Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 103-112).

During development of epithelial organs, epithelial cells collectively migrate and invade into their surroundings to form complex 3D structures, such as the tubular ducts and alveoli of the mammary gland. A prerequisite for coordinating such collective movement is apicobasal cell polarity. This polarity divides the plasma membrane of epithelial cells into an apical domain towards the lumen of structures and a basal domain facing the matrix. Polarity is essential for the functionality of epithelial tissues and signals from the surrounding matrix are known to participate in its establishment. In contrast, loss of polarity has been associated with progression of diseases such as cancer. During epithelial tubulogenesis, the pro-invasive enzyme MT1-MMP is regulated according to apicobasal polarity. This regulation is essential for tubulogenesis to occur and restricts MT1-MMP activity to the tip of protruding tubules by ensuring that the enzyme only localises to the basal, matrix-abutting cell surface in this location. Signals from fibrillar collagen I contribute to regulating the polarised distribution of MT1-MMP. How such signals are transmitted and influence polarised trafficking is however not understood. In this study, I found that inhibition of the collagen receptor DDR1 disturbed the apicobasal distribution of MT1-MMP in MDCK cells. In 3D environments, DDR1 inhibition blocked MT1-MMP-dependent tubulogenesis of epithelial cells, which instead formed compact, multi-layered aggregates. Furthermore, polarisation of the epithelial cell membrane into an apical and a basal domain failed in absence of DDR1 signalling, suggesting that DDR1 affects establishment of epithelial polarity. In support of this, the effects of DDR1 signalling on apicobasal polarity were not limited to MT1-MMP- dependent morphogenesis, but also proved essential for polarisation of cells during 3D morphogenesis that did not require ECM degradation. An investigation of signalling downstream of DDR1 in establishment of apicobasal polarity revealed this to involve modulation of cytoskeletal tension. Inhibition of DDR1 in 2D culture of MDCK cells thus increased ROCK-dependent phosphorylation of MLC along cell-cell junctions, suggesting that DDR1 can suppress ROCK activity. Importantly, the ROCK-suppressing function of DDR1 contributed to establishment of polarity in MDCK cells in 3D matrices, where inhibition of ROCK activity rescued the formation of polarised cysts in absence of DDR1 signalling. The role of DDR1 in epithelial organisation was reflected in the epithelium of the mammary gland of lactating DDR1-null mice, which had smaller alveoli with a diffuse distribution of basement membrane components compared to wild type mice. Furthermore, DDR1 inhibition attenuated formation of milk-producing mammospheres during lactogenic differentiation of mammary epithelial cells in vitro in a ROCK- dependent manner. This suggests that the ROCK-suppressing function of DDR1 observed in MDCK cells is important for morphogenesis of other epithelial cell types as well. Overall, this study suggests that DDR1 signalling contributes to epithelial polarisation and morphogenesis in a manner involving regulation of cytoskeletal organisation, at least partly through regulation of ROCK activity.

M. Ing. / Projects are generally undertaken because they are part of the plans to advance organisations to new levels of performance and to operationalise business strategies. Projects are however constrained by conflicting demands and competing priorities within the project environment. Project success is a topic of much debate, but it is generally agreed that successful projects meet the strategic objectives and higher purpose of the endeavour. The processes and methods of project management provide the structure, focus, flexibility and control to help guide significant project investments to beneficial change. The project management body of knowledge (PMBOK) endorses that every project is governed by the triple constraint, which reflects a framework for evaluating competing demands. The triple constraint is a critical project management concept that originates from the project basis and provides direction for framing the project. The triple constraint comprises the three key elements of scope, time and cost. Quality is an inherent objective of the project specification that takes root in all three properties of the triple constraint. The current literature in the project management domain suggests that there exists a lack of appropriate (and consistent) scholarship on the triple constraint and its dynamics. Project managers often create an illusion of tangible progress by relying heavily upon traditional on-time, on-budget and on-target measures – yet this tactic fails to address the strategy ambiguity or establish appropriate project goals. The triple constraint trade-offs are also often perceived as organisational problems that have a definitive solution – yet this tactic fails to effectively negotiate the triple constraint and leads to destructive conflict.

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This research attempt further analyzes implications of statistical correlations regarding specific relationships between the extremity-intensity variables, as defined by the social judgment instrument, and the polarity variable, as defined by the semantic differential scale.

Thesis advisor: David R. Burgess / Establishment and maintenance of cell polarity has become an increasingly interesting biological question in a diversity of cell types and has been found to play a role in a variety of biological functions. Previously, it was thought that the echinoderm embryo remained relatively unpolarized until the first asymmetric division at the 16 cell stage of development. However, there is mounting evidence to suggest that polarity is established much earlier. I analyzed roles of the cell polarity regulators, the PAR complex proteins, and how their disruption in early development affects later developmental milestones such as blastula formation. I found that PAR6 along with aPKC and CDC42 localize to the apical cortex (free surface) as early as the 2 cell stage of development and this localization is retained through the gastrula stage. Interestingly, PAR1 also colocalizes with these apical markers through the gastrula stage, despite the formation of a polarized epithelium and a series of asymmetric divisions. Additionally, PAR1 was found to be in complex with aPKC, but not PAR6, during these developmental stages. PAR6, aPKC, and CDC42 are anchored in the cortex by assembled myosin; however, a clear role for myosin assembly in PAR1 localization could not be determined. Furthermore, myosin assembly was found to be necessary to maintain proper PAR6 localization through subsequent cleavage divisions. Interference with myosin assembly prevented the embryos from reaching the blastula stage, while transient disruptions of either actin or microtubules did not have this effect. Similarly, inhibition of aPKC activity during early cleavage stages impeded blastula formation; however, aPKC is not involved in the regulation of the first asymmetric division at the 16 cell stage in sea urchin embryos. These observations suggest that disruptions of the polarity complex in the early embryo can have a significant impact on the ability of the embryo to reach later critical stages in development. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The ESCRT machinery has a well established role within the endocytic pathway. Studies conducted in Drosophila have identified ESCRT proteins as important regulators of epithelial cell polarity and growth. Consequently ESCRTs have been classified as potential tumour suppressors. Alterations in the expression of various ESCRT components have been observed in human cancers. However, the possible link between ESCRT proteins, mammalian epithelial cell polarity and tumourigenesis has not been investigated. This thesis demonstrates for the first time that the ESCRT-I protein, Tsg101, is required for maintenance of mammalian epithelial cell organisation and polarity. siRNA knockdown of Tsg101 in the human Caco-2 cell line results in the formation of a multilayered epithelium with compromised apicobasal polarity. In addition, Tsg101 depletion impairs differentiation of the epithelial sheet and formation of polarised 3D Caco-2 cysts. Depletion of Tsg101 also results in intracellular accumulation of the tight junction protein, claudin-1. This is shown to be constitutively endocytosed and recycled in Caco-2 epithelial monolayers, suggesting that ESCRT-I is required for claudin-1 recycling to tight junctions. Tsg101 knockdown also impairs epithelial barrier formation and enhances Caco-2 migratory ability. This suggests that tight junction integrity is impaired and may contribute to the loss of Caco-2 cell organisation and polarity observed upon Tsg101 depletion. Finally, Tsg101 depleted Caco-2 cells appear to overproliferate, forming multilayered regions of the epithelial sheet. However, multilayered cells are eventually eliminated via apoptosis. Preliminary results suggest that inhibition of this apoptotic response enhances the aberrant epithelial phenotype, suggesting that the ability to evade apoptosis may be an important factor in determining the tumourigenic potential of ESCRT-I depletion. Therefore, results presented in this thesis suggest that the role of ESCRT-I as a tumour suppressor is conserved from Drosophila to mammals.

Cell polarity is essential for cellular functions, growth, development, and formation of multicellular organisms. Cell polarization is often regulated during the cell division cycle. For instance, many cell types lose polarity and round up during mitosis, and then reestablish polarity after division. The fission yeast Schizosaccharomyces pombe is a model system for studying cell polarization. These unicellular rod-shaped cells grow by extension from their tips, and then stop growth during mitosis. Upon cytokinesis, they initiate growth from the old cell end and later in interphase, initiate growth at the second cell end in a process known as "new end take off" or NETO. NETO is regulated by polarity proteins tea1p and tea4p which are deposited by microtubules at the cell tips. How these proteins regulate cell polarity is not yet well understood. These polarity proteins are thought to function in recruiting other proteins, which leads to localized actin polymerization, membrane trafficking and cell wall assembly, leading ultimately to polarized cell growth at the cell tip. In this thesis, I report the characterization of a new polarity protein tea5p in fission yeast. I identified tea5p in a screen for new NETO mutants. Tea5p is a new component of the tea-protein polarity pathway. It resides at cell tips in complexes with the other polarity proteins tea1p and tea3p, and functions downstream of tea1p. Genetic interactions suggest that tea5p regulates polarized growth by regulating the small GTPase cdc42p and its activator gef1p. Tea5p is a pseudokinase that binds to the plasma membrane with its N terminus, and requires its kinase like domain for function. Together my results begin to establish a pathway that links microtubules to activation of cdc42p for regulation for polarized growth in S. pombe.