Epithelial to Mesenchymal Transition (EMT) is a cellular transformation process that is employed repeatedly and ubiquitously during vertebrate morphogenesis to build complex tissues and organs. Cellular transformations that occur during cancer cell invasion are phenotypically similar to developmental EMT, and involve the same molecular signalling pathways. EMT processes are diverse, but are characterised by: a loss of cell-cell adhesion; a gain in cell-matrix adhesion; an increase in cell motility; the secretion of proteases that degrade basement membrane proteins; an increased resistance to apoptosis; a loss of polarisation; increased production of extracellular matrix components; a change from a rounded to a fibroblastic morphology; and an invasive phenotype. This thesis focuses explicitly on endocardial EMT, which is the EMT that occurs during vertebrate embryonic heart development. The embryonic heart initially forms as a tube, with myocardium externally, endocardium internally, with these tissue layers separated by a thick extracellular matrix termed the cardiac jelly. Some of the endocardial cells in specific regions of the embryonic heart tube undergo EMT and invade the cardiac jelly. This causes cellularised swellings inside the embryonic heart tube termed the endocardial cushions. The emergence of the four chambered double pump heart of mammals involves a complex remodelling that the endocardial cushions play an active role in. Even while heart remodelling is taking place, the heart tube is operating as a single-circulation pump, and the endocardial cushions are performing a valve-like function that is critical to the survival of the embryo (Nomura-Kitabayashi et al. 2009). As the endocardial cushions grow and remodel, they become the valve leaflets of the foetal heart. The endocardial cushions also contribute tissue to the septa (walls) of the heart. Their correct formation is thus essential to the development of a fully functional, fully divided, double-pump system. It has been shown that genetic mutations that cause impaired endocardial EMT lead to the development of a range of congenital heart defects (Fischer et al. 2007). An extensive review is conducted of existing experimental investigations into endocardial EMT. The information extracted from this review is used to develop a multiscale conceptual model of endocardial EMT, including the major protein signalling pathways involved, and the cellular phenotypes that they induce or inhibit. After considering the requirements for computational simulations of EMT, and reviewing the various techniques and simulation packages available for multi-cell modelling, cellular Potts modelling is selected as having the most appropriate combination of features. The open source simulation platform Compucell3D is selected for model development, due to the flexibility, range of features provided and an existing implementation of multiscale models; that include subcellular models of reaction pathways. Based on the conceptual model of endocardial EMT, abstract computational simulations of key aspects are developed, in order to investigate qualitative behaviour under different simulated conditions. The abstract simulations include a 2D multiscale model of Notch signalling lateral induction, which is the mechanism by which the embryonic heart tube is patterned into cushion and non-cushion forming regions. Additionally, a 3D simulation is used to investigate the possible role of contact-inhibited mitosis, upregulated by the VEGF protein, in maintaining an epithelial phenotype. One particular in vitro investigation of endocardial EMT (Luna-Zurita et al. 2010) is used to develop quantitative simulations. The quantitative data used for fitting the simulations consist of cell shape metrics that are derived from simple processing of the imaging results. Single cell simulations are used to investigate the relationship between cell motility and cell shape in the cellular Potts model. The findings are then implemented in multi-cell models, in order to investigate the relationship between cell-cell adhesion, cell-matrix adhesion, cell motility and cell shape during EMT.

The FRIABLE1 (FRB1) gene is identified to be a novel glycosyltransferase involved in cell adhesion, based on reverse genetics and immunocytochemistry studies. A total of 31 FRB1 paralogues were found in Arabidopsis thaliana using a bioinformatics approach. The following expression analysis has revealed 6 FRB1 paralogues to be pollen-specific. One pollen-specific FRB1 paralogue, At1g14970, exhibits longer silique lengths when exposed to higher than normal temperature at 28oC in its T-DNA insertional knockout when compared to Columbia wildtype plants. This may be due to the loss of temperature sensing and the continuous stimulated pollen tube cell wall growth or the up-regulation of genes that encode other glycosyltransferases. Thus, the identification of FRB1 paralogues and homologues in both rice and poplar may have tremendous potential to increase their yield in global warming for agricultural and industrial benefits.

The skin-implant interface of percutaneous devices is generally weak and can fail when excessive loading disrupts the sealing of the interface by dermal and epidermal cells and tissue. As such, the formation of a stable implant-skin junction is a major factor in determining percutaneous implant success. In this study, we used functionalized self-assembled monolayers (SAMs) with discrete surface properties as model systems to assess the effects of biomaterial surface properties on controlling fibronectin (FN) adsorption and keratinocyte spreading and adhesion. The surface properties investigated were charge (positive and negative) and wettability (hydrophobic and hydrophilic). Gold slides prepared with SAMs were incubated with FN overnight. The cell binding sites were quantified on each surface using an antibody that targets the synergy binding site of the cell binding domains (HFN7.1) and the topography of the FN on the surfaces was evaluated with atomic force microscopy. The topography data demonstrated that the availability of cell binding domains is dependent on surface-mediated FN binding orientation. Cell spreading was assessed using a lipid membrane stain, maleimide. The cells were imaged by fluorescence microscopy and the cell area calculated. The percentage of cell adhesion was determined using a centrifugal force assay. Both keratinocyte assays suggested that the charge of the surface was the prominent factor in determining cell function on the surface over the surface wettability. The findings of this study strongly suggest that a positively charged implant surface with a FN coating will enhance the strength of the cutaneous seal around percutaneous implants over an unmodified surface.

O carcinoma espinocelular é uma neoplasia maligna que representa aproximadamente 94% de todas as ocorrências presentes em boca e uma das suas principais características celulares é a migração de suas células para formar metástases. A adesão celular é considerada um dos eventos determinantes da migração celular. Para as células formarem uma estrutura tecidual tridimensional as adesões entre células e entre células e matriz extracelular são de grande importância. As junções de adesão celulares surgem, caracteristicamente, pela interação entre receptores adesivos, vias de sinalização e elementos do citoesqueleto. A proteína E-caderina está presente em adesões entre células no tecido epitelial. A proteína FAK está envolvida na maioria dos eventos relacionados à adesão celular estimulada por integrinas. A Vinculina é uma proteína de adesão que se liga ao citoesqueleto de actinomiosina como uma proteína de adesão focal através das integrinas. Estudos recentes sugerem que há alteração na expressão e atividade de proteínas de adesão em tumores malignos. O objetivo deste trabalho foi descrever o padrão de expressão e de regulação da atividade de proteínas de adesão em amostras de tumores de carcinoma espinocelular. Foram realizadas reações de imunoistoquímica para verificar o padrão de distribuição das proteínas E-caderina, Vimentina e FAK-y397 em amostras de tumores de carcinoma espinocelular oral. Verificou-se a diminuição da expressão de E-caderina e de Vinculina em regiões de adesão célula-célula e em contrapartida constatou-se aumento na marcação citoplasmática de Vinculina bem como na marcação de FAK-y397 em todas as amostras de tumores. Apesar dos avanços, ainda são necessários mais estudos observacionais que averiguem não apenas o grau de expressão dessas proteínas de adesão, mas também o seu nível de regulação. A partir dos resultados deste estudo, pode-se sugerir que o controle do nível de expressão e de atividade da adesão celular podem ser considerados como potenciais alvos para a aplicação de terapias coadjuvantes que visam a diminuir ou impedir a progressão tumoral, bem como o desenvolvimento de metástases. / Squamous cell carcinoma is a malignant neoplasm that accounts for approximately 94% of all occurrences present in mouth and one of its main characteristics is the cellular migration of its cells to form metastases. Cell adhesion is considered one of the defining events of cell migration. For a three-dimensional tissue structure, adhesions between cells and between cells and the extracellular matrix is of great importance. Cell adhesion junctions arise characteristically by interaction between adhesive receptors, signaling pathways and cytoskeletal elements. The protein E-cadherin is present in cells in the adhesion between epithelial tissue. The Focal Adhesion Kinase (FAK) protein is involved in most events related to cell adhesion stimulated by integrins. The vinculin is an adhesion protein that binds cytoskeletal protein through integrins activaion. Recent studies suggest that there are alterations in the expression and activity of adhesion proteins in malignant tumors. The aim of this study was to describe the pattern of expression and regulation of the activity of adhesion proteins in tumor samples of squamous cell carcinoma. Immunohistochemical reactions were performed to check the distribution pattern of the protein E-cadherin, vimentin and FAK-y397 in tumor samples of oral squamous cell carcinoma. There was a decrease in the expression of E-cadherin and vinculin in regions of cell-cell adhesion but, on the other hand, it was found to increase in cytoplasmic as well as unscheduled vinculin FAK-y397 in all tumor samples. Despite progress, it is necessary more observational studies that examine not only the degree of expression of these adhesion proteins, but also its level of regulation. From the results of this study it is suggested that the control of the expression level and activity of cell adhesion may be considered as potential targets for application adjuvant therapies that aim to reduce or prevent tumor progression and the development metastases.

Integrin recycling has been generally assumed to be important for cell migration but the trafficking pathways and the molecules regulating integrin trafficking remain poorly characterized. Furthermore, little is known about the activation status of endocytosed integrins and how it affects the recycling of these receptors. It is likely that FA-engaged integrins will follow different trafficking pathways than bulk integrins and here I sought to study the endocytic fate of this particular integrin pool using the MT-induced FA disassembly assay. I found that integrins previously resident at FAs travel through different Rab compartments after FA disassembly and that their return to the plasma membrane is Rab11- and Src-dependent. Strikingly, I unveiled new functions for FAK and Src family kinases in this process by showing that these kinases are critical to keep integrins active during endocytic trafficking. This finding is unprecedented since it was not known whether endocytosed integrins were kept active during their trafficking. Interestingly, reassembly of FAs from endocytosed integrin occurred preferentially at the leading edge of migrating cells suggesting that integrins are trafficked in a polarized fashion. Furthermore, the recycling of integrins from the Rab11-positive compartment to the plasma membrane is a long-range transport implying the existence of a MT motor committed to this task. Consistently, I identified that a kinesin-II motor, Kif3AC, is engaged in this process. My work establishes a FAK- and Src family kinases-based mechanism for integrin "adhesion memory" during endocytic trafficking and identifies a direct link between FA disassembly and reassembly through an endocytic recycling pathway involving Rab5 and Rab11 and a kinesin-II family member.

Dysregulation of protein expression, in particular expression of proto-oncogenes and tumor-suppressor genes whose function play key roles in cell growth, adhesion and migration, are hallmarks of human malignancies. Transmembrane and immunoglobulin-containing domain 1 (TMIGD1) was recently discovered as a cell adhesion molecule (CAM) that plays an important role in epithelial cell function by regulating epithelial cell polarity and adhesion. The extracellular domain of TMIGD1 contains two Ig domains that are involved in cell-cell interaction, followed by a transmembrane region and short cytoplasmic domain with potential to relay signal transduction. Our further investigation demonstrated TMIGD1 is downregulated in human colon cancer, suggesting a potentially important role for TMIGD1 in the regulation colorectal cancer. However, the molecular mechanisms of TMIGD1-mediated signal transduction, which could relay its function in epithelial cells, are not known. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we have identified moesin as a possible TMIGD1 binding protein. Moesin, a member of the Ezrin/Radixin/Moesin (ERM) family of proteins, is upregulated in human tumors. Moesin stimulates cell migration, tumor invasion, adherence and modulates cytoskeletal actin assembly. Similar to other ERM family proteins, moesin contains an N-terminal FERM domain, which binds to transmembrane proteins, and a C-terminal C-ERMAD domain, which binds F-actin. The overall goal of this study was to determine the binding of moesin with TMIGD1 and the specific domain involved in mediating the binding of moesin with TMIGD1. Our study in vitro and in vivo binding assays demonstrate that moesin interacts with the cytoplasmic domain of TMIGD1 via its FERM domain. Moreover, we demonstrate TMIGD1 interaction with moesin inhibits phosphorylation of moesin, indicating that perhaps TMIGD1 inhibits tumor cell migration through inhibition of phosphorylation of moesin. Additionally, TMIGD1 alters cellular localization of moesin, suggesting that altered cellular localization by TMIGD1 could account for inhibition of phosphorylation of moesin. We propose that TMIGD1 sequesters moesin near the cell membrane, preventing its interaction with PIP2, which is required for its phosphorylation and hence inhibits moesin activation. Altogether, the data presented in this work identifies moesin as a key signaling component of TMIGD1. Moesin directly interacts with TMIGD1 via its FERM domain. Recruitment of moesin to TMIGD1 blocks phosphorylation of moesin, suggesting that TMIGD1 exerts its effect in tumor cells in part by inhibition of moesin activation. / 2018-07-11T00:00:00Z

Metastatic melanoma is unusually lethal with a ten year survival rate of less than 10%. Conventional DNA-damaging agents produce little improvement in patient survival. Vemurafenib (Zelboraf), a targeted therapeutic that inhibits the oncogenic BRAF demonstrates significant survival benefit. Unfortunately, it is now evident that there is both intrinsic and acquired resistance. Consequently, new strategies for sensitizing melanomas to vemurafenib are needed. Melanoma resistance to therapy is fueled in part by the integrins, the major cell surface adhesion receptors which are highly over-expressed in melanoma. Both integrin antagonists and agents that engage defective integrins increase the sensitivity of melanomas to chemotherapy. Our laboratory has identified a novel peptide, denoted vinculin activating peptide or VAP that targets integrins from within the cell and brings aberrant integrin function intact. VAP sensitizes melanoma to dacarbazine in vitro and in vivo. The effect VAP has on overcoming resistance to targeted therapies like vemurafenib, as well as the mechanism for its effects are not well understood. The goals of this project are to determine if VAP can be employed to improve sensitivity and/or overcome resistance to vemurafenib and to identify the cell surface target of VAP. Our results show that VAP not only improves melanoma sensitivity to vemurafenib but also decreases intrinsic resistance to this promising drug. In addition, we present evidence that β1 and β3 integrins are the target of VAP's effects. Since peptide-based therapies are not stable in the clinic, we explored another integrin binding partner, kindlin-2. We found that kindlin-2 is over expressed in resistant melanomas. The inhibition of kindlin-2 increases β1 integrin activation and decreases β3 integrin functions. Agents that bring aberrant β1 and β3 integrin function intact can be employed to improve sensitivity and overcome resistance to vemurafenib suggesting that combinatorial therapies that employ vemurafenib and integrin-based agents might be efficacious in combatting resistance in melanoma patients.

The Exocyst is a hetero-octameric complex involved in tethering of post-Golgi vesicle transport to sites of membrane expansion. In budding yeast, the Exocyst targets vesicles to bud site resulting in bud emergence and abscission of the daughter cell. Mammalian Exocyst is recruited to developing lateral membranes after cadherin mediated adhesion and then is segregated to adherens junctional complexes (AJC). In polarized epithelia, the Exocyst is required for basal-lateral transport of LDL receptor. Additional Exocyst subunit localizations and functions have been identified. It is not known whether these supplementary roles can be attributed to the Exocyst or other unidentified Exocyst subcomplexes. Sec3, an Exocyst subunit, is hypothesized to be a landmark of polarization in yeast. In polarized epithelia, GFP tagged Sec3 remained cytosolic in polarized epithelia unlike Sec6/8. Sec3-GFP was recruited to lateral membranes only after dual over expression of heterologous GLYT1. Little is known about endogenous mammalian Sec3. Our work suggests Sec3 defines an Exocyst subcomplex that is required for desmosome integrity. Sec3 and additional subunits (Sec6, Sec8, Sec15, Exo70, and Exo84) were present at desmosomes, but Sec3 failed to localize to AJC. Only antibodies to Sec6 and Sec8 labeled AJC. Reduction of Sec3 protein expression resulted in the impairment of desmosome morphology and function with no detrimental effect on adherens junctions. These data suggest the existence of functionally different Exocyst subcomplexes. Sec3-exocyst recruited minus-end directed microtubule motor KifC3 to desmosomes. KifC3 was previously shown to be recruited with a microtubule anchoring complex to basal-lateral membrane. This suggests Sec3 may recruit KifC3 to organize microtubules at desmosomes. This would establish a pathway to efficiently transport newly synthesized basal-lateral cargo. These results suggest a novel mechanism of the Exocyst to regulate post-Golgi vesicular transport and intercellular adhesion.

Tumor angiogenesis, the ability of tumor cells to stimulate blood vessel growth, is one the most critical steps of tumor progression. To support the growth of the expanding tumor, the “angiogenic switch” is turned on, which is often triggered by hypoxia (i.e., low oxygen)-mediated events such as expression of vascular endothelial growth factor (VEGF), causing normally quiescent endothelial cells to proliferate and sprout. An emerging picture of angiogenesis suggests that while governed by complex mechanisms, cell adhesion molecules (CAMs) plays a pivotal role in the regulation of angiogenesis. Our laboratory recently identified multiple previously unknown proteins including, transmembrane and immunoglobulin domain containing 1 (TMIGD1) and immunoglobulin-containing and proline-rich receptor 1 (IGPR1). Immunoglobulin-containing and cysteine-rich receptor 1 (IGCR1) represents the third remember of IGPR-1 family proteins. To investigate the expression and function of IGCR1, we have developed a rabbit polyclonal anti-IGCR1 antibody and demonstrated that IGCR1 is expressed in the endothelial cells of human blood vessels. To examine possible function of IGCR1, we have generated porcine aortic endothelial (PAE) cells over-expressing IGCR1. We demonstrate that IGCR1 expression in PAE cells inhibited cell proliferation and capillary tube formation as measured by colorimetric MTT and matrigel tube formation assays, respectively. In contrast, over-expression of IGCR1 in PAE cells inhibited cell migration as measured by wounding assay. Taken together, this study identifies IGCR1 as a novel regulator of angiogenesis. Given, angiogenesis is a highly coordinated cellular processes controlled spatially and temporally by a myriad of cell surface receptors and ligands, IGCR1 by modulating the rate of endothelial cell proliferation and migration, plays a significant role in the formation of blood vessels. / 2018-07-11T00:00:00Z

Includes list of publications by the author. Includes bibliographical references (leaves 103-123) Evaluates the nature of the interactions occurring between semen and cells of the uterus that occur following mating in pigs. Describes a novel ability of porcine seminal plasma to induce dose dependent cell-cell adhesion and mitogenesis amongst peripheral blood lymphocytes in vitro.