Interdisciplinary Biological Sciences
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Item Open Access The Differential Roles of pH sensing G-Protein Coupled Receptors in Inflammation and Cancer(East Carolina University, 2022-12-13) Marie, Mona Abdou AhmedIntestinal inflammation is a burdening disease that may occur due to an aberrative immune response to certain drugs, infections, genetics, or unknown environmental factors. Inflammatory bowel disease (IBD) is of special concern as long-standing chronic intestinal inflammation may increase the risk for colitis-associated colorectal cancer (CAC) development due to cellular transformation to neoplastic lesions. Both the inflamed and tumor microenvironments are complex in nature and are characterized by an acidic environment. Bacterial byproducts, leukocytes respiratory bursts, and tissue ischemia and glycolysis are the main sources of protons produced in the inflamed environment, which cause local tissue acidosis. Tissue acidosis may alter both the immune and vasculature responses and subsequent cytokines and chemokines production. The pH sensing G-protein coupled receptors (GPCRs), GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A) have emerged as a new class of proton sensing receptors that are expressed by immune and non-immune cells. GPR65 is mainly expressed in immune cells and is functionally critical for intestinal homeostasis, as identified by Genome-Wide Association Study (GWAS), for being a genetic risk factor in patients with IBD. We and others observed an anti-inflammatory role for GPR65 in pre-clinical mouse models, possibly through modulating the innate immune response towards a less inflammatory phenotype. GPR4, on the other hand, is mainly expressed in endothelial cells and confers a proinflammatory role that our group had previously uncovered the mechanism for. Upon acidic activation of GPR4, a proinflammatory program in the endothelium is activated, upregulating cytokines, chemokines, adhesion molecules, and ER stress responses. This activation is particularly crucial in the process of immune cell extravasation to the site of inflammation which is a critical step in IBD pH homeostasis and chronic inflammation. We and others observed reduced inflammatory response in IBD preclinical mouse models using both genetic and pharmacological inhibition for GPR4. Additionally, a biological, as well as a pathological proangiogenic role for GPR4 has been previously described. Therefore, based on the differential roles observed for GPR65 and GPR4 in inhibiting and mediating intestinal inflammation, respectively, we sought to investigate their functional roles in CAC development. To this end, we utilized the well-established azoxymethane/dextran sodium sulfate (AOM/DSS) CAC mouse model, using GPR65-null, and GPR4-null mice, to study their functional roles in CAC. Our observations indicate an anti-inflammatory role during chronic intestinal inflammation and an anti-tumoral role of GPR65, leading to less tumor development in CAC mice. Thus, we propose that the use of GPR65 agonist will be of therapeutic benefit in IBD treatment and CAC prophylaxis. Conversely, our results indicate a proinflammatory role for GPR4 during intestinal inflammation in addition to protumorigenic and proangiogenic roles, contributing to CAC development. Hence, we propose the use of GPR4 antagonism as a strategy for IBD and CAC treatment. Finally, we observed that abolishing GPR4 alleviates another type of colitis, immune checkpoint inhibitors-mediated colitis (IMC). This type of colitis occurs as a side effect for the use of immunotherapy treatment in cancer patients. Using an IMC mouse model, our results indicate that abolishing GPR4 reduces disease activity, macrophage clusters, and fibrosis, suggesting that inhibiting GPR4 may provide an effective treatment for IMC. Collectively, this dissertation work provides new insights into the roles of GPR4 and GPR65 in intestinal inflammation and cancer development.Item Open Access The Functional Roles of pH-sensing G protein-coupled receptors in Intestinal Inflammation(East Carolina University, 2018-12-06) Sanderlin, Edward JosephThe inflammatory microenvironment in inflammatory bowel disease (IBD) is complex, replete with microbial byproducts, complement, leukocytes, and resulting inflammatory cytokines. Parallel to these microenvironmental factors are protons, which are produced in excess due to altered metabolism of infiltrated leukocytes and local ischemia. Immune cells and intestinal microvasculature exist in the acidic, inflamed microenvironment and in turn alter their function in response to the acidic pH. Currently, only little is known how cells sense extracellular acidity and subsequently alter the inflammatory response. Recently, a class of proton-sensing G protein-coupled receptors (GPCRs) have emerged as functional pH-sensors, expressed in either leukocytes or vasculature, and are capable of altering immune cell inflammatory programs in response to acidic pH. These family members include GPR4, OGR1 (GPR68), TDAG8 (GPR65), and G2A (GPR132). Our group has uncovered a novel role for GPR4 in mediating endothelial cell (EC) inflammation in response to acidic pH. GPR4 activation in ECs have resulted in increased vascular adhesion molecule expression and functionally mediates leukocyte-EC interactions which are essential for the leukocyte extravasation process. Proton-sensors GPR65 and GPR68, however, are not expressed in ECs but are highly expressed in myeloid and lymphoid cells. GPR65 and GPR68, therefore, has been shown to mediate both pro- and anti-inflammatory responses in leukocytes in response to acidic pH. GPR132, however, has been described as a promiscuous GPCR, capable of responding to protons, bioactive lipids, and oxidized free fatty acids. Evidence suggests GPR132 is highly expressed in immune cells and plays important roles in immunity and the inflammatory response. Several lines of evidence suggest loss of pH homeostasis is associated IBD and could correspond to the degree of inflammation. For these reasons we sought to investigate the functional roles of pH- sensors GPR4, GPR65, and GPR132 in the regulation of intestinal inflammation. We utilized the acute and chronic dextran sulfate sodium (DSS)- induced experimental colitis mouse model with GPR4-null, GPR65-null, or GPR132-null mice. Our results indicate GPR4 contributes to intestinal inflammation in both acute and chronic DSS-induced colitis models likely though mediating leukocyte infiltration into the intestinal mucosa. Furthermore, a novel GPR4 antagonist was capable of inhibiting acute intestinal inflammation, suggesting GPR4 could be a valuable therapeutic target in colitis. Conversely, GPR65 reduces intestinal inflammation in the chronic DSS-induced colitis model. In vitro studies using bone marrow derived macrophages suggest GPR65 regulates macrophage functions. Lastly, GPR132 genetic deficiency was evaluated in two generations of GPR132 knockout mice in intestinal inflammation. Our results suggest GPR132 functions to reduce intestinal inflammation in the DSS-induced colitis mouse model. Further studies need to be performed to fully evaluate the role and mechanism of GPR132 in intestinal inflammation. Overall, this dissertation work highlights the emerging roles of pH-sensing GPCRs in the regulation of intestinal inflammation and implicates these receptors as valuable therapeutic targets in the remediation of intestinal inflammation.Item Open Access THE ROLE OF THE ALVEOLAR MACROPHAGE IN CARBON NANOTUBE ELICITED MURINE MODEL OF PULMONARY GRANULOMATOUS INFLAMMATION(East Carolina University, 2018-11-29) McPeek, MatthewPulmonary sarcoidosis is a debilitating inflammatory condition characterized by the presence of granulomatous lesions throughout the lung. Granulomas are a physiological response to inhaled antigens or particulate matter which cannot be properly degraded. To explore mechanisms of granuloma formation and maintenance our laboratory developed a murine model of pulmonary granulomatous inflammation elicited by multi-wall carbon nanotubes (MWCNT). We have found the MWCNT model bears striking similarities to pulmonary sarcoidosis pathophysiology, including increased expression of inflammatory mediators and decreased expression and activity of peroxisome proliferator activated receptor-gamma (PPARy) in alveolar macrophages. PPARy is a known regulator of macrophage activation and serves a crucial role in pulmonary lipid homeostasis through the regulation of macrophage ATP-binding cassette (ABC) lipid transporter-G1 (ABCG1). Further studies demonstrated that alveolar macrophages obtained from sarcoidosis patients and MWCNT instilled animals have decreased gene expression and protein levels of ABCG1 and ABCA1, a complementary cholesterol transporter. These studies aim to further define the role of alveolar macrophage PPARy, ABCA1 and ABCG1 in pulmonary granulomatous inflammation. We hypothesized that deficiency of ABCA1 and ABCG1 would exacerbate MWCNT induced granuloma formation. To test this hypothesis, macrophage-specific ABCA1, ABCG1 or combined ABCA1/ABCG1 knockout mice were developed and evaluated following MWCNT instillation. We found that deficiency of ABCG1 but not ABCA1 leads to a significant upregulation of pro-inflammatory mediators and promotes pulmonary granuloma formation. Interestingly, the combined deficiency of ABCA1/ABCG1 leads to an exacerbated pulmonary inflammatory phenotype. We further hypothesized that upregulation of the PPARy-ABCG1 pathway would limit MWCNT induced granuloma formation and inflammation. To test this hypothesis, we administered the PPARy-specific ligand rosiglitazone to MWCNT instilled animals and evaluated the effect on granulomatous inflammation. We found that the administration of rosiglitazone promotes the expression of alveolar macrophage ABCG1, limits the severity of MWCNT induced granuloma formation and reduced alveolar macrophage pro-inflammatory gene expression. These studies suggest that the PPAR-ABCG1 pathway, specifically the deficiency of alveolar macrophage ABCG1 plays a significant role in pulmonary granulomatous inflammation.Item Open Access Reproductive Toxicity of Crude oil-Dispersant Mixture in Caenorhabditis elegans(East Carolina University, 2016-05-05) Zhang, YanqiongAs crude oil remains a vital natural resource for the energy need of the world, environmental crude oil spills continue to be a health risk to human beings and ecological systems. During clean-up efforts, surfactant-like dispersants are used to break down big oil slicks into small droplets. Therefore it is necessary to investigate the health impacts of dispersed oil as a mixture entity rather than based on the toxicological profile of individual chemicals. Since reproductive stages of organisms are generally being more sensitive to the effects of toxicants than other stages, investigation of crude oil/dispersant exposure effects on reproduction is critically important. However, studies on the reproductive effects of crude oil-dispersant mixture exposure and its mechanism remain insufficient. The nematode Caenorhabditis elegans (C. elegans) has been a useful tool for environmental toxicity studies, and it is a well-known animal model to study the reproduction system. Therefore in this study, we employed the nematode C. elegans to test impacts of crude oil/dispersant exposure on basic biological processes growth, reproduction, microRNAs and protein-coding gene expression and its underlying mechanisms. In Chapter 1, we employed parallel experiments to test the effects of crude oil from the DWH oil well, chemical dispersant Corexit 9500A, and dispersant-oil mixture on growth and reproduction in the model organism Caenorhabditis elegans. Both the crude oil and the dispersant significantly inhibited the reproduction of C. elegans. Dose-dependent inhibition of hatched larvae production was observed in worms exposed to both crude oil and dispersant. Importantly, the chemical dispersant Corexit 9500A potentiated crude oil effects; the dispersant-oil mixture induced more significant effects than oil or dispersant-alone exposures. While oil-alone exposure and dispersant-alone exposure have none to moderate inhibitory effects on hatched larvae production, respectively, the mixture of dispersant and oil induced much more significant inhibition of offspring production. The production of hatched larvae was almost completely inhibited by several high concentrations of the dispersant-oil mixture. We also investigated the effects of crude oil/dispersant exposure at the molecular level by measuring the expressions of 31 functional genes. Results showed that the dispersant and the dispersant-oil mixture induced aberrant expressions of 12 protein-coding genes. These 12 genes are associated with a variety of biological processes, including egg-laying, oxidative stress, muscle contraction, and neurological functions. In Chapter 2, we showed that crude oil-dispersant mixture affected reproduction by inducing abnormal sperm during the process of spermatogenesis. Results showed that the abnormal immature sperm were significantly increased in the gonad arms of Dis-Oil mixture treated animals compared to controls (K-medium). We further explored the oil-dispersant mixture toxicity effects on spermatogenesis by using a male C. elegans strain. After 48h exposure to Dis-Oil mixture, spermatids appeared abnormal morphology including irregular shape of the spermatid membrane and unexpected tails induced by dispersed oil. Moreover we utilized puf-8; lip-1 tumor sensitive strain to test the cell fate of immature sperm induced by Dis-Oil mixture treatment. We found increased tumor occurrence in dispersed oil treatments compared to control. Results also suggest that the immature sperm may undergo dedifferentiation and become tumor-like cells in puf-8; lip-1 mutants through the MAPK-independent pathway. Based on the genome-wide investigation of microRNA profile, in Chapter 3, we found that the aberrant expression of miRNAs was induced. The KEGG pathway enrichment analyses indicated that those significantly changed miRNAs regulate many biological processes in C. elegans. Many affected pathways are related to environmental information processing, such as ABC transporters, MAPK signaling pathway, Erbb signaling pathway, JAK-STAT signaling pathway, MTOR signaling pathway and calcium-signaling pathway. Some pathways are related to oil uptake and metabolism, such as endocytosis, fatty acid biosynthesis and phosphatidylinositol signaling system. In summary, based on our studies, both crude oil and dispersant can induce the reproductive toxicity, and the dispersant enhanced the crude oil toxicity. Since the currently identified proteins and microRNAs in C.elegans show remarkable conservation with mammals including humans, the oil/dispersant may also induce similar change at physiological and molecular levels and affect many biological processes in mammals.Item Open Access Tight junction claudin-7 protein modulates multiple processes of cancer progression in human lung cancer cells(East Carolina University, 2016-05-03) Kim, Do HyungCarcinogenesis usually consists of cancer initiation, promotion, and progression. Several tight junction claudin proteins have been identified as tumor suppressors or activators during cancer development. As apical proteins, tight junction claudins seal the apical sides of neighboring epithelial cells in regulating the transport of ions and fluids from the extracellular environment. However, claudin-7, which is believed to direct cell-matrix adhesion, has been found at the basal side of several human organs, including the lung. It has also been clinically reported that the low survival rate of lung cancer patients is closely associated with their low claudin-7 expression. The molecular mechanism of claudin-7 that regulates lung cancer progression is not clearly understood. In order to understand how claudin-7 is involved in lung carcinogenesis, this dissertation presents the molecular and cellular changes in human lung cancer cells upon the suppression of claudin-7 expression to study how claudin-7 modulates lung cancer cell progression, including cell proliferation, migration, and invasion, cell-matrix attachment, and cell metabolism.Item Open Access Changes in zebra finch central nervous system morphology associated with developmental cannabinoid exposure(East Carolina University, 2012) Gilbert, Marcoita TerreenAdolescent CNS development is a highly organized, coordinated process that is both genetically and environmentally influenced, and is characterized by a period of dynamic, activity-dependent changes in synaptic connectivity. Growing evidence suggests that adolescent cannabis use is a risk factor for the development of persistent alterations in brain function. Taeniopygia guttata, the zebra finch, was used as a model of cannabinoid-altered vocal learning. We explored effects of cannabinoid-altered signaling during normal late-postnatal CNS development, as well as lasting morphological changes following exogenous cannabinoid exposure. In these studies, the cannabinoid agonist WIN 55,212-2 was administered to developing male zebra finches during sensorimotor song learning and dendritic spine densities measured following Golgi-Cox impregnation. Within HVC, a region necessary for songbird vocal production, and Area X, a striatal region essential for song learning, dendritic spines were inappropriately elevated by an average of 25% following developmental cannabinoid treatment. Treatments of adults that had already learned song were not associated with spine density changes. Cannabinoid-altered song and neuronal morphology were correlated with changes in levels of proteins related to cell signaling and morphology, including axonal Nf-200 and dendritic MAP2. After sensorimotor developmental cannabinoid treatment, anti-Nf-200 and -MAP2 antibodies were used to immunohistochemically confirm Golgi-Cox staining results. In the same brain areas where dendritic spines were elevated following CB agonist treatment, Nf-200 and MAP2 immunoreactivity (ir) were also elevated. To investigate mechanisms of cannabinoid-induced changes in neuronal morphology, we measured expression of the cytoskeletal protein Arc in NCM. A single exposure to novel song increased the postsynaptic densities of Arc protein. Two exposures to song were sufficient to produce habituation of this response. Habituation was prevented by pretreatment with WIN. These findings suggest there is a persistent, developmentally-restricted condition during periadolescence, and that cannabinoid agonism interferes with sensory integration and encoding necessary for accurate formation of memories.Item Open Access Recruitment of Estuarine-Dependent Alosines to Roanoke River and Albemarle Sound, North Carolina(East Carolina University, 2012) Riley, Kenneth Lee PickrellThe deleterious effects of dams on alosine populations are widely documented in many rivers along the Atlantic coast. Alterations to the natural hydrologic regime can disrupt spawning, egg dispersal, and recruitment of larvae to nursery habitats. The goal of this study was to investigate the ecological processes that influence recruitment of river herring (blueback herring Alosa aestivalis and alewife A. pseudoharengus) to nursery habitats within lower Roanoke River and Albemarle Sound, North Carolina. It was hypothesized that variability in abiotic conditions and fluctuations in food abundance could structure nursery habitat and severely restrict recruitment. Ichthyoplankton and zooplankton samples were collected concurrently March through June 2008-09 at 19 stations, within three areas: River, Delta, and Sound. Significant spatial and temporal differences were observed for river herring abundances. Abundances (number/100m³ ± SD) were significantly higher in 2009 (30.8 ± 149.8) than in 2008 (4.1 ± 20.9). Across both years, abundances within the River (21.0 ± 127.6) were significantly higher than those in Delta (7.4 ± 35.4) and Sound (4.6 ± 24.8). Yolk-sac larvae were prevalent throughout samples (32%); however, larvae collected were predominantly preflexion stage (66%). Fish ages ranged from 4 to 19 days after hatch. Growth rates were similar for blueback herring (0.29 ± 0.16 mm/d) and alewife (0.30 ± 0.14 mm/d). Growth estimates were indicative of habitat quality and suggested riverine habitats supported the highest growth rates. Mortality estimates for blueback herring (0.76 ± 0.23 per day) were significantly higher than mortality estimates for alewife (0.64 ± 0.17 per day). High mortality for both years was probably related to larval dispersal and advective loss. Larvae do not appear to be food limited in this system as indicated by diet analyses and the spatiotemporal overlap between river herring and zooplankton. Decreasing zooplankton abundance was correlated with larval abundance and suggests foraging by larval alosines could negatively alter the structure of the zooplankton community. Diets varied little with early ontogeny and the smallest taxa (copepod nauplii and rotifers) accounted for over 85% of the diet. Because of a high-level of dietary overlap, intraspecific and interspecific competition is substantial for anadromous alosines. The result of long-term data analysis (1984 - 2009) for larval and juvenile river herring confirms Roanoke-Albemarle stocks are in decline. Larval fish abundance was negatively affected by spring river flow (r² = 0.62). High flows (> 300 m³/s) resulted in larval advection from Roanoke River. Spring river flow was positively correlated with juvenile abundance (r = 0.95) and best recruitment of juveniles occurs in years with moderate spring river flow (141 - 311 m³/s).Item Restricted HIGHER-LEVEL SYSTEMATICS OF THE MILLIPEDES (ARTHROPODA: MYRIAPODA: DIPLOPODA) EVALUATED USING TRANSCRIPTOMIC DATA, MITOCHONDRIAL GENOMICS, AND CLASSIFICATION ANALYSES(East Carolina University, 2012) Brewer, Michael S.Knowledge of deep level phylogenetic relationships within and between many animal groups is currently lacking. Past attempts to reconstruct these ancient evolutionary relationships once relied chiefly on morphological characters and consequently suffered from deficiencies inherent to these data (homoplasy, few diagnostic characters, etc.). Advances in molecular biology over the last 20 years (namely PCR and high-throughput sequencing) have provided a whole other facet of potential characters via a vast array of loci, both nuclear and mitochondrial, which represent unlinked regions spanning entire genomes. The class Diplopoda (the millipedes) is a diverse group, 12,000 described species, which has suffered a long and convoluted taxonomic and systematic history. Containing many overinflated groups (numerous monotypic taxa) while other groups are vastly understudied, the current scheme of classification does not reflect the evolutionary history of the group but instead exists primarily for identification purposes. A classification constructed in such a manner can be characterized as an "unnatural classification". This work represents an attempt to: 1) ascertain the uniformity of millipede taxa at the ordinal level by evaluating species diversity contained in higher taxa (orders, families, genera); 2) achieve a robust and empirically derived estimate of millipede global diversity; 3) reconstruct the higher level relationships among all major millipede taxa using genomic scale molecular data; 4) map the ancestral states of key millipede characteristics; and 5) date the divergences of major diplopod lineages. Full mitochondrial genomes and transcriptomic data obtained via second-generation sequencing for exemplar taxa representing the ordinal taxa were used to reconstruct the relationships between the higher millipede taxa. Our work uncovered interesting and potentially alarming trends in millipede taxonomy. Additionally, we demonstrated the utility, or lack thereof, of using mitochondrial sequence data to reconstruct deep evolutionary relationships. Transcriptomic data analyses yielded well- supported trees containing relationships that have not been suggested previously. The transcriptome-based phylogeny was used to reconstruct the evolutionary changes of key millipede characteristics and, consequently, to assess their utility in delineating millipede taxa. This information provides a basis for future work within the Diplopoda including but not limited to: character evolution, biogeography, divergence time estimations, and comparative genomics.Item Open Access THE EVOLUTION OFHOX [sic] PARALOG GROUP 2 GENE EXPRESSION AND REGULATION IN THE JAPANESE MEDAKA(East Carolina University, 2011) Davis, AdamHox paralog group 2 (PG2) genes are evolutionarily conserved developmental regulatory genes that function to specify rhombomere (r) and pharyngeal arch (PA) identities in animal embryos. Several rounds of whole genome duplications in animals, including one specific to ray-finned fishes, and post-genome duplication independent gene loss have resulted in divergent Hox PG2 gene complements across evolutionarily divergent teleost fishes. Divergence in gene complements may have, in part, been responsible for generating divergent Hox PG2 gene expression patterns and specification of hindbrain and PA-derived structures during the evolution of osteichthyan embryogenesis. In this dissertation, I describe the cDNA cloning and expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. I show that there are only two functional canonical genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, [psi]hoxa2b. The canonical genes, hoxa2a and b2a, were expressed in developing rhombomeres and PAs in a manner that was generally conserved throughout the osteichthyans. By contrast, [psi]hoxa2b was expressed at detectable levels only in noncanonical Hox PG2 gene expression domains, including the ventral-most aspect of the neural tube, the pectoral fin buds and the caudal-most region of the embryonic trunk, indicative that regulatory control elements needed for spatiotemporal specification of expression have diverged from the canonical orthologs. In order to understand whether sequence divergence within cis-regulatory control elements are linked to the divergent expression patterns of the medaka hoxa2 paralogs, conserved genomic sequences upstream of the medaka hoxa2a and [psi]hoxa2b genes were tested functionally using a transgenic GFP reporter system. The medaka hoxa2a r3/5 enhancer region (r3/5ER) was shown to direct reporter gene expression in r4, PA2 and the posterior PAs, while the r3/5ER of [psi]hoxa2b directed reporter gene expression in r3-7, PA2 and the posterior PAs, which is different from transgenic mapping studies of the orthologous regions tested in chick and mouse embryos. These analyses provide evidence for significant post-genome duplication divergence in cis-regulatory element function in the r3/5ER of osteichthyans. Further, they question the ancestral nature of the r3/5ER prior to the evolutionary split of sarcopterygians (lobe-finned fishes) from the actinopterygians (ray-finned fishes).Item Open Access Thermodynamic and Spectroscopic Investigations of Novel Antimicrobial Peptides containing Unnatural Amino Acids with Model Membrane Systems(East Carolina University, 2011) Russell, Amanda LauraWith the over prescription and use of antibiotic drugs the evolution of drug resistant bacteria strains has become a world wide health crisis. In response to this crisis, the search for new drugs to kill bacteria via a novel mode of action has become of critical importance to modern medicine. Natural and synthetic antimicrobial peptides (AMPs) have exhibited a broad spectrum of activity against various infectious microorganisms while exhibiting little to no mammalian cell toxicity. It's because of this selectivity that AMPs are considered as a potential new source of therapeutics for drug-resistant bacteria. It is believed that the selectivity for bacteria cell membranes over host cells and the potency between different bacteria strains is derived from the ability of the AMPs to exploit the differences in chemical composition of various organism's membranes. The hypothesis guiding this research states: The physicochemical surface properties of the target cell's membrane interact with the 3D physicochemical surface properties of the approaching AMP inducing a conformational change onto the AMP maximizing the interactions between the two to facilitate AMP-membrane binding and the formation of pores leading to intracellular leakage and cell death. There are two binding interactions that are of interest, the S-state and I-state. The S-state, or surface state, refers to the binding of the peptide to the surface of the membrane, while the I-state involves the insertion of the peptide into the bilayer. By understanding the interactions of various AMPs with different target cell membranes one can develop novel analogs with increased potency and selectivity for a particular strain of bacteria. This knowledge would eventually lead to the design of antimicrobial peptides used as pharmaceuticals to treat bacteria resistant to current antibiotics. In our laboratory we have developed a series of antimicrobial peptides that incorporate unnatural amino acids to impart specific physicochemical properties onto these peptides. Circular Dichroism (CD), isothermal titration calorimetry (ITC) and calcein leakage assays were conducted and it was determined that there are two distinctly different mechanisms of binding occurring between this series of peptides and zwitterionic and anionic membrane models. By understanding the interactions of various AMPs with different target cell membranes one can develop novel analogs with increased potency and selectivity.Item Open Access Molecular characterization of the activation domain of the HTLV-1 HBZ protein and its interaction with the KIX domain of the cellular transcriptional coactivators p300 and CBP(East Carolina University, 2010) Cook, Pamela ReneeThe complex retrovirus human T-cell leukemia virus type I (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Deregulation of cellular transcription is thought to be a key event in virally-mediated T-cell transformation. The HTLV-1 basic leucine zipper factor (HBZ) originates from the 3' long terminal repeat (LTR) and is transcribed from the sense DNA strand relative to other viral genes. HBZ is the only viral gene known to be consistently expressed in ATL cells. HBZ is able to repress HTLV-1 transcription, in part, by binding to the homologous cellular coactivators p300 and CREB-binding protein (CBP). HBZ binds the kinase-inducible interaction (KIX) domain of p300/CBP via two φXXφφ motifs in the activation domain of HBZ (HBZ-AD). In this study, it was determined that HBZ-AD binds with high affinity to the KIX domain of p300/CBP with an apparent equilibrium dissociation constant (Kd) of 3.2 ± 0.5 nM. The KIX domain contains two binding surfaces that are differentially targeted by various transcriptional regulatory proteins. One surface of KIX can bind the activation domain of the mixed lineage leukemia (MLL-AD) protein and the HTLV-1 transactivator Tax, among others. The other surface of KIX can contact the cellular transcription factors c-Myb or cyclic AMP (cAMP) response element binding protein (CREB). HBZ-AD was determined to bind the MLL-AD-binding surface of KIX and to compete with MLL-AD, Tax, and p53 for KIX binding. HBZ-AD was additionally found to significantly enhance the binding of the activation domain of c-Myb (c-Myb-AD) and CREB to KIX. HBZ was also shown to inhibit transcriptional activation mediated by MLL-AD and enhance activation mediated by c-Myb-AD. These results indicate that HBZ is able to alter gene expression by competing with one group of transcription factors for occupancy of KIX while enhancing the binding of other factors to a different surface on KIX.Item Open Access The Poxvirus A35 Protein Promotes Virulence by Regulating the Host Adaptive Immune Response(East Carolina University, 2010) Rehm, Kristina ElizabethPoxviruses are large double-stranded DNA viruses with a broad host range and worldwide distribution. The most infamous poxvirus is Variola virus, the causative agent of smallpox, which caused over 500 million deaths in the twentieth century before it was declared eradicated in 1980 by the World Health Organization. Nevertheless, more than 25 years after its eradication from nature, smallpox remains a threat, as there is now concern for its use in an act of bioterrorism. Other poxviruses have emerged in recent years. In 2003, there was a monkeypox outbreak in the United States, with nearly 80 cases. This disease is similar to smallpox, but can be spread not only from human to human, but also from animal to human, which emphasizes the fact that many poxviruses are a zoonotic threat. The current poxvirus vaccine (Vaccinia virus, VACV), while highly effective, is unsafe and its use is contraindicated for an estimated 25% of the population due to its virulence. The virulence of VACV is attributed to the production of numerous proteins that function to evade the host immune response, including inhibitors of interferons, regulators of apoptosis, and cytokine receptor homologs. Despite the large amount of work that has been done to characterize poxvirus virulence mechanisms, there are more than 25 highly conserved genes that remain uncharacterized. The poxvirus A35R gene is highly conserved in all sequenced mammalian tropic poxviruses and has little similarity to any non-pox protein, suggesting an important and novel function. The A35 protein was not required for viral replication in eight cell lines from various hosts, but removal of A35 from the viral genome resulted in a 1000-fold decrease in virulence in the mouse model. We therefore hypothesized that the poxvirus A35 gene product promotes virulence by affecting viral replication in certain cell types in vivo or by regulating aspects of the host adaptive immune response. We show that A35 is not required for viral replication in various cell types from six different mammalian hosts and also in mouse tissues early after infection. Using a model major histocompatibility complex (MHC) class II-restricted antigen presentation system, we have shown that VACV and A35 inhibit the amount of numerous cytokines that are produced from both the T cell and the antigen presenting cell (APC) as a result of specific antigen presentation, including IL-2 and nitric oxide. Further data implicated the APC as the viral target, as the T cells used in the assays were unaffected by VACV and A35. The A35 protein did not induce apoptosis of the APC or affect antigen-independent NO production. Furthermore, A35 localized to the endosomal compartments of the APC and decreased MHC class II expression and the amount of peptide presented in the cleft of MHC class II molecules. To determine if A35 would decrease the host adaptive immune response, mice were vaccinated with 500 plaque forming units (pfu) of either VACV (Western Reserve strain, WR) or A35 deletion mutant virus. Our data indicate that A35 decreased VACV- specific immune response including interferon- (IFN)-gamma production, cytotoxic T lymphocyte (CTL) killing, serum antibody, and neutralizing antibody. The decreased immune response was shown to be important as it resulted in increased viral replication in target organs. The A35 deletion mutant virus was also an effective vaccine, as it protected mice as well as WR from a lethal challenge. We also made a recombinant A35 deletion mutant virus in the Modified Vaccinia Ankara (MVA) replication-deficient vaccine strain, which has been shown to be safe when used in immunocompromised individuals. Results with MVA were similar to studies performed with WR: A35 was not required for virus replication and decreased VACV-specific immune responses. Furthermore, the MVA35 deletion mutant vaccine protected mice as well as MVA from a lethal respiratory challenge. Our data expands the knowledge of what is known regarding poxvirus virulence mechanisms and is applicable to the development of safer and highly immunogenic vaccines. Since poxviruses are now being used widely as a platform for a number of vaccines for various diseases, including human immunodeficiency virus (HIV) and cancer, our work with A35 is broadly applicable to vaccine development.Item Open Access The role of peroxisome proliferator-activated receptor-gamma in surfactant catabolism in the alveolar macrophage(East Carolina University, 2009) Baker, Anna DeLanePulmonary alveolar proteinosis (PAP) is a lung disease characterized by surfactant accumulation in the alveolar spaces and alveolar macrophages. Although PAP is rare, surfactant abnormalities occur in many lung diseases including acute respiratory distress syndrome, sarcoidosis, and asthma. Studies have shown that surfactant accumulation in PAP patients results from insufficient catabolism by alveolar macrophages. Research in PAP patients and granulocyte-macrophage colony-stimulating factor knockout (GM-CSF KO) mice revealed deficiencies in the transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ) and downstream cholesterol transporter ATP-binding cassette G1 (ABCG1). PPARγ regulates lipid metabolism in macrophages and is a prominent target of research in the fields of atherosclerosis and diabetes. This study tested the hypothesis that PPARγ promotes catabolism of surfactant in alveolar macrophages through the transcriptional regulation of ABCG1. Alveolar macrophages from macrophage-specific PPARγ knockout (PPARγ KO) mice accumulate surfactant and exhibit reduced expression of ABCG1 and reduced ABCG1-mediated cholesterol efflux. These results directly link PPARγ-deficiency to surfactant accumulation and demonstrate that PPARγ regulates cholesterol efflux in alveolar macrophages. We next investigated the expression of genes involved in the uptake and biosynthesis of cholesterol in PPARγ KO alveolar macrophages. Expression of key cholesterol biosynthesis genes was suppressed, and cholesterol influx genes (scavenger receptors) were up-regulated. These results suggested PPARγ regulates cholesterol metabolism in alveolar macrophages. We next investigated the up-regulation of PPARγ in the GM-CSF KO alveolar macrophages by instilling mice with a Lentivirus vector containing PPARγ (Lenti-PPARγ). Reconstitution of PPARγ promoted ABCG1 expression and ABCG1-mediated cholesterol efflux in the alveolar macrophages of GM-CSF KO instilled with Lenti-PPARγ. Taken together, these observations support the hypothesis that PPARγ-mediated transcriptional regulation of ABCG1 is critical to cholesterol metabolism and the maintenance of surfactant homeostasis overall. Understanding the role of PPARγ in normal surfactant homeostasis provides insight into the pathophysiology of PAP and identifies a potential therapeutic target.Item Open Access The serine protease from the venom of polistes dominulus contains allergenic epitopes(East Carolina University, 2010) Fitch, Christina D.The protection introduced by immunotherapy to patients with allergies to paper wasps may be partial depending on the sensitizing species. Partial cross-reactivity between major paper wasp groups may be the reason for partial protection. Understanding patient response to venom allergens from the paper wasp groups is important to improve and develop effective treatments. Research has shown that the allergenic serine protease may be important in this partial cross-reactivity. In this study we investigated the importance of epitopes on the allergenic serine protease (Pol d 4) from Polistes dominulus . Recombinant Pol d 4 was expressed in prokaryotic and eukaryotic expression systems. The expression products from the eukaryotic system yielded a protein of larger size than predicted size. Two constructs of Pol d 4 were produced in the prokaryotic expression system and were of expected size. Re-folding of the prokaryotic expressed recombinant proteins was not successful. The major amino acid epitopes on Pol d 4 were predicted. Three mutant constructs were chosen, each containing two amino acid substitutions within a predicted epitopes. IgE binding was assayed by immunoblot assays using sera from European and North American patients. The majority of European patient sera had IgE that bound to the recombinant form of Pol d 4. Statistical analysis showed that there was no significant difference in IgE binding to the various proteins. IgE from American patients bound recombinant Pol d 4. Bromelain was used in immunoblot inhibition to assay possible carbohydrate epitopes. The inhibition assay showed evidence of IgE binding. The serine protease from Polistes gallicus was characterized. Identity between the two serine proteases was high. Evidence supports the presence of carbohydrate on the recombinant protease and carbohydrate epitopes may be present. Visually, all three mutant proteins bound IgE; mutant 2 bound IgE from the least number of patients suggesting it may be an important epitope on Pol d 4. Analysis of densitometric data showed no significant difference between the proteins tested. The data suggests that there may be amino acid epitopes present. Further, the high sequence identity between Pol d 4 and Pol g 4 support high cross-reactivity between sister species.