Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.

Chow, Wai Ming Alison. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 138-153). / Abstracts in English and Chinese.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 130-150).

Acute injury of major epithelial organ systems (kidney, liver, lung, etc.) is collectively a principal cause of death worldwide. Regenerative medicine promises to meet these human health challenges by harnessing intrinsic cellular processes to repair or replace damaged tissues. Epithelial morphogenesis is a hard-wired, multicellular differentiation program that dynamically integrates microenvironmental cues to coordinate cell fate processes including adhesion, migration, proliferation, and polarization. Thus, epithelial morphogenesis is an instructive mode of tissue assembly, maintenance, and repair. Three-dimensional epithelial cell cultures in natural basement membrane (BM) extracts produce hollow, spherical cyst structures and have indicated that the BM provides the critical cell adhesion ligands to facilitate cell survival, stimulate proliferation, and promote polarization and lumen formation. However, the utility of natural BMs for detailed studies is generally limited by lot-to-lot variations, uncontrolled cell adhesive interactions, or growth factor contamination. The goal of this thesis was to engineer bioartificial extracellular matrices (ECM) that would support and modulate epithelial cyst morphogenesis. We have engineered hydrogels, based on a multi-arm maleimide-terminated poly (ethylene glycol) (PEG-4MAL), that present cell adhesive molecules and enzymatic degradation substrates and promote polarized epithelial cyst differentiation in vitro. To investigate the influence of matrix physical and biochemical signals on cyst morphogenesis, we independently varied the polymer weight percentage (wt%), the density of a cell adhesion ligand (RGD), and crosslink degradation rates of the hydrogels. Then, we evaluated functional outcomes including Madin-Darby canine kidney (MDCK II) epithelial cell survival, proliferation, cyst polarization, and lumen formation. We found that cell proliferation, but not cell survival, was sensitive to the polymer wt%, which is related to elastic modulus and crosslink density. This result defined a working range of PEG-4MAL concentration (3.5% - 4.5%) that promotes robust proliferation. Analysis of mature cysts indicated that 4.0% and 4.5% gels produced cysts resembling those typically grown in type I collagen gels while 3.5% gels produced cysts with higher incidence of inverted polarity and multiple lumens. Perturbation of matrix degradability using a slow-degrading crosslink peptide or matrix metalloproteinase inhibitors showed that the rate of matrix degradation exerts major influence on cyst growth in PEG-4MAL gels. We employed 4.0% PEG-4MAL hydrogels with RGD ligand density ranging over 0 – 2000 uM to discover that (1) lumen formation was eliminated in the absence of RGD, (2) extent of lumen formation increased with increasing RGD concentration, and (3) cyst polarity was inverted below a threshold of integrin binding to RGD. Together, these results show that the biochemical and physical properties of the matrix, particularly integrin binding and matrix degradability, effectively modulate establishment of apico-basal polarity and lumen phenotypes in MDCK II epithelial cyst structures. Furthermore, these studies validate PEG-4MAL hydrogels as a powerful culture platform to enable detailed investigation of matrix-directed modulation of epithelial morphogenesis.

Thesis (M.S.)--University of Michigan, 1964.

Thesis (M.S.)--University of Michigan, 1964.

Thesis (M.S.)--University of Southern California, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Southern California, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Adherens junctions are a main cell-cell adhesion structure found in epithelial cells. The stability of adherens junctions is attributed to various protein-signaling cascades and importantly the interaction between the transmembrane protein E-cadherin and cytoplasmic p120 catenin. This interaction is critical for cell adhesion and prevention of uncontrolled growth in normal cells. The interaction interface between these two binding partners was previously determined to comprise p120's Armadillo repeat domain (p120Arm) and Ecadherin's cytoplasmic juxtamembrane domain (Ecadc). Based on this information, peptide aptamers were derived from p120Arm and their interaction with Ecadc was tested in vitro. We reasoned that those could be expressed in vivo to stabilize adherens junctions at the cell-cell junction. In this study, we established protein-protein interaction assays to demonstrate p120Arm's ability to bind Ecadc and then used these assays to determine if p120Arm-derived peptides may competitively bind Ecadc. We demonstrated the interaction between p120Arm and Ecadc using assays that were not previously used such as: co-precipitation, analytical gel filtration and the bacterial-2-hybrid assay. However, the p120Arm-derived peptides did not bind to Ecadc or compete its interaction with p120Arm. This may be due to the nature of the assays that may not reflect competitive binding or the aptamers may not adopt the native conformation preventing binding to Ecadc. / Thesis / Master of Science (MSc)

The influenza virus has a large impact on global health; however, it is difficult to formulate vaccines and influenza therapies that are effective against influenza. The influenza virus mutates rapidly, has the ability to emerge as novel strains with pandemic potential and can quickly become resistant to any given drug. Therefore, the generation of novel anti-influenza therapeutics that are effective against multiple strains would be highly beneficial. To date, the majority of anti-influenza research has focused on targeting specific components of the virus in order to interfere with its replication. However, it has been proposed that host proteins and signaling pathways may be essential components to viral replication and could also become novel anti-influenza drug targets. Therefore, this study utilized a large proteomic screen to identify host proteins that were up- and down-regulated in response to influenza infection. Collectively, these proteins clustered into five specific cell pathways and processes including interferon signaling, purine metabolism, cell death, ubiquitin-like signaling and mitochondrial oxidoreductases. Overall, this project identified potential novel anti-influenza targets in primary airway epithelial cells. / May 2015