Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

Prader-Willi syndrome (PWS) is a recurrent microdeletion syndrome characterized by severe obesity, hyperphagia, hypotonia, and developmental delay, and is caused by the loss of expression of four protein-coding genes and set of small nucleolar RNAs on chromosome 15. NDN, encoding the protein necdin, is one of these genes, and a large body of literature supports the theory that necdin is important for the differentiation and survival of neurons. Given that necdin is also abundant in developing muscle and that hypotonia is a cardinal feature of PWS, I hypothesize that necdin promotes normal skeletal muscle development. I provide two lines of evidence demonstrating that loss of necdin impairs muscle development in mice. First, necdin interacts with the inhibitor of muscle differentiation EID-1 to relieve inhibition of MyoD-dependent transcription by sequestering this protein in the cytoplasm in over-expression assays. Unexpectedly, the presence of necdin increases EID-1 protein abundance in transfected cells and endogenous EID-1 is less abundant in Ndn-null embryonic mouse tissue compared to controls. Finally, conversion from MyoD+ to Myosin Heavy Chain+ cells is impaired in limb bud cultures from Ndn-null embryos, consistent with the hypothesis that loss of necdin impairs muscle differentiation by failing to relieve EID-1-dependent transcriptional inhibition. Second, loss of necdin impairs polarization of muscle progenitors in vitro and in vivo due to failed activation of the actin-myosin cytoskeleton, and reduces the proportional area of forelimb extensor muscles in Ndn-null mice at birth. This conclusion is supported by defective centrosome re-orientation due to impaired nuclear rearward movement and failed Cdc42 activation in Ndn-null mouse embryonic fibroblasts (MEFs), impaired myosin activation in Ndn-null MEFs and cortical neurons, and excessive branching and failure of hippocampal neurons to polarize with respect to a growth factor. Additionally, PWS patient fibroblasts display centrosome re-orientation defects and impaired myosin activation identical to Ndn-null MEFs, indicating that loss of necdin produces a similar phenotype in both mice and humans. These results provide strong evidence that necdin is critical for both the migration and primary differentiation of skeletal muscle, and validates the Ndn-null mouse as a model for hypotonia in PWS.

Thesis (Ph.D.) -- University of Western Sydney, Hawkesbury, 1999. / Thesis submitted for the degress of Master of Science (Hons.). Includes bibliographical references (leaves 123-125).

Thesis (Ph. D.)--University of Texas at Austin, 2009. / Title from PDF title page (University of Texas Digital Repository, viewed on Sept. 15, 2009). Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Sc.)--University of Canterbury, 2005. / Typescript (photocopy). "October 2005." Includes bibliographical references (leaves 60-62). Also available via the World Wide Web.

The Par complex protein, atypical protein kinase C (aPKC), plays an instrumental role in diverse cell polarities. aPKC is able to restrict substrate localization through a phosphorylation-induced cortical exclusion mechanism, allowing for the generation of molecularly distinct cortical domains. Thus, controlling the localization of aPKC is central to Par-mediated polarity but the mechanism by which aPKC is polarized remains poorly understood. In this dissertation I investigated the restriction of aPKC to the apical cortex of Drosophila neural stem cells, neuroblasts, as these cells dynamically polarize aPKC through repeated asymmetric cell divisions. The polarity created through aPKC phosphorylation must be tightly regulated in order to ensure proper balance between self-renewal and differentiation. To begin, I investigated whether or not aPKC’s so called ‘maturation’ by PDK1 phosphorylation is required for aPKC activity and localization. We found that aPKC’s phosphorylation by PDK1 is required for both polarity and full activity. An aPKC containing an unphosphorylatable activation loop mutation localizes symmetrically around the cortex in a manner independent of its binding partner, Par-6, suggesting that aPKC could interact with the cortex by an unknown mechanism. To investigate how aPKC is able to localize to the cortex independent of Par-6, I used an in vivo structure function analysis of domains within aPKC, accompanied by biochemical approaches. I identified a necessity for the aPKC C1 domain for binding to the neuroblast cortex. This interaction is mediated by negatively charged phospholipids. Neither aPKC interaction, with phospholipids or Par-6, is sufficient to restrict aPKC to the apical cortex. Thus, aPKC polarization utilizes a dual interaction mechanism that takes advantage of both protein-lipid and protein-protein interactions, and proper control of each of these signals is required to prevent neuroblast division defects. One interaction, mediated by the C1, is a general cortical targeting mechanism, whereas the other specifies polarization mediated by Par complex interactions. We conclude that a conformational change induced by these interactions activates aPKC’s catalytic activity, thereby coupling localization and activity. This dissertation includes unpublished co-authored material.

abstract: This thesis explores the distribution of certain lexical items in Modern Standard Arabic (MSA) and their relationship with two linguistic phenomena, negative concord (NC) and negative polarity items (NPIs). The present study examines two central questions: the first question investigates whether or not MSA shows the patterns of negative concord languages. The second question concerns the distribution of N-words and NPIs in MSA, and in which environments they appear. To answer the research questions, the thesis uses the framework of generative grammar of Chomsky (1995) and The (Non)veridicality Approach by Giannakidou (1998, 2000, 2002). The data reveal that MSA shows the patterns of strict negative concord languages that are suggested by Giannakidou (2000) in the sense that the negative particle obligatorily co-occurs with the N-words which strengthen the degree of negation, and never lead to a double negation interpretation. Moreover, the data show that there is only one pure NPI which appears optionally in two environments, antiveridical and nonveridical environments, and it is disallowed in veridical environments. On the other hand, the investigated indefinite nouns show a mixed picture since they work differently from their counterparts in Arabic dialects. Their descendants in Arabic dialects appear as NPIs while they tend to be indefinite nouns rather than NPIs in MSA. / Dissertation/Thesis / M.A. English 2013

In this thesis I discuss the problem of negative polarity items (NPls). NPis are items that have to be licensed by a certain group of expressions. In this group of expressions which can trigger NPIs we find, among other things: negations, adversative expressions, questions and conditionals. I show that there is an important problem for a grammatical approach to negative polarity: the group of expressions which can licence NPls can't be adequately defined in a grammatical way. There is, however, a semantic way of defining the group of expressions that can licence NPIs. In semantics the group is often referred to as the group of "triggers". It can be proven logically that the group of triggers can be divided into four subgroups: a group of downward-entailing expressions, antimultiplicative expressions, anti-additive expressions and antimorphic expressions. By carrying out a corpus study I find evidence for the hypothesis that the way in which NPIs are licenced by the triggers with different logical properties originates from the different grammatical classes of NPIs (negative polarity nouns, negative polarity adjectives and negative polarity verbs). Since there is evidence for this causal relation, I argue that a grammatical approach to NPI-triggering is necessary from a formal point of view. I give a Minimalist account of NPI-triggering. To make the Minimalist Program suitable for NPI-triggering I have to assume, however, that the semantic information about triggers is available in the lexicon of the MP.