranial neural crest migration is predominately based on the adhesive function. In order for CNC migration to proceed, the amount of cadherin-11 mediated cell-cell adhesion is tightly regulated throughout this process. These cell-cell interactions are likely important for “sheet” and “branch” migration where CNC cells maintain a lot of cell-cell cohesion.

No description available.

No description available.

No description available.

No description available.

<p>Adhesion of the blastocyst to the uterine wall is a highly sensitive phenomenon referred to as implantation. Novel-males are capable of disrupting the success of this process (the Bruce effect). A leading hypothesis invokes the transfer of estradiol from the male to the female via urine. This estradiol has direct effect on the uterus which may include morphology and molecular dynamics. Estradiol has been related to closure of the uterus around the blastocyst during implantation, which may assist in bringing the blastocyst close to the uterine wall for strong adhesion. E-cadherin, a cellular adhesion molecule, is found on both blastocyst and uterine surfaces and has been suggested to be involved in their interaction during implantation. Estradiol has been observed to reduce e-cadherin expression in hormonally sensitive tissues like the mammary glands, ovaries and uteri. Here, male-induced disruption of implantation was examined across days 2-8 of gestation. Luminal area was quantified in isolated and male-exposed females as a measure of extent of luminal closure. This area was larger in male-exposed animals. E-cadherin was found to have reduced expression on luminal epithelial cells. I suggest that the reduction in e-cadherin may lead to weaker attachment of the blastocyst to the uterine wall as well as reduced adhesion between opposing uterine walls leading to the “opening” of the uterus observed in male exposed animals. Together, these data may in part explain the blastocyst implantation failure observed in male-exposed animals during the Bruce effect.</p> / Master of Science (MSc)

Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). The virally encoded X antigen, HBx, contributes importantly to the development of HCC through its trans-activating role in various signal transduction pathways. Pathways implicated in stem cell self-renewal also contribute to carcinogenesis. Thus, experiments were designed to test if HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HBx expressing (HepG2X) and control (HepG2CAT) human cell lines were assayed for phenotypic and molecular characteristics of "stemness." Western blotting of protein extracts from HepG2X and HepG2CAT cells as well as immunohistochemical staining of HCC and adjacent liver tissue sections from HBV infected patients showed up-regulation of "stemness"-associated (EpCAM and beta-catenin) and "stemness" (Oct-4, Nanog, Klf-4) markers by HBx. Moreover, HBx stimulated cell migration and spheroid formation. HBx expression was also associated with depressed levels of E-cadherin and subsequent activation of beta-catenin and EpCAM. Results from ChIP-chip data performed previously in this lab suggest an associative link between HBx and the expression of epigenetic co-repressor, mSin3A, which is known to repress E-cadherin when complexed with histone deacetylases. Thus, experiments were also designed to test if HBx represses the E-cadherin gene (CDH1) through histone deacetylation by the mSin3A/HDAC complex. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was showed by immunofluorescent staining. Chromatin immunoprecipitation revealed that HBx mediated the recruitment of the mSin3A/HDAC complex to the CDH1 promoter. HDAC inhibition by Trichostatin A treatment restored E-cadherin expression. Thus, HBx-associated epigenetic repression of E-cadherin and up-regulated expression of multiple "stemness" markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristic of CSCs. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. / Biology