The asymmetric segregation of fate determinants is a conserved process by which differential cell fate can be acquired upon cell division. In this thesis we investigate how the asymmetric localisation of fate determinants is achieved in Drosophila neuroblasts (NBs, Neural Stem Cells). In particular we focus on the localisation of the fate determinant Miranda, which is segregated to the basal pole of the NB cell cortex in mitosis and carries a series of signalling molecules into one of the two daughter cells, promoting differentiation. The most widely accepted model for how Miranda becomes polarised at mitosis is based on its phosphorylation by the apically localised kinase, aPKC (atypical protein kinase C). This model proposes that aPKC localises to the apical cortex and phosphorylates Miranda, excluding it from the apical domain by phosphorylation of Miranda’s membrane binding motif. However, earlier work demonstrated that the acto-myosin cell cortex is essential for asymmetric Miranda localisation. Thus far these two models have not been successfully integrated. In this thesis we generated flies carrying fluorescent reporters for apical and basal polarity proteins and imaged their localisation live. We reveal that localisation appears to happen in two stages. Firstly, Miranda is localised uniformly to the plasma membrane, from where it is cleared by aPKC at the onset of prophase in an actin independent manner. After NEB, Miranda returns to the cell cortex, localising to a basal crescent in an acto-myosin dependent manner. Furthermore, the size of the basal domain to which Miranda localises appears to be under the control of Rho kinase, and linked to cell size asymmetry. Together these data suggest that in mitosis, Miranda localisation is under structural control. Therefore, we reveal that aPKC and Actin-myosin activity contribute to Miranda localisation at distinct time points in the cell cycle.

Cell polarity is the asymmetric distribution of organelles that almost all cells use to separate individual processes and perform complex functions. Although the manner in which cells are polarized is very diverse, the processes necessary to assume polarized phenotypes are similar in many cell types. Epithelial cell polarization is of particular importance, as these cells serve form linings of organs and act as barriers distinguishing different compartments. Furthermore, loss of epithelial polarization occurs in some disease states and may result in cell invasion through underlying matrix. During initial polarization, vesicle trafficking is indispensible for assembly of structures, including apical junctional complex formation. Trafficking of new membrane and associated proteins to leading edges is also necessary for cell migration. RalA and RalB are members of the Ras superfamily of GTPases and have been implicated in several processes, including vesicle trafficking. Only 5 Ral effectors have been identified, two of which are members of the Exocyst complex, a hetero-octameric complex also involved with vesicle trafficking. I hypothesized that Ral GTPases were necessary for several aspects of cell polarization, and that they engage the Exocyst complex to mediate these processes. Initial investigation of tight junction assembly found that both RalA and RalB antagonistically affect paracellular permeability. Knockdown of RalA and RalB resulted in decreased and increased incorporation of components into assembling tight junctions, respectively. Furthermore, both RalA and RalB engaged the Exocyst in order to mediate tight junction assembly. I next examined the role of RalA-Sec5 and RalA-Exo84 interactions during tumor cell migration and invasion. Both interactions were necessary for invasion and single cell migration, although disruption of each interaction affected different aspects of migration. Furthermore, significant differences in cytoskeleton organization occurred in response to disruption of RalA-Sec5 and RalA-Exo84 interactions. Finally, I investigated the effects of RalA and RalB knockdown on growth of primary cilia and cyst formation. RalA decreased primary cilia growth and reduced average cilia length, while RalB increased cilia length. Knockdown of RalA and RalB also affected lumen formation during cystogenesis, as RalA knockdown prevented lumen formation and RalB knockdown caused formation of multiple lumens. Taken together, data presented here show that Ral engages the Exocyst to mediate distinct processes during tight junction assembly and cell migration, and implicates Ral GTPases in several different aspects of cell polarity.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Dec. 19, 2007). Vita. Includes bibliographical references.

The Jurassic Quiet Zone (JQZ) is a region of low amplitude, difficult-to-correlate magnetic anomalies located over Jurassic oceanic crust. We collected 1200 km of new deep-tow magnetic anomaly profiles over the Pacific JQZ that complement 2 deep-tow profiles reported in Sager et al. (1998). Our primary goals were to extend the correlation of deep-tow magnetic anomalies farther back in time, to evaluate the correlatability and repeatability of anomalies, and to refine the Jurassic geomagnetic polarity reversal time scale (GPTS). Correlations of anomalies were excellent over M34 and over supposedly older seafloor to the south of ODP Site 801. In contrast, the correlation in the region between M34 and Site 801 was difficult. Using anomaly correlation models, we made magnetic polarity block models to establish a revised Jurassic GPTS extending until 169.4 Ma. Age calibration was accomplished with radiometric dates from two ODP holes. Systematic changes in anomaly amplitudes occur along the survey lines with the amplitudes decreasing backward in time and then increasing again in the oldest part of survey area. The zone of the most difficult to correlate anomalies corresponds to a period of ~4 m.y. that appears to have an abrupt end. This low amplitude zone suggests unusual magnetic behavior during the Jurassic. It has been said that many of the larger anomalies are likely caused by changes in polarity, whereas smaller anomalies may be intensity fluctuations. Although it is impossible to identify which anomalies are caused by reversals and which are not, magnetization structures observed in ODP Hole 801C suggest that many of the smallest anomalies, particularly around Hole 801C indicate polarity reversals. We concluded that (1) the new data demonstrates repeatability and correlatability of the JQZ magnetic anomalies implying that they are seafloor spreading lineations and (2) good correlations made new GPTS models extending back to 169.4 Ma; and (3) the origin of the JQZ may be a combination of rapid polarity reversals in the Jurassic low magnetic dipole field and closely spaced, tilted magnetization structure in the oceanic crust.

Through his work in ludics and Geometry of Interaction, Jean-Yves Girard invites us to a change of paradigm in the study of logic: the quest for a transcendental syntax, some kind of idealized language that emerges from the rules of logic. Amongst these rules, "testing" plays a leading role in defining a duality for the interpretation of negation. The present work focuses on a notion of polarity which is a central technique used throughout Girard's work to express linear negation. We describe some properties and illustrate them with examples with the purpose of getting acquainted with the technique. We also highlight how the classical connectives (conjunction and disjunction) arise from an interpretation based on testing. In a sense, this work is intended to provide an alternative introduction to Girard's ideas and we hope it can have some pedagogical value.

Partitioning-defective protein 6 (Par6), a regulator of cell polarity, is emerging as a mediator of cell differentiation. Herein I sought to assess the contribution of Par6 to trophoblast fusion in normal and pathological human placentae. I hypothesized that Par6 regulates fusion in response to oxygen and transforming growth factor 3 (TGF3) and that this process is altered in preeclampsia (PE). Using silencing and overexpression strategies in choriocarcinoma BeWo cells, my results demonstrate Par6 negatively regulates trophoblast fusion via its roles on tight junctions and cytoskeleton dynamics. Additionally, Par6 expression is elevated in PE, a pathology characterized by placentalhypoxia, increased TGF3, and altered trophoblast fusion. Using low O2 conditions to model PE in BeWo and primary trophoblast cells, Par6 levels increased, and thisassociated with maintenance of tight junctions at cell boundaries and decreased fusion. Overall, my data provides insight into the mechanisms involving Par6 in contributing to the pathogenesis of PE.

Partitioning-defective protein 6 (Par6), a regulator of cell polarity, is emerging as a mediator of cell differentiation. Herein I sought to assess the contribution of Par6 to trophoblast fusion in normal and pathological human placentae. I hypothesized that Par6 regulates fusion in response to oxygen and transforming growth factor 3 (TGF3) and that this process is altered in preeclampsia (PE). Using silencing and overexpression strategies in choriocarcinoma BeWo cells, my results demonstrate Par6 negatively regulates trophoblast fusion via its roles on tight junctions and cytoskeleton dynamics. Additionally, Par6 expression is elevated in PE, a pathology characterized by placentalhypoxia, increased TGF3, and altered trophoblast fusion. Using low O2 conditions to model PE in BeWo and primary trophoblast cells, Par6 levels increased, and thisassociated with maintenance of tight junctions at cell boundaries and decreased fusion. Overall, my data provides insight into the mechanisms involving Par6 in contributing to the pathogenesis of PE.

The Jurassic Quiet Zone (JQZ) is a region of low amplitude, difficult-to-correlate magnetic anomalies located over Jurassic oceanic crust. We collected 1200 km of new deep-tow magnetic anomaly profiles over the Pacific JQZ that complement 2 deep-tow profiles reported in Sager et al. (1998). Our primary goals were to extend the correlation of deep-tow magnetic anomalies farther back in time, to evaluate the correlatability and repeatability of anomalies, and to refine the Jurassic geomagnetic polarity reversal time scale (GPTS). Correlations of anomalies were excellent over M34 and over supposedly older seafloor to the south of ODP Site 801. In contrast, the correlation in the region between M34 and Site 801 was difficult. Using anomaly correlation models, we made magnetic polarity block models to establish a revised Jurassic GPTS extending until 169.4 Ma. Age calibration was accomplished with radiometric dates from two ODP holes. Systematic changes in anomaly amplitudes occur along the survey lines with the amplitudes decreasing backward in time and then increasing again in the oldest part of survey area. The zone of the most difficult to correlate anomalies corresponds to a period of ~4 m.y. that appears to have an abrupt end. This low amplitude zone suggests unusual magnetic behavior during the Jurassic. It has been said that many of the larger anomalies are likely caused by changes in polarity, whereas smaller anomalies may be intensity fluctuations. Although it is impossible to identify which anomalies are caused by reversals and which are not, magnetization structures observed in ODP Hole 801C suggest that many of the smallest anomalies, particularly around Hole 801C indicate polarity reversals. We concluded that (1) the new data demonstrates repeatability and correlatability of the JQZ magnetic anomalies implying that they are seafloor spreading lineations and (2) good correlations made new GPTS models extending back to 169.4 Ma; and (3) the origin of the JQZ may be a combination of rapid polarity reversals in the Jurassic low magnetic dipole field and closely spaced, tilted magnetization structure in the oceanic crust.

Thesis (M.A.)--St. Stephen's Priory, Dominican House of Philosophy, 1964.

Dishevelled (Dvl) proteins are key transducers of Wnt signaling and are encoded by members of a multi-gene family in vertebrates. We report here divergent, tissue-specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression in the mouse. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, one of the outgroups to chordates, we find that the one Dvl gene is dynamically expressed in a tissue-specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation. Most strikingly, we report a novel role for Dvl3 in the maintenance of differentiated muscle and the development of the Xenopus sclerotome. Together, these data demonstrate that that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution. The planar cell polarity (PCP) signaling pathway is essential for embryonic development because it governs diverse cellular behaviors, and the "core PCP" proteins, such as Dishevelled and Frizzled, have been extensively characterized. By contrast, the "PCP effector" proteins, such as Intu and Fuz, remain largely unstudied. These proteins are essential for PCP signaling, but they have never been investigated in a mammal and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice display neural tube defects, polydactyly, and skeletal dysmorphologies that stem from defective ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we establish a central role for Fuz in membrane trafficking, showing that Fuz is essential for apical trafficking of ciliogenesis factors in ciliated cells and also for exocytosis in secretory cells. We identify a novel, Rab-related small GTPase as an interaction partner for Fuz, and this GTPase also is essential for ciliogenesis and secretion. These results are significant because they provide novel insights into the mechanisms by which developmental regulatory systems like PCP signaling interface with fundamental cellular systems such as the vesicle trafficking machinery. / text