Thesis (Ph. D.)--Georgia State University, 2008. / Title from title page (Digital Archive@GSU, viewed July 1, 2010) Jenny J. Yang, committee chair; Edward Brown, Giovanni Gadda, Zhi-ren Liu, committee members. Includes bibliographical references (p. 230-258).

In animals immunized with 2,4 dinitrophenyl (DNP) hapten-carrier protein conjugates, no in vitro cellular response is elicited by DNP, either alone, or when coupled to a heterologous carrier. In contrast, animals immunized with haptenic peptide-carrier conjugates do mount an in vitro cellular response towards the haptenic peptide. This apparent inconsistency led us to compare the in vivo and in vitro cellular immune responses to a synthetic peptide antigen and its DNP derivative to determine the activation specificity of the cells evoking this response. Guinea pigs were immunized with either the DNP substituted immunogen (DNP-N-10-C) or its unsubstituted form (N-10-C) and subsequent in vivo or in vitro cellular activation was evaluated for DNP alone, DNP coupled to the homologous determinant, and DNP coupled to heterologous carriers. The data suggests that in DNP-N-10-C immune guinea pigs, DNP substitution opens a new determinant exhibiting, in antigen reactive cells, a unique specificity towards the DNP moiety as well as a portion of the peptide to which it is conjugated. However the DNP group by itself does not have the configurational requirement to evoke cellular activation. It therefore plays a minor role in activation of the cellular immune response; the major contribution being supplied by the peptide portion of the 'shared' determinant. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

<p> The development of metabolic dysfunctions like diabetes and insulin resistance in mammals is regulated by a myriad of factors. Oxidative stress seems to play a central role in this process as recent evidence shows a general increase in oxidative damage and a decrease in oxidative defense associated with several metabolic diseases. These changes in oxidative stress can be directly correlated with increased fat accumulation, obesity and consumption of high calorie/high fat diets. Modulation of oxidant protection through either genetic mutation or treatment with antioxidants can significantly alter oxidative stress resistance and accumulation of oxidative damage in laboratory rodents. Antioxidant mutant mice have previously been utilized to examine the role of oxidative stress in other disease models, but have been relatively unexplored as models to study the regulation of glucose metabolism. </p><p> Our studies have focused on MsrA in mammalian systems and have utilized mice that lack MsrA (<i>MsrA^-/-</i>) and that over express MsrA (MsrA^mitoTg and MsrA^cytoTg). Under normal conditions, our lab has shown that <i>MsrA^-/-</i> mice are phenotypically similar to wildtype (WT) mice, but are susceptible to oxidative stress [244]. MsrA^mitoTg and MsrA^cytoTg are also phenotypically similar to WT (though oxidative stress resistance has not yet been tested). This indicates that excess methionine oxidation may not occur at basal ROS levels, which is supported by yeast studies [202]. In vivo, increasing adiposity has been associated with increases in oxidative stress, altered redox signaling and increased oxidative damage to cellular macromolecules in several disease models. It is also thought that adipose tissue-induced oxidative stress may be a primary factor in the etiology of obesity-induced metabolic diseases. When placed on a high fat (HF) diet to induce an increase in oxidative stress, <i>MsrA^-/-</i> mice become more insulin resistant than WT mice whereas MsrA^mitoTg mice are protected. The increase in insulin resistance in <i> MsrA^-/-</i> mice is not due to further exacerbation of pathways thought to link oxidative stress and insulin resistance (JNK signaling or pro-inflammatory cytokine expression). These results suggest that oxidative damage to proteins may play an important role in obesity-induced insulin resistance. </p>

<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>

published_or_final_version / Architecture / Doctoral / Doctor of Philosophy