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A developing vertebrate embryo can only subsist for a finite time without a vasculature. The vascular system is one of the earliest organ systems to develop and allows continued rapid maturation of complex multicellular organisms. Failure of the cardiovascular system to develop results in early termination of the fetus. Together, my thesis research demonstrates the important implications that development and cell biology have on vascular function and repair. First, a thorough evaluation of characteristics of vascular smooth muscle cells from differing regions of the adult and embryonic aorta revealed that differences between the ascending and descending embryonic aortae converge in the adult. These data have important implications for vascular development and disease. The second story examines the cell biological implications of the loss of centromere protein F (CENP-F). A cardiac specific deletion of CENP-F results in dilated cardiomyopathy and by understanding how CENP-F alters cell biology in a model cell line, we now know how loss of CENP-F modifies cardiac biology. Ultimately, these seemingly disparate studies further our understanding of vascular biology and disease.

The ubiquitinâproteasome system (UPS) influences gene transcription in multiple ways. One way in which the UPS impacts transcription centers on transcriptional activators, the function of which can be stimulated by components of the UPS that also trigger their destruction. Activation of transcription by the yeast activator Gcn4, for example, is attenuated by mutations in the ubiquitin-ligase that mediates Gcn4 ubiquitylation or by inhibition of the proteasome, leading to the idea that ubiquitin-mediated proteolysis of Gcn4 is required for its activity. Here, I probe the steps in Gcn4 activity that are perturbed by disruption of the UPS. I show that the ubiquitylation machinery and the proteasome control different steps in Gcn4 function, and that proteasome activity is required for the ability of Gcn4 to bind to its target genes in the context of chromatin. Curiously, the impact of proteasome inhibition on Gcn4 activity is suppressed by mutations in the ubiquitin-selective chaperone Cdc48, revealing that proteolysis per se is not required for Gcn4 activity. My data highlights the role of Cdc48 in controlling promoter occupancy by Gcn4 and support a model in which ubiquitylation of activatorsânot their destructionâis important for function.

<p>Many marine invertebrates have larvae that must feed to complete development to metamorphosis. Larval feeding performance affects the amount of time larvae spend in the plankton, which affects larval mortality and dispersal, and juvenile quality. Larval feeding performance is partly determined by the abundance of edible particles in the plankton. However, the plankton also contains particles that are too large for ingestion. In this thesis I show that: 1) echinoderm larval feeding performance is reduced in environmentally realistic concentrations of inedible particles in simplified laboratory feeding environments, 2) larval feeding performance of at least one echinoid is reduced in the presence of natural inedible particles in plankton, and 3) larval feeding performance may be affected by inedible beads in the mouth. My results suggest that in nature, rates of food acquisition by larvae may depend not only on food abundance, but also on the abundance of potentially interfering non-food particles.

Eukaryotic cells respond to stress by reprogramming their gene expression program to inhibit global protein synthesis and direct translationally silenced mRNAs to cytoplasmic foci known as stress granules (SGs). SGs function as sites where mRNAs are sorted for storage, decay or translation. SGs are linked with neurodegeneration, cancer and viral infections. However, the molecular mechanisms underlying SG function during stress and in diseases are poorly understood. DEAD-box proteins (Dbps) are RNA-dependent ATPases that mediate changes in mRNA-protein complex (mRNP) structure. Several Dbps, including DDX3, are recruited to SGs and regulate mRNP entry and exit. But, it is unclear how the Dbp activity is regulated in SGs. In the work presented here, we show that human (h) Gle1 is a novel factor of SGs and it regulates SGs by modulating the dynamic equilibrium between SGs and translation through its regulation of DDX3. We further show that two alternatively spliced isoforms of the GLE1 gene perform distinct and non-overlapping functions in a cell; hGle1A is required for SG function, whereas hGle1B functions in SGs. Interestingly, our results also show that an amyotrophic lateral sclerosis (ALS)-linked mutation in GLE1 disrupts functional specificity of hGle1 isoforms and the resulting protein variant is bifunctional. Overall, this study provides insight into the critical roles of hGle1 in SG biology and also gives clues as to how mutation of GLE1 contributes towards ALS pathogenesis.

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. In this dissertation we show that polymeric immunoglobulin receptor-deficient (pIgRâ/â) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodeling and emphysema in pIgRâ/â mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-kappa B activation, leukocyte infiltration, and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgRâ/â mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodeling, while repetitive inhalation of bacterial products exacerbates disease. In addition, we demonstrate that p73 is required for PIGR expression in mice, and that mice lacking p73 also develop airway remodeling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodeling and emphysema. Based on this data, we propose that altered mucosal immunity due to SIgA deficiency contributes to chronic inflammation and airway remodeling in COPD.

<p>Male mammals must simultaneously produce prodigious numbers of sperm and maintain an adequate reserve of stem cells to ensure continuous production of gametes throughout life. Failures in the mechanisms responsible for balancing germ cell differentiation and spermatogonial stem cell (SSC) self-renewal can result in infertility. We discovered a novel requirement for Androgen Receptor Trapped clone-27 (ART-27) in spermatogenesis by developing the first knockout mouse model for this gene. Constitutive deletion of ART-27 is embryonic lethal between e5.5 and 7.5 due to defects in extra-embryonic tissues. Conditional knockout in the male germline results in a rapid decline in pre-meiotic germ cell number that starts around day 6-7 post-partum, eventually leading to a Sertoli cell-only phenotype that does not recover in the adult. Gene expression analysis revealed that ART-27 deletion downregulates the transcription of genes governing SSC self-renewal, differentiation, and meiosis. These data are consistent with spermatogenic arrest before meiotic entry and the total lack of germ cells after day 23. Sertoli cell-specific knockout of ART-27 also results in germ cell loss, and we hypothesize this is due to disruption of androgen receptor signaling. Our study has revealed the first in vivo function for ART-27 in the mammalian germline as a regulator of distinct transcriptional programs in SSCs and differentiating spermatogonia.

Programmed cell death (PCD) is an important process necessary for the maintenance of tissues in adult organisms and the crafting of distinct tissues in development. The two main types of PCD, apoptosis and necroptosis (i.e. programmed necrosis), are characterized through differing morphologic presentations and outcomes. Death receptor signaling is a context in which both apoptotic or necroptotic outcomes can occur. Several recent studies implicate proteins involved in apoptotic signaling in the inhibition of necroptosis including Caspase-8, FADD, and cFlipL. Bid, a member of the BCL-2 family of proteins, is cleaved by Caspase-8 which promotes its activation and translocation to the mitochondrion, promoting apoptosis. To evaluate what role Bid might play in the necroptotic arm of death receptor signaling we developed a mouse with Bid and its apoptotic arm of function (Bax and Bak) removed in hematopoietic cells. Loss of these three proteins leads to loss of restraint of necroptosis leading to increased necroptotic death, inflammatory signaling, and perturbation of tissue homeostasis in the hematopoietic and gastrointestinal organ systems. These findings in mice have implications for Myelodysplastic Syndrome, a bone marrow failure disorder characterized by increased PCD, and Inflammatory Bowel Diseases, inflammatory diseases characterized by overwhelming inflammation in the gastrointestinal system.

The Wnt pathway is a highly-conserved pathway that controls many developmental processes and is mutated in many human diseases (e.g., cancer). The tumor suppressor adenomatous polyposis coli (APC) is a critical negative regulator of Wnt signal transduction. Mutations in the APC gene resulting in constitutive activation of the Wnt pathway occur in over 80% of human colorectal cancers (CRC). Despite its critical role in the Wnt pathway, the exact mechanism of APC function in Wnt signal transduction is not clear. The lab developed a monoclonal antibody (mAb7E5) that targets the co-receptor LRP6 and inhibits Wnt signaling in APC-mutant CRC cells. Using the antibody mAb7E5, I found that APC regulates Wnt receptor activation. Furthermore, I found that, in APC-depleted cells, the co-receptor LRP6 is constitutively active in a manner independent of Wnt ligands and that LRP6 is internalized by the clathrin-dependent endocytic machinery. Finally, I demonstrate that APC, clathrin, and the AP-2 adaptor protein interact as a complex. Thus, my studies reveal a new role for APC function in Wnt signal transduction and provide insight into the development of therapeutic agents targeting APC-mutant tumors.

Type 2 diabetes is characterized by failure of pancreatic β-cells to secrete adequate insulin to meet the needs of the body. This β-cell failure is thought to be caused by increased metabolic load due to mounting insulin resistance, but the molecular mechanisms by which this dysfunction occurs are not fully understood. To better understand how β-cells fail, we took a whole-transcriptome approach, collecting RNA-sequencing datasets from purified β-cell populations from several mouse models of β-cell stress. First, we used mice lacking Abcc8, a key component of the β-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on β-cell identity and gene expression. We found that chronically elevated β-cell [Ca2+]i results in the dysregulation of over 4,200 genes, as well as modest loss of β-cell identity, characterized by decreased expression of key functional genes, increased expression of genes associated with β-cell dedifferentiation, increased β-cell transdifferentiation to PP-expressing cells, and decreased β-cell function. These studies prompted us to propose a model by which chronically elevated β-cell [Ca2+]i acts through a putative Ca2+-regulated transcription factor, ASCL1, to disrupt a network of genes, contributing to β-cell failure. In addition to exploring the effects of chronically elevated [Ca2+]i on β-cell gene expression, we analyzed β-cells from mice ectopically expressing human growth hormone (hGH), mice made insulin resistant by feeding a high-fat diet (HFD), and mice of different sexes. We found that both ectopic hGH and HFD have beneficial effects (induction of β-cell proliferation genes) as well as deleterious effects (increased expression of ER stress genes) on β-cell function. Ultimately, the collection of 17 RNA-sequencing datasets allowed us to perform weighted gene correlation network analysis (WGCNA) to generate modules of similarly-expressed genes. Several of these initial modules have meaningful correlations to specific β-cell stresses. Overall, these studies highlight the power of using whole transcriptome datasets from highly-pure cell populations and have allowed us to elucidate how stress alters the β-cell gene regulatory network.