Amyotrophic lateral sclerosis (ALS) is a progressive adult neurodegenerative disease that causes death of both upper and lower motor neurons. Approximately 90 percent of ALS cases are sporadic (SALS), and 10 percent are inherited (FALS). Mutations in the PFN1 gene have been identified as causative for one percent of FALS. PFN1 is a small actin-binding protein that promotes actin polymerization, but how ALS-linked PFN1 mutations affect its cognate functions or acquire gain-of-function toxicity remains largely unknown. To elucidate the contribution of ALS-linked PFN1 mutations to neurodegeneration, we have characterized these mutants in both mammalian cultured cells and Drosophila models. In mammalian neuronal cells, we demonstrate that ALS-linked PFN1 mutants form ubiquitinated aggregates and alter neuronal morphology. We also show that ALS-linked PFN1 mutants have partial loss-of-function effects on actin polymerization in growth cones of mouse primary motor neurons and larval neuromuscular junctions (NMJ) in Drosophila. In Drosophila, we also observe that PFN1 level influences integrity of adult motor neurons, as demonstrated by locomotion, lifespan, and leg NMJ morphology. In sum, the work presented in this dissertation has shed light on PFN1- linked ALS pathogenesis by demonstrating a loss-of-function mechanism. We have also developed a Drosophila PFN1 model that will serve as a valuable tool to further uncover PFN1-associated cellular pathways that mediate motor neuron functions.

Cells must regulate their metabolism in order to grow, adapt to changes in nutrient availability and maintain homeostasis. Flux, or the turnover of metabolites, through the metabolic network can be regulated at the allosteric and transcriptional levels. While study of allosteric regulation is limited to biochemical examination of individual proteins, transcriptional control of metabolism can be explored at a systems level. We endeavored to elucidate transcriptional mechanisms of metabolic flux regulation in the model organism Caenorhabditis elegans (C. elegans). We also worked to create a visual tool to explore metabolic pathways that will support future efforts in the research of metabolic gene regulation. C. elegans is a small, free-living nematode that feeds on bacteria and experiences a high level of diversity in nutrient level and composition. Previously, we identified a mechanism by which the essential cofactor, vitamin B12, regulates the expression of genes involved in the degradation of propionate, referred to as B12‑mechanism‑I. This mechanism functions to prevent the toxic accumulation of propionate and requires the TFs NHR-10 and NHR-68. Using genetic screens as well as transcriptomic and metabolomic approaches, we discover a second mechanism by which vitamin B12 regulates metabolic gene expression: B12-mechanism-II. Unlike B12-mechanism-I, B12-mechanism-II is independent of propionate, requires the transcription factor NHR-114 and functions to maintain the metabolic activity of the Methionine/S-adenosylmethionine cycle in a tightly regulated regime. We also present WormPaths, an online resource that allows visualization of C. elegans metabolic pathways and enables metabolic pathway enrichment of user-uploaded transcriptomic data.

Background: The mood stabilizer lithium has long been reported to be associated with reduced suicide risks, but many studies reporting associations between lithium and reduced suicide risks also have been nonrandomized and lacked adjustment for many potential confounders, active controls, uniform follow-up, or intent-to-treat samples. Concerns also have been raised that medications being considered as potential suicide preventative might increase risks of nonsuicide mortality while reducing risks of suicide. Methods: Three studies of Veterans Health Administration (VHA) patients were conducted combining high-dimensional propensity score matching with intent-to-treat analyses to examine the associations between lithium and valproate and one-year suicide and nonsuicide mortality outcomes. Results: In intention-to-treat analyses, initiation of lithium, compared to valproate, was associated with increased suicide mortality over 0-365 days among patients with bipolar disorder (Hazard Ratio (HR) 1.50 [95% Confidence Interval 1.05, 2.15]) Nonsuicide mortality among VHA patients with or without bipolar disorder was not significantly associated with the initiation of lithium compared to valproate ( HR 0.92 [0.82-1.04]). Rates of treatment discontinuation, however, were very high (≈ 92%). Longitudinal analyses revealed that the increased suicide risks associated with initiating lithium among patients with bipolar disorder occurred exclusively after discontinuation of lithium vii treatment. In secondary analyses restricted to patients still receiving their initial treatment, there was no difference in suicide risk between the initiation of lithium or valproate. Conclusions: Significantly increased risks of suicide were observed at one year among VHA patients with bipolar disorder initiating lithium compared to valproate, related to risks observed after the discontinuation of lithium treatment Since these studies are nonrandomized, confounding may account for some or all of our findings, including the risks observed after lithium discontinuation. Nevertheless, these results suggest that health systems and providers consider steps to minimize any potential lithium discontinuation-associated risk. Approaches might include educating patients about possible risks associated with discontinuation and closely monitoring patients after discontinuation if feasible. Given the obvious importance of any substantive difference between lithium and valproate in suicide or nonsuicide mortality risk, our studies also suggest that further research is needed, especially research that can further minimize the potential for confounding.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

Integrins have the ability to impact major aspects of epithelial biology including adhesion, migration, invasion, signaling and differentiation, as well as the formation and progression of cancer (Hynes 2002; Srichai and Zent 2010; Anderson et al. 2014). This thesis focuses on how integrins are regulated and function in the context of mammary epithelial biology and breast cancer with a specific focus on the α6 integrin heterodimers (α6β1 and α6β4). These integrins function primarily as receptors for the laminin family of extracellular matrix (ECM) proteins and they have been implicated in mammary gland biology and breast cancer (Friedrichs et al. 1995; Wewer et al. 1997; Mercurio et al. 2001; Margadant and Sonnenberg 2010; Muschler and Streuli 2010; Nistico et al. 2014). The first project investigates how alternative splicing of the α6 subunit impacts the genesis and function of breast cancer stem cells (CSCs). This work revealed that the α6Bβ1 splice variant, but not α6Aβ1, is necessary for the function of breast CSCs because it activates the Hippo transducer TAZ (Zhao et al. 2008a), which is known to be essential for breast CSCs (Cordenonsi et al. 2011). My work also led to the discovery that laminin (LM) 511 is the specific ligand for α6Bβ1 and that autocrine LM511, which is mediated by TAZ, is needed to sustain breast CSCs by functioning as a ‘ECM niche’. An important aspect of this study is the finding that surface-bound LM511 characterizes a small population of cells in human breast tumors with CSC properties. The second project of my thesis concentrated on identifying transcription factors that regulate expression of the β4 subunit. The expression of the α6β4 integrin is repressed during the epithelial-mesenchymal transition (EMT) (Yang et al. 2009) but the contribution of specific transcription factors to this repression is poorly understood. This study revealed that Snai1 is a transcriptional repressor of β4, which is responsible for establishing the PRC2 (Polycomb complex 2)- associated repressive histone mark H3K27Me3. However, I also found that the ability of Snai1 to repress transcription is abrogated by its interaction with Id2. Specifically, I identified the biochemical mechanism for how Id2 regulates Snai1. Id2 binds the SNAG domain of Snai1 that is the docking site for several corepressors (Peinado et al. 2004; Lin et al. 2010b; Dong et al. 2012a). One important consequence of Id2 interacting with Snai1 on the β4 promoter is that it prevents repressive epigenetic modifications. This finding may explain why some epithelial cells express Snai1 and β4 because they also express Id2 (Vincent et al. 2009; Bastea et al. 2012). The repression of the α6β4 integrin during the EMT is consistent with data indicating that this integrin is not expressed in CSCs (Mani et al. 2008; Goel et al. 2012; Goel et al. 2013; Goel et al. 2014). An important question going forward is to understand how the α6β4 integrin contributes to tumor formation. In summary, my thesis provides novel insights into the biology of the α6 integrins that has important implications for the function of these integrins in mammary gland biology and breast cancer, especially our understanding of breast CSCs.

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability in eukaryotes and for driving developmental processes in higher organisms. A hallmark of heterochromatin is the presence of specialized architectural proteins that alter chromatin structure to inhibit transcription and recombination. Although it is generally assumed that heterochromatin is highly condensed, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, heterochromatin assembly at telomeres and the HM silent mating type loci requires the Sir proteins: Sir3, believed to be the major structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for SIR recruitment to silencing regions and deacetylation of lysine 16 of the histone H4 tail, a mark associated with active chromatin. A combination of sedimentation velocity, atomic force microscopy, and nucleosomal array capture was used to characterize the stoichiometry and conformation of SIR nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome, and that Sir2/4 may additionally bind at a ratio of one per nucleosome. Despite Sir3’s ability to repress transcription in vivo and homologous recombination in vitro in the absence of Sir2/4, Sir3 fibers were found to be significantly less compact than canonical magnesium-induced 30 nanometer fibers. However, heterochromatin fibers composed of all three Sir proteins did adopt a more condensed, globular structure. These results suggest that heterochromatic silencing is mediated both by the creation of more stable nucleosomes and by the steric exclusion of external factors.

Maintaining cellular homeostasis is a complex task, which involves monitoring energy states and essential nutrients, regulating metabolic fluxes to accommodate energy and biomass needs, and preventing buildup of potentially toxic metabolic intermediates and byproducts. Measures aimed at maintaining a healthy cellular economy inherently depend on the composition of nutrients available to the organism through its diet. We sought to delineate links between dietary composition, metabolic gene regulation, and physiological responses in the model organism C. elegans. As a soil-dwelling bacterivore, C. elegans encounters diverse bacterial diets. Compared to a diet of E. coli OP50, a diet of Comamonas aquatica accelerates C. elegans developmental rate, alters egg-laying dynamics and shortens lifespan. These physiological responses are accompanied by gene expression changes. Taking advantage of this natural, genetically tractable predator-prey system, we performed genetic screens i) in C. elegans to identify regulators of diet-responsive genes, and ii) in E. coli and Comamonas to determine dietary factors driving transcriptional responses in C. elegans. We identified a C. elegans transcriptional program that regulates metabolic genes in response to vitamin B12 content in the bacterial diet. Interestingly, several B12- repressed metabolic genes of unknown function are highly activated when B12- dependent propionyl-CoA breakdown is impaired, and inactivation of these genes renders animals sensitive to propionate-induced toxicity. We provide genetic and metabolomic evidence in support of the hypothesis that these genes form a parallel, B12-independent, β-oxidation-like propionate breakdown shunt in C. elegans, similar to the pathway utilized by organisms like yeast and plants that do not use vitamin B12.

Evolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner. We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness. In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution. Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’. In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that most notably influence dopamine (DA) release. In this thesis, I examine the role of nAChRs in mediating DA-related behaviors such as movement and drug dependence. To accomplish this, I utilized a “gain-offunction” knock-in mouse (the Leu9’Ala line) containing agonist-hypersensitive α4* nAChRs (* indicates other nAChR subunits in addition to α4 are within the receptor complex) that renders receptors 50-fold more sensitive to nicotine and acetylcholine than wild-type (WT) receptors. I found that DHβE, a selective antagonist for α4β2* nAChRs, induced reversible and robust motor dysfunction characterized by hypolocomotion, akinesia, catalepsy, tremor, and clasping in Leu9’Ala but not WT mice. Reversal of the phenotype was achieved by targeting dopamine signaling. Blockade of mutant α4* nAChRs elicited activation of brain regions in the basal ganglia including dorsal striatum and substantia nigra pars reticulata indicated by c-Fos immunoreactivity. These data indicate that blocking α4* nAChRs in Leu9’Ala mice activates the indirect motor pathway resulting in a motor deficit. We also determined that α4* nAChRs involved in motor behaviors did not contain the α6 subunit, a nAChR subunit highly expressed in DAergic neurons suggesting that different nAChR subtypes modulating striatal DA release have separate functions in motor output. Conditioned place aversion and hypolocomotion, behaviors elicited during nicotine withdrawal, were also induced by DHβE in nicotine-naïve Leu9’Ala but not WT mice. Together these data suggest that DHβE globally reduces DA release in the CNS. In a separate project, I determined that α4* and α6* nAChRs modulate drug-induced hypnosis. Activation of nAChRs increased sensitivity to ketamine-induced hypnosis; whereas antagonizing nAChRs had the opposite effect. Additionally, α4 knockout (KO) mice were less sensitive to the hypnotic effects of ketamine, but α6 KO were more sensitive. High doses of ethanol induce an anesthesia-like state characterized by immobility, analgesia, and hypnosis. Testing the effects of ethanol hypnosis in α4 KO revealed that α4* nAChR do not play a large role in the acute effects of ethanol-induced hypnosis, but are involved in tolerance to this ethanol-induced behavior. The mechanisms of anesthetic-induced hypnosis are still largely unclear, despite the wide use of anesthesia. Future work on these receptors and their involvement in the anesthetic response will help to define a mechanism for hypnosis and improve the use of anesthetic drugs.

Background: Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in the U.S. Acute myocardial infarction (AMI), with or without ST-segment elevation, is a common presentation of coronary heart disease and affected more than 800,000 American adults in 2010. The overall goal of this dissertation was to examine decade-long trends in the extent of delay in the receipt of a primary percutaneous coronary intervention (PCI) among patients hospitalized with ST-segment elevation myocardial infarction (STEMI), 30-day hospital readmission rates in patients having survived an AMI, and multiple decade long trends in 1-year post-hospital all-cause mortality, as well as factors associated with these outcomes, among patients hospitalized with AMI. Methods: Data from the Worcester Heart Attack Study, a population-based chronic disease surveillance project that has been carried out among adult residents of the Worcester, MA, metropolitan area, hospitalized with AMI on a biennial basis from 1975 through 2009 at all medical centers in central MA, were used for this dissertation. Results: Between 1999 and 2009, among patients hospitalized with STEMI, the likelihood of receiving a primary PCI within 90 minutes after emergency department arrival increased dramatically from 1999/2001 (11.6%) to 2007/2009 (70.5%). Between 1999 and 2009, among hospital survivors of an AMI, the 30-day all-cause rehospitalization rates decreased from 1999/2001 (20.3%) to 2007/2009 (16.7%). The overall cause-specific 30-day rehospitalization rates due to CVD, non-CVD, and AMI were 10.1%, 7.1%, and 1.8%, respectively, during the years under study. Between 1975 and 2009, among hospital survivors for a first AMI, the 1-year post-discharge mortality rates remained relatively stable from 1975-1984 (12.9%) to 1986-1997 (12.5%), but increased during 1999-2009 (15.8%). We identified several demographic, clinical and in-hospital treatment factors associated with an increased risk of failing to receive a primary PCI within 90 minutes after emergency department arrival, 30-day readmissions, and 1-year post-discharge mortality. Conclusions: Our findings can hopefully lead to the enhanced development of innovative, patient-centered, intervention strategies which can further improve the treatment and transitions of care, as well as short and long-term prognosis, of men and women hospitalized with AMI.