How do embryos develop with such poise from a single zygote to multiple cells with different identities, and yet survive? At the four-cell stage of the C. elegans embryo, only the blastomere EMS adopts the endo-mesoderm identity. This fate requires SKN-1, the master regulator of endoderm and mesoderm differentiation. However, in the absence of the RNA binding protein POS-1, EMS fails to fulfill its fate despite the presence of SKN-1. pos-1(-) embryos die gutless. Conversely, the RNA binding protein MEX-5 prevents ectoderm blastomeres from adopting the endo-mesoderm identity by repressing SKN-1. mex-5(-) embryos die with excess muscle at the expense of skin and neurons. Through forward and reverse genetics, I found that genes gld-3/Bicaudal C, cytoplasmic adenylase gld-2, cye-1/Cyclin E, glp-1/Notch and the novel gene neg-1 are suppressors that restore gut development despite the absence of pos-1. Both POS-1 and MEX-5 bind the 3’UTR of neg-1 mRNA and its poly(A) tail requires GLD-3/2 for elongation. Moreover, neg-1 requires MEX-5 for its expression in anterior ectoderm blastomeres and is repressed in EMS by POS-1. Most neg-1(-) embryos die with defects in anterior ectoderm development where the mesoderm transcription factor pha-4 becomes ectopically expressed. This lethality is reduced by the concomitant loss of med- 1, a key mesoderm-promoting transcription factor. Thus the endo-mesoderm identity of EMS is determined by the presence of SKN- 1 and the POS-1 repression of neg-1, whose expression is promoted by MEX-5. Together they promote the anterior ectoderm identity by repressing mesoderm differentiation. Such checks and balances ensure the vital plurality of cellular identity without the lethal tyranny of a single fate.

BACKGROUND: Of the 22 known measures of nicotine dependence (ND), none capture the overall disease severity of physical dependence alone. Instead, they capture constructs related to dependence, such as perceived risk, psychological addiction, smoker motivations, or smoking related behaviors, but none of the measures include only physical withdrawal symptoms to capture physical dependence on nicotine. AIM: To develop a range of nicotine dependence measures that capture physical dependence on nicotine. METHODS: The final measures were developed in a cross-sectional study conducted in three phases: 1) candidate item development through literature review and cognitive interviews, 2) developing and pre-testing the survey, and 3) survey administration and psychometric evaluation to validate three distinct measures. The final survey was conducted at four health clinics and three high schools. Psychometric tests used to select the final measure items included inter-item correlations, sensitivity analyses done by subgroup, item-total correlations, convergent validity tests, and confirmatory factor analysis. The final measures were evaluated using confirmatory factor analysis (CFA), internal reliability, total score distributions, and convergent validity correlations. Relative validity analyses were also conducted using a ratio of F-Statistics to compare the ability of each new measure to differentiate dependent smokers as compared previous measures. RESULTS: The final sample included 275 smokers ranging from 14 to 76 years old (mean=30.9, SD=16.2), who smoked an average of 11.5 cigarettes per day (range=0-50, SD=9.4). The sample was 86.5% white and 57.5% male. The three new measures developed included: 1) the 4-item Withdrawal-Induced Craving Scale (WICS) used to capture severity of craving, the most common physical withdrawal symptom; 2) the 12- item Nicotine Withdrawal Symptom Checklist (NWSC), which measures both overall disease severity and the severity of a comprehensive list of individual physical withdrawal symptoms including withdrawal-induced craving, anger, anxiety, depression, headache, insomnia, loss of focus, restlessness, and stress; and 3) the 6-item brief NWSC (NWSC-b), a short measure which only captures overall disease severity. All of the new measures exhibited a unidimensional factor structure loading highly on a single factor (thought to be physical dependence). They also correlated highly (over 0.6) and significantly (p<0.001) to a battery of convergent validity indices including four widely used nicotine dependence measures: Hooked on Nicotine Checklist (HONC), the Autonomy Over Tobacco Scale (AUTOS), the Fagerström Test for Nicotine Dependence (FTND), and self-rated addiction. CONCLUSION: The WICS, NWSC, and NWSC-b provide three distinct validated tools that can be used by researchers, clinicians, and educators to track the progression of physical dependence on nicotine across a range of smoking behaviors and histories.

The primary amino acid sequence of a protein governs its specific cellular functions. Since the cracking of the genetic code in the late 1950’s, it has been possible to predict the amino acid sequence of a given protein from the DNA sequence of a gene. Nevertheless, the ability to predict a protein’s function from its primary sequence remains a great challenge in biology. In order to address this problem, we combined recent advances in next generation sequencing technologies with systematic mutagenesis strategies to assess the function of thousands of protein variants in a single experiment. Using this strategy, my dissertation describes the effects of most possible single point mutants in the multifunctional Ubiquitin protein in yeast. The effects of these mutants on the essential activation of ubiquitin by the ubiquitin activating protein (E1, Uba1p) as well as their effects on overall yeast growth were measured. Ubiquitin mutants defective for E1 activation were found to correlate with growth defects, although in a non-linear fashion. Further examination of select point mutants indicated that E1 activation deficiencies predict downstream defects in Ubiquitin function, resulting in the observed growth phenotypes. These results indicate that there may be selective pressure for the activity of the E1enzyme to selectively activate ubiquitin protein variants that do not result in functional downstream defects. Additionally, I will describe the use of similar techniques to discover drug resistant mutants of the oncogenic protein BRAFV600E in human melanoma cell lines as an example of the widespread applicability of our strategy for addressing the relationship between protein function and biological fitness.

Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.

Obesity has been on the rise over the last 30 years, reaching worldwide epidemic proportions. Obesity has been linked to multiple metabolic disorders and co-morbidities such as Type 2 Diabetes Mellitus (T2DM), cardiovascular disease, non-alcoholic steatohepatitis and various cancers. Furthermore, obesity is associated with a chronic state of low-grade inflammation in adipose tissue (AT), and it is thought that insulin resistance (IR) and T2DM is associated with the inflammatory state of AT. Endothelial cells (ECs) mediate the migration of immune cells into underlying tissues during times of inflammation, including obesity- and cardiovascular disease-associated inflammation. Cytokines and chemoattractants released from inflamed tissues promote EC activation. Upon activation, ECs increase the expression of leukocyte adhesion molecules (LCAMs) including intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), E-selectin (E-sel) and P-selectin (P-sel). Increased expression of these LCAMs and increased infiltration of inflammatory cells such as macrophages, have been linked to IR, diabetes and atherosclerosis in obese individuals. Preliminary data from our lab suggests that lipolysis induced by the β-adrenergic receptor agonist CL 316,243 causes an increase in endothelial LCAM gene expression. In addition, histological analyses show increased content of immune cells within AT after the ECs become activated. Here, we demonstrate that CL 316,243-induced lipolysis causes infiltration of neutrophils in wild type (WT) but not E-sel knockout (KO) mice. Following EC activation, there was also a marked increase in cytokine gene expression including IL-1β, MCP-1, and TNF-α in an E-sel-dependent manner. In contrast, fasting-induced lipolysis was associated with increased macrophage infiltration into AT in the absence of EC activation in an E-sel-independent manner. We also examined the role of mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potential contributor to endothelial activation and atherosclerosis. Here we demonstrate that deletion of MAP4K4 in ECs in vitro diminishes TNF-α-induced EC activation. Additionally, MAP4K4 depletion in primary ECs derived from lungs of mice expressing MAP4K4 shRNA decreases EC activation. Finally, endothelial specific depletion or loss of MAP4K4 reduced atherosclerotic plaque formation in vivo. Taken together, these results highlight the importance of the endothelium in modulating obesity-associated comorbidities. Furthermore, these data implicate endothelial MAP4K4 as a novel regulator of EC activation and consequently AT inflammation and atherosclerosis.

The following work examines the mechanisms by which Tip60-p400 chromatin remodeling complex regulates gene expression in embryonic stem cells (ESCs). Tip60-p400 complex has distinct functions in undifferentiated and differentiated cells. While Tip60-p400 is often associated with gene activation in differentiated cells, its most prominent function in ESCs is to repress differentiation-related genes. I show that Tip60-p400 interacts with Hdac6 and other proteins to form a unique form of the complex in ESCs. Tip60-Hdac6 interaction is stem cell specific and is necessary for Tip60-p400 mediated gene regulation, indicating that Tip60- p400 function is controlled in part through the regulation of Hdac6 during development. Furthermore, I find that Hdac6 is required for the binding of Tip60- p400 to many of its target genes, indicating Hdac6 is necessary for the unique function of Tip60-p400 in ESCs. In addition to accessory proteins like Hdac6, Tip60-p400 also interacts with thousands of coding and noncoding RNAs in ESCs. I show that R-loops, DNA-RNA hybrids formed during transcription of many genes, are important for regulation of chromatin binding by at least two chromatin regulators (Tip60-p400 and PRC2). This finding suggests that transcripts produced by many genes in ESC may serve as a signal to modulate binding of chromatin regulators. However, R-loops might also function to regulate chromatin architecture in differentiated cells as well. Future studies based on this work will be necessary to understand the full repertoire of cell types and chromatin regulators regulated by these structures.

Drosophila is host to the steroid hormone ecdysone, which regulates development and immune functions using a common group of transcription factors. Developmentally-induced ecdysone pulses activate the expression of the EcR, BR-C, HR46, Eip74EF, Eip75B, Eip78C, and Eip93F, which assume control of hundreds of other genes involved in the transition from larva to pupa stage. Many of the transcription factors are related to mammalian nuclear hormone receptors by homology. In addition to these transcription factors, the ecdysoneregulated GATA factors SRP and PNR are required for the proper expression of the peptidoglycan sensor PGRP-LC, which belongs to a conserved class of proteins in innate immunity. Although the transcriptional network has been elucidated in development, it is unclear why ecdysone control of PGRP-LC gene activity involves these nine transcription factors and how ecdysone is regulated in the context of an infection in vivo. An ecdysone-activated enhancer was located upstream of the PGRP-LC locus using a reporter plasmid. Female flies that lacked the enhancer had reduced PGRP-LC expression, but survived infection. Male flies did not experience these changes. Therefore, PGRP-LC enhancer appears to be a female-specific cis-regulatory element. The lack of survival phenotype could be caused by using an improper injection site. Bioinformatics software was used to identify putative individual and overlapping binding sites for some transcription factors. Site-directed mutations of the motifs reduced PGRP-LC promoter activity without abolishing the signal. These results suggest that the transcription factors assemble at multiple locations on the PGRP-LC enhancer and form strong protein-protein bonds. Septic injury led to elevated ecdysone in whole flies, which could be a neuroendocrine response to stress similar to the mammalian system. Steroid hormone regulation of immune receptors is a common theme in humans and flies, and these results could advance our understanding of the transcriptional regulation of related genes and gender differences observed in innate immune responses at the transcriptional level.

Background: Mothers’ diets impact their health and the health of their children, but diet quality is suboptimal among women with young children. Evening snacking among women with young children, especially consumption of high-calorie, high-carbohydrate snacks, may impact overall diet quality and glucose metabolism. Short sleep duration and poor sleep quality may be potential risk factors. We examined whether sleep duration and poor sleep quality were associated with diet quality and evening snacking among women with young children. Methods: Data were from the National Health and Nutrition Examination Survey (NHANES) 2005-2012, nationally representative cross-sectional surveys of noninstitutionalized U.S. population. Eligible participants were non-pregnant women aged 20-44 years within 5 years of childbirth who completed two 24-hour dietary recalls and completed questions on sleep duration and quality. Results: Among US women with young children, sleep duration was not associated with diet quality. However, overall sleep quality was associated with poorer diet quality. Short sleep duration was not associated with the consumption of neither evening snacks, nor energy intake from or nutrient consumption of evening snacks. Conclusion: The findings of this dissertation provide information useful for informing the direction of future research of dietary quality and eating behaviors of U.S. women with young children. Studies are needed to explore whether improvement in sleep quality may improve diet quality among women with young children, which has the potential to improve both maternal and children’s health. Research may be better focused on identifying other psychosocial and behavioral risk factors for unhealthy dietary behaviors among US women with young children.

Background. Diabetes requires significant disease management, patient-provider communication, and interaction between patients, family members, caregivers, and care teams. Emerging patient-facing technologies, such as cellular-enabled glucose meters, can facilitate additional care support and improve diabetes self-management. This study evaluated patient acceptability, feasibility, and efficacy of a diabetes care support program facilitated by cellular-enabled glucose meters. Methods. A two-phase study approach was taken. Get In Touch – Phase 1 (GIT-1) was a 1-month pilot involving patients with type 1 and type 2 diabetes. Get In Touch – Phase 2 (GIT-2) was a 12-month randomized controlled crossover trial involving patients with poorly-controlled type 2 diabetes. Results from GIT-1 and preliminary results from GIT-2 are presented. Results. GIT-1 participants with type 1 (n=6) and type 2 (n=10) diabetes reported the intervention and cellular-enabled glucose meter were easy to use and useful while identifying potential areas of improvement. GIT-2 participants in both the intervention (n=60) and control (n=60) groups saw significant improvements in treatment satisfaction and A1c change, with intervention participants experiencing slightly greater improvements in each after 6 months (p=0.09 and p=0.16, respectively) compared to control participants. Conclusions. Patients reported favorable acceptability of the intervention. Preliminary results from a randomized trial demonstrated potential of intervention to improve patient-reported and physiological health outcomes. Future studies should evaluate feasibility and efficacy over a longer period of time, with a greater number of participants, and targeting different populations of patients with diabetes. Provider perspectives and changes in provider behavior, clinical work flow, and caregiver burden should also be assessed.

Epithelial cells are necessary building blocks of the organs they line. Their apicalbasolateral polarity, characterized by an asymmetric distribution of cell components along their apical-basal axis, is a requirement for normal organ function. Although the centrosome, also known as the microtubule organizing center, is important in establishing cell polarity the mechanisms through which it achieves this remain unclear. It has been suggested that the centrosome influences cell polarity through microtubule cytoskeleton organization and endosome trafficking. In the first chapter of this thesis, I summarize the current understanding of the mechanisms regulating cell polarity and review evidence for the role of centrosomes in this process. In the second chapter, I examine the roles of the mother centriole appendages in cell polarity during cell migration and cell division. Interestingly, the subdistal appendages, but not the distal appendages, are essential in both processes, a role they achieve through organizing centrosomal microtubules. Depletion of subdistal appendages disrupts microtubule organization at the centrosome and hence, affects microtubule stability. These microtubule defects affect centrosome reorientation and spindle orientation during cell migration and division, respectively. In addition, depletion of subdistal appendages affects the localization and dynamics of apical polarity proteins in relation to microtubule stability and endosome recycling. Taken together, our results suggest the mother centriole subdistal appendages play an essential role in regulating cell polarity. A discussion of the significance of these results is included in chapter three.