The intestinal brush border serves as the sole site of nutrient absorption within the body, and also acts as an important barrier against luminal pathogens. The brush border is comprised of membrane protrusions called microvilli that are found on the apical surface of enterocytes. These protrusions are supported by a core bundle of 20 to 30 parallel actin filaments with the plus ends oriented towards the lumen. Our laboratory showed that protocadherin-24 and mucin-like protocadherin play a key role in the assembly and organization of the brush border. These protocadherins interact to form a trans-heterophilic adhesion complex that physically connects the distal tips of microvilli and regulates the tight packing. How these complexes are targeted to microvillar tips remains unknown. Microvillar protocadherins interact with several cytoplasmic binding partners, including the actin based motor, myosin-7b (Myo7b). Given the role of other myosin motors in building actin-based protrusions, this work focuses on elucidating the function of Myo7b in brush border assembly. We show that Myo7b is highly enriched at the tips of microvilli in both kidney and intestinal brush borders. Loss of Myo7b results in the mislocalization of the intermicrovillar adhesion links along the microvillar axis, disrupting brush border assembly. We also found that Myo7b motor domains are capable of supporting tip-directed transport. However, motor activity is supplemented by other passive targeting mechanisms, which together drive highly efficient accumulation of the adhesion complex at the tips. Additionally, we have established an in-cell reconstitution assay using filopodial protrusions that can be used to study protein interactions within the adhesion complex. Using this assay, we have begun to characterize the morphological effects of tip-enriched adhesion on actin-based protrusions. Initial experiments suggest adhesion at the distal tips regulates the stability and length of these protrusions. This work provides evidence that the actin-based motor Myo7b promotes the accumulation of adhesion complexes at microvillar tips, which may alter the morphology and dynamics of actin-based protrusions. These findings hold important implications for understanding apical morphogenesis in transporting and sensory epithelial tissues.

Post-translational modification of proteins are events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). While the mitotic substrates of the APC/C have been well characterized, it is not clear what role deubiquitinating proteases (DUBs) play in antagonizing APC/C function. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast Schizosaccharomyces pombe. Our evidence demonstrates that deletion of the SAGA associated DUB Ubp8 (ubp8Î) suppresses the temperature sensitive phenotypes of APC/C mutants and this suppression is specific to the SAGA DUB module. Furthermore, mutations abolishing histone H2B ubiquitylation were not able to suppress the APC/C temperature sensitive phenotype. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function utilizing a mechanism dependent on H2B ubiquitination. Hhp1/2 are the soluble casein kinase 1 (CK1) family members in S. pombe. One of their functions is to inhibit the septation initiation network (SIN) during a mitotic checkpoint arrest. Hhp1/2 phospho-prime the SIN scaffold protein Sid4 for ubiquitination by the E3 ligase Dma1; Sid4 ubiquitination delays SIN activation and thus cell division. The SIN is assembled at spindle pole bodies (SPBs), and though Hhp1/2 also localize to SPBs, it is not known if their SPB localization is required for SIN inhibition. Here, we establish that Hhp1/2 localize constitutively to SPBs, the nucleus, cell tips, and division site. We find that their catalytic domains but not enzymatic function are used for SPB targeting and that this targeting strategy is conserved in human CK1δ/ε localization to centrosomes. Further, we pinpoint amino acids in the Hhp1 catalytic domain required for SPB interaction; mutation of these residues disrupts Hhp1 association with the core SPB protein Ppc89, and the inhibition of cytokinesis in the presence of spindle stress. Taken together, we have defined a molecular mechanism used by CK1 enzymes to target to a specific cellular locale for compartmentalized signaling.

<p> Celsr1 is an atypical cadherin central to the asymmetric cell-cell complexes that define planar cell polarity (PCP). Previous work has shown that Celsr1 undergoes bulk endocytosis during cell division in the basal layer of the mouse skin. Here, we report the unexpected finding that Celsr1-mediated intercellular complexes remain intact during mitotic internalization, resulting in uptake of Celsr1 and associated PCP components into dividing cells from their neighbors in a process known as trans-endocytosis. Our observations suggest that the bulk of this internalized pool of Celsr1 is targeted for degradation. Furthermore, Celsr1 internalized from neighboring cells carries with it additional core PCP proteins, including the posteriorly-enriched Fzd6 and anteriorly-enriched Vangl2. However surprisingly, Vangl2 originating from the dividing cell is excluded from mitotic endosomes and remains associated with the membrane. Overexpression of Vangl2 in vitro is sufficient to interfere with Celsr1 internalization, and mitotic internalization of Celsr1 within the skin is delayed at anterior cell surfaces. We propose that Vangl2 stabilizes Celsr1 at the membrane and its dissociation from Celsr1 is a prerequisite for Celsr1 turnover. Together our results indicate that mitotic turnover of Celsr1 depends on the displacement of Vangl2 from PCP complexes and results in the non-autonomous turnover of PCP proteins from neighboring cells.</p><p>

<p> Through a mixed methodology of hermeneutic and heuristic inquiry, this thesis examines the influence of early childhood numinous, or profound spiritual, experience on the developing psyche and its relationship to repetitive compulsion over the lifespan. A dual lens of depth psychology and neuroscience is employed to explore the interstitial spaces between theories on the numen, early psychological development, and the repetition compulsion, comparing the literature with personal and clinical examples. Findings illustrated that numinous experiences are intimately connected with repetitive symptomology and that certain individuals are genetically predisposed to numinous experience. The hypothesis was proposed that repetition compulsion engendered by early numinous experiences, and perhaps all repetition phenomena, might reflect an unconscious <i> telos</i>, or the aim of an integral part of one&rsquo;s psyche seeking expression, integration, and relationship in the world.</p><p>

<p> Echinoid larvae exhibit phenotypic plasticity, where development of long ciliary bands in low-food conditions is considered advantageous for improved algal particle capture. Conversely, larvae in concentrated algal conditions redirect growth to persisting structures (i.e. not lost during metamorphosis) and develop quickly. This study seeks to understand the organismal growth efficiency of sand dollar larvae (<i>Dendraster excentricus</i>) with different phenotypes by comparing larvae developing in low and high algal concentrations (1,000 and 10,000 algal cells ml^&ndash;1, respectively). I measured ingestion, metabolism, and biomass growth rates (protein and lipid) during larval development in three independent cultures. Resulting phenotypes demonstrated morphological divergence, where high-fed larvae grew smaller arms relative to stomach size (a remaining post-metamorphic feature) compared to low-fed larvae. Physiological data were converted to energetic units (mJ) to determine assimilation and growth efficiencies. Low-fed larvae proportionally allocated more energy to metabolism while the high-fed larvae allocated more toward growth. This resulted in different assimilation and growth efficiencies in low- and high-fed treatments. The energetic demands and organismal growth efficiencies of these contrasting phenotypes is important for understanding the constraints on population connectivity of adult populations in rapidly changing marine conditions.</p><p>

Tuberous Sclerosis Complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. TSC causes refractory epilepsy and intellectual disability but the pathogenesis of the neurological symptoms is not understood. Identifying when and in what cell types mutations in TSC1 or TSC2 lead to neurological dysfunction is the first step to better and more targeted treatments. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprograming plasmid containing a shRNA against TP53. Loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. Further, we show that homozygous loss of TSC2 leads to increased mTORC1 in neural progenitors and impaired neural progenitor formation. These results support the contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.

<p> Skeletal muscle generates contractile forces that allow the body to execute movements for walking, speaking and breathing. Although we understand a great deal about the steps of muscle formation, the mechanisms that control muscle size are poorly understood. Even less is known about how muscles interact with skeletal elements, including connective tissue, tendon and bone. This dissertation describes a novel role for Abelson tyrosine-protein kinase 2, a non-receptor tyrosine kinase, during muscle development. First, I characterize the defects in skeletal muscle of <i>abl2</i> mutant mice and show that muscle fibers in the diaphragm and other muscles are extraordinarily long in <i>abl2</i> mutant mice. As a consequence of expansion of the diaphragm muscle, the central tendon of the diaphragm is proportionally reduced in size. Second, I demonstrate that <i>abl2</i> controls muscle size by regulating myoblast proliferation. Third, I show that Abl2 acts in myoblasts to attenuate their proliferation, thereby limiting myoblast fusion and muscle fiber size. Fourth, I show that the exercise endurance of <i> abl2</i> mutant mice is diminished, likely due to the compensatory reduction in size of the diaphragm central tendon. Finally, I provide evidence for signaling between muscle cells and tendon cells that induces tendon cell differentiation. </p><p>

In Schizosaccharomyces pombe cytokinesis requires assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single essential formin, Cdc12, that localizes to the cell middle upon mitotic onset. The molecular mechanisms dictating its divison site targeting during cytokinesis are unknown. We defined that a Cdc12 N-terminal motif directly binds the F-BAR domain of the scaffolding protein Cdc15 and this interaction is controlled by Cdk1 phosphorylation of Cdc12. Phosphorylation of Cdc12 inhibits binding to the F-BAR Cdc15. cdc12 alleles that cannot bind Cdc15 or with all six Cdk1 sites mutated to phospho-mimetic residues show reduced Cdc12 cell division site accumulation and delayed CR formation. Thus Cdk1 phosphorylation of Cdc12 antagonizes its interaction with Cdc15 and its division site localization, consistent with a general role for Cdk1 in inhibiting cytokinesis until chromosome segregation is complete. The CR is physically linked to the plasma membrane (PM). Cells lacking efr3, which encodes a conserved PM scaffold for the phosphatidylinositol 4-kinase Stt4, build CRs that can slide away from the cell middle in a myosin-V-dependent manner. The Efr3-dependent CR anchoring mechanism is distinct from previously reported pathways dependent on the F-BAR protein Cdc15 and paxillin. In efr3â, the concentrations of several membrane-binding proteins were reduced in the CR and/or on the PM. Our results suggest that proper PM lipid composition is important to stabilize the central position of the CR and resist myosin V-based forces to promote the fidelity of cell division.

In normal epithelial wound repair, cells across an epithelial sheet begin a coordinated process of re-epithelialization within minutes of wounding. These coordinated behaviors are driven by a calcium wave, a rise in cytosolic calcium expanding away from the wound in a wave-like fashion. The calcium wave is evolutionarily conserved and is the earliest detectable wound response. Understanding the mechanisms of calcium influx and propagation may reveal fundamental aspects of wound detection and of cell coordination. We observed multiple, distinct mechanisms of calcium influx and propagation around reproducible wounds in the Drosophila notum. First, extracellular calcium flows directly into cells through micro-tears on the cell surface. We were able to assess the role of micro-tears in calcium dynamics by using pulsed laser ablation, a common wounding method that generates exaggerated micro-tears. Pulsed laser ablation creates a cavitation bubble, which forms and collapses within microseconds of ablation and damages the plasma membranes of cells tens of microns away from the wound. Once inside the cells, our model predicts calcium diffuses to neighboring cells via gap junctions. Next, we observed a larger, wound-induced calcium wave that is driven by an unknown extracellular signal. This signal activates a Gαq mediated signaling cascade and induces calcium release from intracellular ER stores. Our simple, pulsed laser ablation wounding model recapitulates a complex damage profile and reveals multiple patterns of calcium influx and propagation around a single wound. For this reason, this model has the potential to unite previous, and seemingly contradictory, findings regarding calcium dynamics in the wound healing field.

<p> The purpose of this dissertation was to provide a comprehensive understanding of the social construction of authenticity and how this is experienced throughout the stages of adult development, with the specific intent of augmenting women&rsquo;s leadership development programs, and thus preventing further entrenchment of stereotypes. In a culture that is deeply conflicted by women&rsquo;s authority, where men and women, along with the gendered structure of most organizations, hold unconscious biases and stereotypes, a developmental understanding for women&rsquo;s leadership development is necessary. The research question was: How do women develop and experience authentic leading and leadership throughout the adult stages of development? </p><p> This qualitative study used Moustakas&rsquo;s (1994) transcendental phenomenological approach. The sources of data were women participants who had previously completed a developmental StAGES assessment. There were 33 women who completed an initial survey and, utilizing stratified sampling, 10 women were selected for interviews, spread evenly across different developmental levels. The interviews were coded to determine <i>what</i> and <i>how</i> leading authentically is experienced, summarized, and analyzed, creating 4 distinct developmental syntheses: the socialized mind, the self-authoring mind, the self-transforming mind, and the self-transcending mind. </p><p> The results indicate women experience and understand authentic leading and leadership differently throughout the stages of development. They follow a path toward wholeness, a developmental process that can be described as the heroine&rsquo;s journey, where their definitions of authenticity, authentic leaders and leadership, and their experience of leading authentically becomes more complex with ever widening perspectives and understanding, initially intellectual and then embodied. The socialized mind has a theoretical understanding with momentary experiences of the phenomenon, the embodied experience of authentic leading arose in the self-authoring mind, and the awareness of gender and leader biases emerged within the self-transforming mind, thus indicating the very subtle nature of these stereotypes, and the correlation between later developmental levels and greater awareness. </p><p> This study contributes to understanding authenticity as a developmental journey and the pervasiveness of gender and leader biases. This study can augment women&rsquo;s authentic leadership development programs to facilitate authentic leading and vertical development. Deeper inquiry into understanding and preventing the entrenchment of these stereotypes is necessary.</p><p>