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Dysregulated mTOR signaling and tissue-specific phenotypes in Tuberous Sclerosis ComplexArmour, Eric Andrew 27 November 2013 (has links)
Tuberous Sclerosis Complex (TSC) is a multi-organ hamartomatous disease caused by loss of function mutations in either the TSC1 or TSC2 genes. Despite involvement of multiple organs such as the kidneys, lungs, and skin, neurological aspects are usually the most severe due to a very high prevalence of cognitive impairment, autism and epilepsy. The protein products of TSC1 and TSC2, hamartin and tuberin respectively, regulate the mTOR kinase signaling pathway. Current models of TSC propose that hamartoma formation is secondary to a loss of heterozygosity at either the TSC1 or TSC2 loci, and subsequent hyperactivation of mTOR Complex 1 (mTORC1). In this dissertation I explore the underlying mechanisms of organ specific pathogenesis in TSC.
In the first half of my dissertation, I demonstrate that loss of Tsc1 in the distal convoluted tubule of the kidney results in cystogenesis. Cyst formation in these kidneys is due to a mTORC1 but not mTORC2 dependent process. I then show that cystic changes in these kidneys may be due to ciliary defects.
While a loss of heterozygosity has clearly been reported in the kidney and other organ system, second hit mutations in neural lesions have only rarely been identified. Thus, to begin to define the role of the heterozygosity of TSC1 or TSC2 during the pathogenesis of TSC in the brain, we generated induced pluripotent stem cells (iPSC) from patients with TSC. Deep sequencing of these patents revealed that all of our patient derived lines are heterozygous for TSC2 mutations. I then provide evidence that these heterozygous iPSCs are abnormal with increased cell survival and enhanced maintenance of pluripotency. These changes may be due to slight changes in mTORC1 signaling.
The work presented in this dissertation increases our understanding of the tissue specific phenotypes and underlying mechanisms of TSC pathogenesis. This research may lead to the identification of new therapeutic targets for TSC and associated comorbidities.
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Nuclear Structure in Budding yeast: Impacts of Chromatin Organization and Gene ExpressionBurns, Laura Titus 04 December 2013 (has links)
The genome of a eukaryotic cell tightly packed within the nucleus with a high degree of structural organization. Two mechanisms accounting for nuclear structure and the dynamics of subnucler organization in S. cerevisiae are presented within. First, two powerful genetic screens identify requirements for the RSC chromatin-remodeling complex in maintaining nuclear morphology. The major NE-malformations observed in rsc mutants likely result from aberrant transcription and lipid homeostasis. Second, nuclear organization of transcriptional events in response to osmotic stress in S. cerevisiae involves the relocalization of the Hot1 transcription factor to foci that overlap with corresponding target genes. Casein Kinase II negatively regulates Hot1 localization to foci, and also leads to a reduced transcriptional response. These results suggest that the nuclear organization of transcription events impact the stochastic activity of environmentally induced genes. In conclusion, both chromatin organization and transcription events result in dynamic alterations in nuclear structure impacting the output of the genome.
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Investigating the roles of Notch and Vascular Endothelial Growth Factor in Hepatic Cell Fate Decisions and Architectural Establishment in Development and Disease.Walter, Teagan Jo 09 December 2013 (has links)
During liver development, precise signaling is required to direct cell fate decisions and architectural establishment. It is hypothesized that the same signaling pathways are important in directing liver regeneration. To examine the signals important in liver development and disease, we genetically manipulated Recombination signaling binding protein J kappa (Rbpj) and Hepatocyte nuclear factor 6 (Hnf6), or Vascular endothelial growth factor (Vegf) within the liver epithelium embryonically in mice. Along with these genetic models, we also examined several non-genetic rodent liver injury models and assessed the protein localization and tissue architecture of both epithelial and endothelial structures in the liver. We find that Rpbj and Hnf6 are together required for the embryonic formation of the intrahepatic bile duct, but dispensable for its regeneration postnatally. In both genetic and non-genetic cholestatic liver injuries in rodent models and in human liver disease, the expression of Sry-related HMG box 9 (Sox9) was found to be a marker of non-proliferative hepatobiliary intermediate cells that may aid the regenerative process. We also determined that the epithelial secretion of Vegf is required for proper endothelial and epithelial cell maturation and tissue architecture. These studies implicate a number of signaling relationships required for proper liver development and regeneration.
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Regulation of dynein localization and ciliogenesis by ASUNDER via its role in the RNA processing complex, IntegratorJodoin, Jeanne Nicole 10 December 2013 (has links)
Cytoplasmic dynein is a large, multimeric complex that walks along microtubules to perform multiple functions within the cell. This motor is commonly found associated with the dynein-activating complex, dynactin. Dynein is required for a variety of essential functions such as cargo transport, organelle positioning, centrosome assembly and coupling to the nuclear envelope at the G2/M transition, mitotic spindle positioning, and ciliogenesis. Due to the vast number of roles within the cell, dynein complexes are subject to multiple layers of regulation, including binding of accessory proteins, phosphorylation, variations in subunit composition, and subcellular localization. Additionally, dynein roles are often cell cycle dependent.
At the G2/M transition, a subpopulation of dynein is found anchored to the nuclear envelope in multiple species. In cultured human cells, this pool facilitates centrosome coupling to the nuclear envelope, centrosome splitting and migration to the presumptive poles, and nuclear envelope breakdown. Bicaudal D2 and Centromere protein F are required for anchoring this pool of dynein on the nuclear envelope. Another cell cycle-dependent role for dynein is primary ciliogenesis during G1 in cultured human cells. Dynein is required for promoting assembly of the primary cilium as well as regulating its length. For both functions, the complete mechanism for regulating dynein remains unclear.
Asunder has been previously shown to regulate perinuclear dynein at G2/M in Drosophila spermatogenesis. Additionally, Asunder has been shown to be an essential subunit in the small nuclear RNA-processing complex, Integrator. Herein, I present data showing that Asunder has a conserved role in regulating perinuclear dynein in cultured human cells via its role in the Integrator complex. I additionally report that Integrator is required for primary ciliogenesis in G1-arrested cells. My work supports the model that Integrator independently regulates these cellular events at the RNA-processing level. This works expands our understanding of the mechanisms for dynein anchoring to the nuclear surface at G2/M and ciliogenesis by adding an RNA-processing component.
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Modulation of Sonic Hedgehog signaling alters cerebellar development and medulloblastoma formationCheng, Frances Yun 10 December 2013 (has links)
Cerebellar development is a complex process involving the tightly regulated proliferation, specification, migration, and connectivity of thousands of neurons and glia. Perturbations in signaling pathways important for these processes can have drastic consequences, including medulloblastoma formation. In this dissertation work I have focused on Sonic Hedgehog (Shh) signaling and its role in cerebellar development and formation of medulloblastoma. Our studies have identified a novel contribution of the multipotent hindbrain roof plate cell to diverse lineages in the cerebellum and its susceptibility to oncogenic transformation by deregulated Shh signaling, which leads to medulloblastoma formation. In addition, we have determined a previously unappreciated role for Shh signaling in specialized cerebellar glial cells, which functions to sustain proliferation of neighboring neuronal precursors. Last, we identify a small molecule as a novel and potent Hh pathway antagonist in multiple cell types, including Hh-responsive medulloblastoma cells. My work therefore offers insight into the diverse roles of Shh activity in the cerebellum during development and disease, which can provide insight into brain growth and the development of targeted therapies for disease processes.
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Domain general versus domain specific mechanisms in theory of mind : a comparison of individuals with autism, developmental delay, and typical developmentBoseovski, Janet J. January 1999 (has links)
Domain specific theories of development postulate that mental state reasoning (i.e., theory of mind; ToM) develops independently of general problem solving ability. In contrast, domain general theories suggest that the abilities develop concurrently. To assess the relation between mental state reasoning and problem solving, children with autism, developmental delay, and typical development were administered 2 ToM tasks, and 3 problem solving tasks that did not require mental state reasoning. Typically developing children performed better than both groups on the problem solving tasks, each of which required the use of embedded rules. For all groups, positive within-group correlations emerged between the tasks that required mental state reasoning and the general problem solving tasks, suggesting that a common mechanism underlies both abilities, and challenging the notion of domain specificity. The current findings are discussed in the context of a domain general versus domain specific account of ToM acquisition in typical and atypical populations.
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Gender public regard and approach towards masculinity in 6-year-oldsBryant, Danielle N. 05 May 2015 (has links)
<p> From early to middle childhood, girls normatively begin to show a shift towards masculinity. Preschools are filled with "girly girls" whereas elementary schools show a high prevalence of girls self-identifying as tomboys. In contrast, boys' masculinity remains stable without a similar shift towards femininity. One possible explanation for this discrepancy is children's awareness of male prestige. As children become more aware that males are valued over females, I hypothesize that children may be motivated to approach masculinity and possibly avoid femininity. The current study uses archival data and examines whether awareness of male prestige is associated with an approach towards masculinity exhibited by children's gender attitudes. Participants included 217 six-year-old children who were interviewed. As hypothesized, the more that children believed that others had a higher regard for boys compared to girls, the more favorable were their attitudes towards boys, and the less favorable were their attitudes toward girls.</p>
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Understanding Positional Information During Zebrafish Fin RegenerationNachtrab, Gregory January 2013 (has links)
<p>Regeneration is a remarkable feat that can only be accomplished by a small number of animals. The regeneration of vertebrate limbs is one such case as certain salamanders and fish regenerate robustly while mammalian ability to regenerate is extremely limited. Successful regeneration requires not just cell proliferation after injury but also the patterning of the new tissue into a suitable replacement structure. The process by which this patterning happens is referred to as positional memory. Identification of factors responsible for positional memory in vertebrate appendage regeneration has remained elusive. This dissertation establishes zebrafish pectoral fins as a model system for studying and defining positional memory factors. This has been accomplished through careful morphological measurements, gene expression profiling, construction of transgenic zebrafish strains, and the use of various chemical reagents. Two stunning examples of positional information in the pectoral fin have been discovered. First is the region-specific defect in male pectoral fin regeneration governed by an androgen's influence on GSK3 activity. The second is the role for hand2 in maintaining restricted vitamin D signaling and thus small bones in the posterior region of the pectoral fin. hand2 is the first defined positional memory factor in a zebrafish fin. However, in spite of this success the tools required for further dissection of positional memory are not available and thus the potential for meaningful future work is slight.</p> / Dissertation
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The process and development of empathy in educators: a phenomenological inquiryStanley, Sharon Anne 14 May 2015 (has links)
Graduate
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Characterization of Lrig1+ colonic stem/progenitor cells and their transformative capacityPoulin, Emily Jean 27 March 2015 (has links)
The intestinal epithelium is a continuously renewing tissue. This rapid renewal is fueled by stem cells that reside in the base of the crypts of Lieberkühn, the functional unit of the intestinal epithelium. The identity of intestinal stem cells has been the subject of over fifty years of ongoing investigation. Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) marks a population of intestinal stem cells, but the identity of Lrig1+ cells has remained controversial due to discrepant reports of Lrig1 expression in the crypt base. We characterized two Lrig1+ populations in the colonic epithelium using two anti-Lrig1 antibodies and a new Lrig1 reporter mouse. We determined that these two populations could be distinguished by reporter expression, Lgr5 expression, and likely the glycosylation status of Lrig1. Since stem cells are widely considered to be tumor-initiating cells in colorectal cancer, we developed new mouse models of colonic neoplasia: Lrig1CreERT2/+;Apcfl/fl and Lrig1CreERT2/+;Apcfl/fl;KrasLSL-G12D/+. We found that Apc loss resulted in high-grade tumors; expression of mutant Kras expression did not significantly affect histological grade of tumors, but may contribute to increased invasion.
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