<p> Bronchopulmonary dysplasia (BPD) is a serious respiratory complication for the preterm newborn characterized clinically by prolonged respiratory distress and histologically by alveolar simplification and decreased pulmonary vasculature. The development of BPD is well linked to oxidative stress suffered by the newborn as a result of a preterm fetal-neonatal transition, supplemental oxygen, infection, increased inflammation, and mechanical ventilation. Damage suffered by oxidative stress may be through direct mechanisms or through alteration of redox&not;sensitive pathways involved in cell death, cell survival, differentiation, and proliferation. Redox&not;sensitive modifications regulating protein function and redox-sensitive pathways have mainly been ascribed to oxidative modification of cysteine thiols. As their modification is critical for protein function, maintenance of the thiol redox status is crucial. Thioredoxin-1 (Trx1) functions in maintenance of thiol redox homeostasis, and its redox activity is intimately linked to antioxidant, cytoprotection, proliferation responses, and cytoprotection. While Trx1 targets of redox regulation have been identified, we hypothesize that additional protein may be redox regulated and that Trx1 target profiles may change during oxidative stress. Therefore a novel immunoprecipitation approach, identified as the substrate trap approach, was developed to identify Trx1 targets. The following demonstrates the use of the substrate trap approach for identification of Trx1 redox targets and further application of the approach to identify alterations in target profiles in response to oxidative stress. Use of nuclear targeted substrate trap was successfully employed to enrich from nuclear Trx1 targets. As a final component the characterization of the Trx1 system in mouse from late embryonic development through the first week of life animals were exposed to room air or hyperoxia (model of BPD). Characterization indicates impairment of the Trx1 system in response to hyperoxic injury. As Trx1 is known to regulate proliferation, cell death, survival, differentiation pathways, impairment of the Trx1 system during early neonatal development may potentiate hyperoxic injury and alterations in lung development. Better understanding of Trx1 interactions occur through the substrate trap in a physiological model of BPD will help elucidate redox-signaling pathways involved in BPD pathogenesis.</p>

Over the past several decades, cell size and its resulting effects on tissue and organ function, as well as on its overall ability of the animal to perform complex tasks, has been studied extensively. Neuronal size (diameter of individual neurons) could have an influence on intelligence, brain capacity, and ability to perform complex behavioral tasks. Furthermore, there appears to be an increase in number of neurons with an increase in brain size in vertebrates. In insects, increased neuron number has also been correlated with more complex behavior. In this thesis, I test the hypothesis that the neuronal number and/or neuronal size correlate with the brain size using an insect model. This may help elucidate the apparent positive correlation between brain size and intelligence. To achieve this goal, I used a species of bumblebee, Bombus impatiens. Bumblebee workers vary extensively in brain and body size and weight, therefore allowing comparison between individuals of the same species. Workers within a colony differ in size and the amount of work a worker does depends on their body size. Larger sized workers have more foraging capability than smaller sized workers and foraging requires a more demanding sensory integration and memory capacity. In my study, it was found that brain volume was positively correlated with bee body size. Three cell body regions of the brain were further analyzed: inside of the mushroom body calyces, a cell body region next to the lobula, and cell bodies associated with the antennal lobe. No significant correlations between neuron number per unit of volume (neuron density) and brain volume were found. Assuming similar neuronal density in large and small brains, increased brain size is thus correlated with an overall increased neuron number.

<p> The use of epigenetic modifying drugs such as DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) is becoming more common in the treatment of cancer. Currently, there is a profound interest in determining predictive biomarkers for patient response and the efficacy of known and novel drugs. There are likely distinct &ldquo;epigenetic profiles&rdquo; defined by the location and abundance of DNA methylation patterns and histone modifications. Here we propose to investigate the response of a selected subset of genes to particular DNMTi and HDACi treatments, in two human cancer cell lines, colorectal carcinoma HCT-116 and liver adenocarcinoma HepG2. In this study we identified unique epigenetic profiles based on microarray and bioinformatics derived epigenetic data that are predictive of the response to epigenetic drug treatment. Microarray studies were used to identify re-activated genes common in two different cancer cell types treated with epigenetic drugs. Bioinformatics data was compiled on these genes and correlated against re-expression to construct the genes&rsquo; &ldquo;epigenetic profile&rdquo;. We then verified the response of the select group of genes in HCT-116 and HepG2 upon treatment at varying concentrations of epigenetic drugs and illustrated that selective reactivation of the target gene. Additionally, two novel genes were introduced and one selectively activated over another. </p><p> Further research would prove invaluable for the medical and drug development communities, as a more extensive model would certainly be of use to determining patient response to drug treatment based on their individual epigenetic profile and leading to more successful novel drug design.</p>

<p>Fibroblast growth factor (FGF) homologous factors (FHFs, FGF11-14) are a family of FGFs that are not secreted, nor activate FGF receptors. Instead, they remain intracellular and bind to the voltage-gated Na<super>+</super> channel C-terminus and modulate function. FGF14 is a locus for the neurodegenerative disease spinocerebellar ataxia 27 and the disease has been attributed to decreased neuronal excitability from changes in Na<super>+</super> channel function. However, several lines of evidence, including data from heterologous expression systems and the distribution of FGF13 within the ventricular cardiomyocyte suggested that it also modulates the Ca_V1.2 voltage-gated Ca<super>2+</super> channel. The central hypothesis to this study is that FHFs modulate both voltage-gated Na<super>+</super> and Ca<super>2+</super> channel channels in the ventricular cardiomyocyte and therefore are loci for cardiac arrhythmia. Using an adult ventricular cardiomyocyte system with adenoviral gene transfer, we manipulated the levels of FGF13 in the cell and performed electrophysiology, biochemistry and immunocytochemistry to analyze the effects on voltage-gated Ca<super>2+</super> channel channel localization and function. We showed that FGF13 is in complex with Junctophilin-2 and modulates Ca_V1.2 current density and localization to the t-tubule, leading to changes in Ca<super>2+</super> channel-induced Ca<super>2+</super> channel release and ultimately a shortened ventricular action potential. Through collaboration with the Mayo Clinic, a mutation in FGF12, the most highly expressed FHF in human ventricle was found in a patient with Brugada syndrome. Using similar methodology, we determined that this mutation results specifically in a Na_V1.5 loss of function without affecting Ca_V1.2 function, resulting in a Brugada-like ventricular action potential. This data shows that FHFs are potent modulators of multiple ion channels and novel arrhythmogenic loci.</p> / Dissertation

The liver has a unique capability for regeneration and is particularly resilient to insult. It plays an essential role in drug disposition and metabolism, regulating numerous pathways involved in ADME (absorption, distribution, metabolism, and excretion) processes. In order for a drug to be effective, it must be able to get to its target site in a timely manner and at an appropriate concentration. Chronic liver disease has been of increasing significance and elucidating the driving forces behind disease progression is key to understanding adverse drug reactions and many cases of liver toxicity. Coordinate regulation of liver transporters and drug metabolism enzymes is essential for maintaining homeostasis and effective liver functionality. Nonalcoholic steatohepatitis, a severe inflammatory disease state that progresses from normal steatosis and Nonalcoholic Fatty Liver Disease has shown significant changes in gene expression as pathological disease progression occurs. Transcription factor binding site analysis proves lucrative in elucidating key signaling pathways in disease progression. Several up and down-regulated genes have enriched transcription factor binding sites in the NASH disease state, including members of the HNF, SOX, and LXR families. These transporters and drug metabolizing enzymes are involved in key processes, including inflammatory signaling, liver cell maintenance, bile acid regulation and other processes that are driving factors in liver repair and insult. By identifying key transcription factors in disease progression and looking at the signaling pathways behind the enriched transcription factors, potential driving factors behind disease progression are discovered. As a major contributor to the progression of the disease state, the significance of driving factors for hepatic fibrosis are discussed. The immune system and inflammatory processes are key drivers of fibrosis and cirrhosis, often mediated by cytokines, such as IL-4 and IL-6.

<p> Sphingolipids (SL) are essential components of eukaryotic cells involved maintaining membrane integrity. They are important for membrane trafficking and function in signaling as messenger molecules. SLs are synthesized <i> de novo</i> from very long chain fatty acids (VLCFA) and sphingoid long-chain bases (LCB), which are amide-linked to form ceramide and further processed by addition of various head-groups. Little is known concerning the regulation of VLCFA levels and how cells coordinate their synthesis with the availability of LCBs for SL synthesis. Here I show that Elo2, a key enzyme of VLCFA synthesis, is controlled by signaling of the guanine nucleotide exchange factor Rom2, initiating at the plasma membrane. This pathway controls Elo2 phosphorylation state and VLCFA synthesis. My data identify a regulatory mechanism for coordinating VLCFA synthesis with SL metabolism and link signal transduction pathways from the plasma membrane to the regulation of lipids for membrane homeostasj.</p>

Although a few regulators of memory and addiction have been identified, the biochemical pathways that mediate the development of addiction and memory remain poorly understood. In addition, important questions remain as to how these two phenomena can persist for so long, sometimes for the entire life of an individual.Signaling molecules and transcription factors are activated in response to stimuli that induce long-term neuronal plastic changes. The transcription factor CREB (cAMP-responsive element binding protein) is clearly involved in triggering processes of addiction and memory, but its sustained activation following a course of chronic drug exposure (or learning) returns to baseline within days [1]. Even the enduring increased levels of deltaFosB (a Fos family transcription factor that couples with other proteins in the AP-1 family to form transcriptional activator/repressor complexes) observed in regions of the mammalian brain following chronic drug exposure, persists for only weeks or months. Thus, although CREB and deltaFosB probably initiate the very stable behavioral changes seen with addiction and memory, their alterations cannot mediate those behavioral changes per se [1]. Long-term up- or down-regulation of molecules downstream of these transcription factors, or others, must be responsible for the enduring modifications in synaptic connectivity and structure believed to be required for the maintenance of these durable behavioral states [2].Many believe that more rapid progress will be made toward understanding the molecular basis of addiction if research efforts proceed hand-in-hand with, rather than in isolation from, the overlapping neurobiological study of learning and memory [1, 2]. The importance and utility of using simple model systems such as Drosophila and Aplysia to identify and characterize genes involved in long-term synaptic plasticity, and hence memory formation, is well documented [3-5]. Identification and functional analyses of neuronal genes transcriptionally regulated by AP-1 and CREB in Drosophila would elaborate on molecular mechanisms of long-term plasticity and hence help us understand, and perhaps manipulate, processes that underlie addiction and memory.

Nitric Oxide (NO) regulates cardiovascular homeostasis by binding to soluble guanylate cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca²⁺]ᵢ) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, I show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition requires an increase in [Ca²⁺]ᵢ. Using digital imaging microscopy on live cells, I further show that E3CaG1 binding results in a substantial increase in [Ca²⁺]ᵢ, up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca²⁺]ᵢ, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca²⁺ remains inhibited, while addition of kinase inhibitors staurosporine, genistein, PP1 or PP2 reverse inhibition, indicating inhibition likely involves a tyrosine kinase, more specifically, a src family kinase. Rat sGC is also inhibited by lysates supplemented with Ca²⁺, suggesting that the site of modification is at an evolutionarily conserved residue. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca²⁺]ᵢ in response to NO, inducing vasodilation, but is also inhibited by high [Ca²⁺]ᵢ, providing a fine balance between signals for vasodilation and vasoconstriction.

<p>The lung is a unique organ, taking a key part physiology but also playing a vital role in host defense. The lung has therefore developed a complex innate immune system that includes ciliated mucus producing cells that trap and remove larger particles in the larger airways and a specialized set of immune cells in the smaller airways. In addition, a variety of host proteins play a vital role in the immune response in the lung. The structural protein caveolin-1 (cav-1) is known to play a role in the uptake of pathogens and controls a variety of signaling pathways, although less is known about its functions in the lung. Surfactant protein A (SP-A) is a secreted protein that is vital in the innate immune response by interacting with microbes and immune cells. The goal of this work was to further elucidate the specific mechanisms by which cav-1 and SP-A affect the host responses, including inflammation and airway hyperreactivity (AHR), to pathogens and particulates. Using a mouse model of environmental lung injury I show that cav-1 is vital in the host response to inhaled lipopolysaccharide (LPS). Although the cav-1 deficient mice had greater lung inflammatory indices compared to wild-type mice, they exhibited reduced AHR following LPS exposure. The uncoupling of these two parameters led me to investigate the role of cav-1 in the contraction of airway smooth muscle and production of nitric oxide, both of which are known to regulate AHR. The bronchi of cav-1 deficient mice contract less than those from wild-type mice although their structure and receptor independent responses were not altered. The absence of cav-1 also resulted in increased nitric oxide levels in the lavage fluid and increased inducible nitric oxide synthase expression in the lung tissue. Administration of the potent and specific inhibitor 1400W increased AHR to levels comparable to wild-type mice. Following intranasal infection with Mycoplasma pneumoniae mast cell numbers increase in the lungs of mice and AHR is dramatically attenuated in SP-A<super>-/-</super> mice when mast cells are absent. Using SP-A<super>-/-</super> Kit<super>W-sh/W-sh</super> mice engrafted with TNF-alpha<super>-/-</super> or TNF receptor (TNF-R<super>-/-</super>) mast cells, I find that TNF-alpha activation of mast cells via the TNF-R and not mast cell derived TNF-alpha, leads to augmented AHR during Mp infection. Additionally, Mp infected SP-A<super>-/-</super>Kit<super>W-sh/W-sh</super> mice engrafted with TNF-alpha <super>-/-</super> or TNF-R<super>-/-</super> mast cells have decreased mucus production compared to those engrafted with wild-type mast cells, while burden was unaffected. Together, these data help to further elucidate the role of cav-1 and SP-A in innate immunity and may lead to the development of more effective human therapies.</p> / Dissertation

<p>Improvements in genomic technology, both in the increased speed and reduced cost of sequencing, have expanded the appreciation of the abundance of human genetic variation. However the sheer amount of variation, as well as the varying type and genomic content of variation, poses a challenge in understanding the clinical consequence of a single mutation. This work uses several methodologies to interpret the observed variation in the human genome, and presents novel strategies for the prediction of allele pathogenicity.</p><p>Using the zebrafish model system as an in vivo assay of allele function, we identified a novel driver of Bardet-Biedl Syndrome (BBS) in CEP76. A combination of targeted sequencing of 785 cilia-associated genes in a cohort of BBS patients and subsequent in vivo functional assays recapitulating the human phenotype gave strong evidence for the role of CEP76 mutations in the pathology of an affected family. This portion of the work demonstrated the necessity of functional testing in validating disease-associated mutations, and added to the catalogue of known BBS disease genes.</p><p>Further study into the role of copy-number variations (CNVs) in a cohort of BBS patients showed the significant contribution of CNVs to disease pathology. Using high-density array comparative genomic hybridization (aCGH) we were able to identify pathogenic CNVs as small as several hundred bp. Dissection of constituent gene and in vivo experiments investigating epistatic interactions between affected genes allowed for an appreciation of several paradigms by which CNVs can contribute to disease. This study revealed that the contribution of CNVs to disease in BBS patients is much higher than previously expected, and demonstrated the necessity of consideration of CNV contribution in future (and retrospective) investigations of human genetic disease.</p><p>Finally, we used a combination of comparative genomics and in vivo complementation assays to identify second-site compensatory modification of pathogenic alleles. These pathogenic alleles, which are found compensated in other species (termed compensated pathogenic deviations [CPDs]), represent a significant fraction (from 3 – 10%) of human disease-associated alleles. In silico pathogenicity prediction algorithms, a valuable method of allele prioritization, often misrepresent these alleles as benign, leading to omission of possibly informative variants in studies of human genetic disease. We created a mathematical model that was able to predict CPDs and putative compensatory sites, and functionally showed in vivo that second-site mutation can mitigate the pathogenicity of disease alleles. Additionally, we made publically available an in silico module for the prediction of CPDs and modifier sites.</p><p>These studies have advanced the ability to interpret the pathogenicity of multiple types of human variation, as well as made available tools for others to do so as well.</p> / Dissertation