Biomedical Sciences
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Item Embargo ASSESSING THE OUTCOME OF PERIPHERAL NERVE INJURY IN A RODENT MODEL: BEHAVIORAL, PHARMACOLOGICAL, IMMUNOHISTOCHEMICAL, AND MASS-SPECTROMETRIC STUDIES(East Carolina University, July 2024) Marshall, Dylan AdlerChronic neuropathic pain (CNP) is a pathophysiological condition and is pain that lasts for longer than 3 months. CNP is usually the result of injury or disease to the central nervous system (CNS) or the peripheral nervous system (PNS). Despite their reputation and their possible side effects, opioids remain a main treatment option for CNP. Preclinical work from the lab has previously shown that an injury to the CNS, in the form of spinal cord injury (SCI), led to CNP in the rat model, and additional data showed that treatment with opioids had an analgesic effect in only ~ 1/3 of the animals tested. SCI is a massive injury inflicted onto the CNS, and here we wanted to probe the outcome of a less severe injury to the PNS, sciatic nerve ligation (SNL) on the development of pain and chronic pain. The aims of this project were to a) determine the behavioral outcome of SNL on pain behaviors, b) assess the analgesic effects of morphine treatment after SNL, c) probe for morphine and dopamine receptor expression in the dorsal root ganglia (DRGs) that receive the sensory information from the periphery, and d) test if the injury potentially led to systemwide changes that could be detected in the blood proteome. We found that, as in centrally-induced pain models, SNL led to a decrease in pain reflex latencies, indicative of a heightened pain sensitivity. Treatment with morphine was also only effective in less than 40% of the animals, again similar to the CNS model. In addition, we found that morphine mu-receptors (MORs) were upregulated in ipsilateral over contralateral dorsal root ganglia (DRGs) at the level of the injury. In contrast, we did not find differences in dopamine D1 receptor (D1R) expression between ipsi- and contralateral sides. Blood samples collected prior to and following SNL were processed for unbiased proteomics. Proteomic analysis identified that unilateral SNL altered the blood proteomic profile. Together, these data suggest that the peripherally-induced unilateral injury mimics the outcomes of the centrally induced CNP model, and that it leads to chronic pain-manifestations that affect both specific neural tissues and blood proteome alike.Item Embargo Kinin B1 Receptor Mediates Bidirectional Interaction between Neuroinflammation and Oxidative Stress(East Carolina University, 2023-05-02) Theobald, DrewHypertension is the leading risk factor for cardiovascular disease and affects nearly half the adults in the United States. It is well established that hypertension is a low-grade inflammatory condition and is associated with increased release of proinflammatory cytokines, elevated oxidative stress levels, and increased activity of the kallikrein-kinin system (KKS). The KKS is a family of vasoactive proinflammatory peptides that play a vital role in regulating blood pressure. Activation of kinin B1 receptor (B1R) results in increased inflammation and vasoconstrictive effects which can ultimately lead to hypertension. Previously, we showed that angiotensin II (Ang II) can upregulate B1R expression and can induce oxidative stress and neuroinflammation in primary neurons. However, the order at which this occurs has not yet been investigated. In this study, we aim to determine the relationships between neuroinflammation, oxidative stress, and activation of B1R in both primary hypothalamic neurons and primary hypothalamic microglia. Following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS), or hydrogen peroxide (H2O2), we were able to identify a significant increase in reactive oxygen species production, inflammation, and B1R expression. Furthermore, we showed that B1R blockade using a B1R specific antagonist can attenuate these effects in both neurons and microglia. Together, these data provide novel evidence that the interaction between neuroinflammation, oxidative stress, and B1R activation in the brain is bidirectional and that blocking B1R may serve as a potential therapeutic target for neuroinflammation and oxidative stress in various disease pathologies.Item Restricted Novel Function of PUF Proteins and Their Partners in Spermatogenesis and Spermatocyte-Derived Germline Tumors in Caenorhabditis elegans(East Carolina University, 2022-04-27) Park, YoungyongConserved PUF (Pumilio and FBF) proteins repress the translation by binding to its target mRNA 3' untranslated region (3'UTR). C. elegans has 11 PUF proteins. Among them, we focused on two FBFs (FBF-1 and FBF-2) and PUF-8. The object of this study is to identify a regulatory network including FBFs and PUF-8 in spermatogenesis and spermatocyte-derived germline tumors in vivo. Specifically, our significant findings are three-fold:1. FBFs and their repression target CYB-1 (B-type Cyclin) promote sperm viability by inhibiting CED-4 (an Apaf1 homolog)-mediated apoptosis (see Chapter 2)2. PUF-8 and its repression target, GLD-2 (a Cytoplasmic poly(A) polymerase) inhibit spermatocyte-derived tumorigenesis by activating GLD-1 (a KH motif-containing RNA-binding protein) and inhibiting MPK-1 (an ERK homolog) (see Chapter 3) 3. CYB-1 loss or X-ray irradiation could induce spermatocyte-derived germline tumors in the absence of PUF-8 and activation of MPK-1 (see Chapter 4). Since the regulators that we propose to study are broadly conserved, we therefore suggest that similar molecular mechanisms may control spermatogenesis and tumorigenesis in other organisms, including humans.Item Restricted SPINAL CORD REFLEXES IN A BRAIN-IRON DEFICIENT MODEL OF RESTLESS LEGS SYNDROME: ROLE OF DOPAMINE AND ADENOSINE RECEPTORS(East Carolina University, 2021-05-04) Woods, SydneyRestless Legs Syndrome (RLS) is a sensorimotor disorder that severely disrupts sleep. RLS patients regularly present with a condition known as Brain Iron Deficiency (BID), and BID is commonly associated with altered dopamine and adenosine neurotransmission in the striatum. Dopamine and adenosine can form receptor heteromers in the striatum, where the adenosine A1 receptor (A1R) modulates dopamine D1 receptor (D1R) function. However, there are no data on the impact of BID in the spinal cord, the ultimate sensorimotor circuitry involved in RLS. We here tested if a diet-induced brain iron-deficient animal model affects spinal cord excitability as reported in other RLS animal models, and we tested the responsiveness of this model to treatment with dopamine and adenosine receptor modulators. Following previously established protocols, C57Bl/6 mice were separated upon weaning into male and female cohorts fed either control iron or iron-reduced diets. The BID diet did not induce an anemic phenotype. To assess spinal cord excitability, we measured thermal pain reflex withdrawal latencies (RWLs) using the Hargreaves system, starting at one-week post-diet exposure. The BID cohorts showed significantly lower RWLs than their respective CTRL cohorts, and these differences remained stable over time. We then tested the responsiveness of this model to dopamine receptor modulators Pramipexole (PPX, D3 receptor agonist, 0.5 mg/kg, i.p.) and SCH 39166 (Ecopipam, D1 receptor antagonist, 0.5 mg/kg + 1.0 mg/kg, i.p.), and adenosine receptor modulators caffeine (A1R/A2R, 50 mg/kg, i.p.) and N6-cyclpentyladenosine (CPA, A1R agonist, 1.0 mg/kg, i.p.). These data indicate that PPX did have significant effects on increasing RWLs but with strong locomotor side effects, SCH 39166 showed significant effects in increasing RWLs in the male BID cohort, but not female BID cohort, and while caffeine did not have significant effects on RWLs in CTRL cohorts, use of CPA led to a significant increase in RWLs in both male and female BID cohorts. Western blot analysis of D1R and A1R expression in the mouse spinal cord revealed an increase in D1R expression and opposing decrease in A1R expression. Finally, the use of proximity ligation assays (PLAs) revealed the presence of A1R-D1R heteromers within the mouse spinal cord, where motoneurons reside, and that these heteromers decrease in number or are no longer functional under BID conditions. Together our data show that diet-induced iron-deficiency leads to a decrease in RWLs, and that use of dopamine and adenosine receptor modulators show significant effects in this model. RLS patients present with BID and are initially highly responsive to dopamine D3 receptor (D3R)-based treatment in the clinic. However, long-term treatment with these compounds can lead to unwanted long-term side effects. We here propose a hypothetical model of RLS and how BID conditions might affect dopamine and adenosine neurotransmission via A1R-D1R heteromers.Item Restricted The Circadian Rhythm and its Role in the Dynamic Dopamine Neuron Phenotype(East Carolina University, 2021-05-04) Barker, SamanthaThe circadian rhythm is strongly implicated in many neuropsychiatric and neurodegenerative disorders, all of which are associated with altered dopamine (DA) neurotransmission in the substantia nigra and ventral tegmental area. Progress has been made in elucidating the circadian rhythm-dopaminergic network and its role in the onset of neuropsychiatric and neurodegenerative disorders. Previous research suggests that circadian rhythm transcription factors are responsible for directly regulating the DA phenotype; however, it is currently unknown what the relationship between the circadian rhythm and dopaminergic genes looks like with respect to age and time of day. Using a transgenic mouse model with Cre recombinase expression under control of dopamine transporter (DATCre) and yellow fluorescent protein (YFP) Cre reporter and immunohistochemistry techniques, we are able to characterize sub-populations of DA neurons in the ventral midbrain (VMB) that are responsive to the circadian rhythm. Here, we demonstrate a dynamic DA neuron phenotype, where classic dopaminergic markers, such as dopamine transporter (DAT) and tyrosine hydroxylase (TH) are not detected in dopaminergic neurons, due to regulation by the circadian rhythm. In this study, mice transgenic for DATCre/YFP were analyzed at postnatal day 0 (P0), P21, P35 and adulthood (>P60). Each time point included mice taken at subjective dawn (circadian time 0) and subjective dusk (CT12), excluding P0 mice. Results revealed that between P21 and P35, there was a significant loss of the DA neuron phenotype at CT12, as compared to CT0. There was no statistical difference between P35 and adults at CT0 or CT12. This suggests that between P21 and P35, DA neurons begin to transition to a 'former' phenotype throughout the circadian rhythm. Additionally, qRT-PCR data revealed abnormal dopaminergic gene mRNA levels at P21. Elucidating the molecular characteristics of these DA neurons is crucial to understanding the biological mechanisms behind the dopaminergic-circadian rhythm network, which will have future implications in understanding neuropsychiatric and neurodegenerative disorders.Item Open Access PUF-8 and MPK-1: Genetic and Chemical Control of Spermatocyte Dedifferentiation in Caenorhabditis elegans(East Carolina University, 2021-05-03) Gaddy, Matthew A.Stem cells face a number of major fate decision during their development: the decision to self-renew or differentiate, and then whether to remain differentiated or dedifferentiate, as occurs in some oncogenesis. A regulatory network controlling these decisions is vital to the development of all multicellular organisms, including humans. Aberrant regulation can result in either loss of specific cell type or uncontrolled cell proliferation, leading to tumors. However, our understanding of how differentiated cell can be reverted to an undifferentiated state remains far more limited.Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) inhibit the formation of germline tumors via repressing the dedifferentiation of spermatocytes into mitotic cells (termed "spermatocyte dedifferentiation") at least in part by inhibiting MPK-1 (an ERK MAPK homolog) activation. To gain insight into the molecular competence for spermatocyte dedifferentiation, we compared the germline phenotypes between two competent mutants -- puf-8(q725); lip-1(zh15) with a high MPK-1 activity and puf-8(q725); fem-3(q20gf) with a low MPK-1 activity. puf-8(q725); lip-1(zh15) mutants developed germline tumors more aggressively than puf-8(q725); fem-3(q20gf) mutants at 25°C with aging. This result suggests that MPK-1 activation is critical to induce the formation of germline tumors via spermatocyte dedifferentiation. This idea was confirmed by treatment of puf-8(q725); fem-3(q20gf) mutant worms with Resveratrol, which stimulates MPK-1 activation. Our results show that 100 mM RSV significantly induced the formation of germline tumors via spermatocyte dedifferentiation at 25°C with aging. Therefore, we conclude that MPK-1 activation is required to promote the formation of germline tumors via spermatocyte dedifferentiation in the absence of PUF-8. Since PUF-8 and MPK-1 are broadly conserved, we therefore suggest that similar molecular mechanisms may control dedifferentiation-mediated tumorigenesis in other organisms, including humans.Item Open Access Borrelia burgdorferi ErpB and ErpQ inhibit C1 complex of the classical pathway of complement through a novel mechanism(East Carolina University, 2021-05-03) Garrigues, RyanThe complement system is an organized proteolytic cascade of dozens of proteins that functions in the recognition, opsonization, and lysis of pathogenic and altered-host cells. Bloodborne pathogens like the etiologic agent of Lyme disease, Borrelia burgdorferi, encounter complement during their bloodmeal and in their dissemination through the body. Therefore, to avoid complement mediated destruction, these pathogens have developed mechanisms that aid in complement evasion and defense. The spirochete B. burgdorferi, has nearly a dozen known complement recruiting or inhibiting surface exposed lipoproteins. Here, we uncover a novel inhibitory mechanism for two surface exposed lipoproteins, ErpB and ErpQ, that were recently identified using a lipoprotein gain of function library. Using surface plasmon resonance, ErpB and ErpQ were found to bind C1 complex proteases C1r and C1s with high affinity. Gel-based biochemical assays showed that ErpB and ErpQ specifically inhibit C1s-mediated cleavage of both C2 and C4 making them the only known bacterial inhibitors of C1s. Furthermore, they were shown to block C1s outside of the active site indicating that they function by a novel mechanism. Additional site-directed mutagenesis of C1s exosites revealed determinants for high affinity inhibitor interactions that have been shown to be important for C1s recognition of C4 outside of the active site. The discovery of a novel mechanism of complement inhibition by a medically-relevant human pathogen expands our knowledge of host pathogens interactions and contributes to previously unknown pathophysiological immune evasion by B. burgdorferi. Mechanistic studies on ErpB and ErpQ also support further understanding of molecular interactions between complement proteases and their substrates, which provides alternative means for the development of specific complement therapeutics toward complement-mediated diseases.Item Restricted Matrix Metalloproteinase 12 is Critical for Granuloma Formation in the Murine Model of Granulomatous disease(East Carolina University, 2019-08-16) Neequaye, Nicole NMatrix Metalloproteinase 12 (MMP12) is a protein produced primarily by alveolar macrophages that degrades elastin in the extracellular matrix (ECM) and enables infiltration of immune cells that participate in the inflammatory response. To our knowledge, few studies have been conducted to clarify the role of MMP12 in granulomatous diseases such as sarcoidosis, a chronic inflammatory disease characterized by granuloma formation primarily in the lungs. Previous studies have shown an increase in gene and protein expression of MMP12 in lung tissue and bronchoalveolar lavage (BAL) of patients with sarcoidosis as well as a correlation between MMP12 elevation and disease severity. Our murine model uses multiwall carbon nanotubes (MWCNT) to mimic the characteristics (gene, protein expression, and granuloma formation) observed in sarcoidosis patients. Based on these observations, we hypothesized that MMP12 plays a role in the acute and late inflammatory response in pulmonary sarcoidosis. MMP12KO mice were used to address this hypothesis. Analysis of gene expression of BAL cells in C57BL/6 (wildtype) mice shows a significant elevation in MMP12 after oropharyngeal instillation of MWCNT at all time points (3, 10, 20, 60, 90 days). We observed similar trends in proinflammatory genes chemokine (C-C motif) ligand 2 (CCL2), matrix metalloproteinase 14 (MMP14), and interferon-gamma (IFNÎđ) at all time points and osteopontin (OPN) at 20, 60, and 90 days. MMP12 protein levels increased in BAL cells at all time points. Evaluation of BAL cells from MMP12KO mice shows a similarity in the expression of all proinflammatory genes explored with wildtype at 10 days. CCL2 and MMP14, identified through gene expression profiling of the wildtype to be directly regulated by MMP12, is significantly reduced at 60 days in MMP12KO MWCNT instilled mice compared to wildtype. Histological analyses at 3, 10, 20, and 60 days shows increasing exacerbation in wildtype and continuous attenuation of granuloma formation in MMP12KO mice after exposure to MWCNT. A proposed mechanism for the reduction of granulomas at 60 days in MMP12KO, lead to an investigation into the relationship between MMP12, a pro-inflammatory mediator and PPAR[gamma], an anti-inflammatory modulator. Gene analysis showed a significant increase in MMP12 in PPAR[gamma]KO mice compared to wildtype and a substantial rise in PPARÎđ in MMP12KO mice compared to wildtype. Interestingly, MMP12 significantly increased, and PPAR[gamma] decreased dramatically in African American sarcoid patient's vs. controls when adjusted for race. MMP12 is seemingly instrumental in driving granuloma pathogenesis during inflammation. Evaluation of genes in MMP12KO mice suggests that the macrophage-secreted cytokines and matrix genes explored are necessary for granuloma formation. The significant increase in PPAR[gamma] intrinsically and after instillation with MWCNT in MMP12KO and its' decrease in wildtype mice after MWCNT instillation at 60 days suggests an inverse relationship between MMP12 and PPAR[gamma]. A reduction in granuloma formation in MMP12KO mice compared to wildtype supports a critical role for MMP12 in granuloma formation.Item Open Access DEVELOPMENT OF SMALL-MODELCULE INHIGITORS OF THE INTIATING PROTEASES, C1r AND C1s, OF THE CLASSICAL COMPLEMENT PATHWAY(2019-12) Rohlik, DeniseComplement is a proteolytic cascade that upon activation plays a key effector role in the innate immune system and acts to prime the adaptive immune response. During normal homeostatic events, complement is tightly regulated for its roles in immune complex clearance, lysis of target cells, opsonization, and recruitment of leukocytes and monocytes to target areas. Several endogenous regulators are responsible for the control of complement activation, however when dysregulation occurs, aberrant complement activation has been linked to autoimmune, proinflammatory, and neurodegenerative diseases, including Alzheimer's disease. Inhibition of the classical complement component C1 may ameliorate hallmarks of autoimmune and inflammatory disease. The serine proteases within the C1 complex, C1r and C1s, are promising therapeutic targets for structure-based small-molecule drug development. We investigated the activity of a series of small-molecule compounds identified in a large-scale fragment library screen and those from a cheminformatics computational docking screen in which hit compounds were predicted to bind the C1r or C1s proteases. Using surface plasmon resonance and ELISA-based assays for hit validation, we analyzed the binding affinities and the inhibitory IC50's of several compounds predicted to bind and inhibit the activation of C1r or C1s in a dose-dependent manner. In this study, we have identified four lead compounds (cmp-1611, cmp-1663, cmp-1696, cmp-1827) and their 10 active structural analogues that target and inhibit C1r activation. Given their abilities to bind and inhibit C1r and favorable physicochemical properties, our lead compounds may provide a starting point for optimizing affinity and specificity necessary for developing novel routes of therapeutic upstream complement inhibition.Item Open Access Method for nerve growth induction(1993-03-16) DeVanzo, John; Paul, Joseph W.A composition comprising nerve growth factor and 2-amino-1,1,3-tricyano-1-propene useful for the induction, stimulation, and maintenance of nerve growth, and methods of potentiating choline O-acetyltransferase and tyrosine hydroxylase by 2-amino-1,1,3-tricyano-1-propene are disclosedItem Restricted Peroxisome Proliferator-Activated Receptor Gamma Deficiency Promotes a T Lymphocyte Response in a Murine Model of Chronic Pulmonary Sarcoidosis(East Carolina University, 2019-07-03) Sanderford, Victoria LSarcoidosis is a chronic granulomatous disease of unknown etiology. Previous studies from our lab have shown a deficiency of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPAR[gamma]) in the alveolar macrophages of sarcoidosis patients. We established a murine model of granuloma formation by instillation with multiwall carbon nanotubes (MWCNT), and the expression of PPAR[gamma] was also decreased in MWCNT instilled mice. There is evidence linking lymphocyte reactivity to mycobacterial antigens in sarcoidosis, so we hypothesized that addition of mycobacterial peptide early secretory antigenic target 6 (ESAT-6) to MWCNT might exacerbate the murine T effector cell response. MWCNT with or without ESAT-6 peptide 14 were instilled into macrophage-specific PPAR[gamma]-KO or wild-type mice. Controls received vehicle or ESAT-6 alone. Bronchoalveolar lavage (BAL) fluid was collected for analysis via qPCR, and lymph nodes were collected for histology, qPCR, in vitro studies, or flow cytometric analysis. PPAR[gamma]-KO mice receiving MWCNT+ESAT-6 displayed markedly increased granuloma formation and exhibited mediastinal lymphadenopathy. Additionally, PPAR[gamma]-KO mice treated with MWCNT+ESAT-6 exhibited exacerbated fibrotic severity at 20- and 60- days post-instillation. Similarly, lymphocyte frequency was elevated in the BAL of PPAR[gamma]-KO mice compared to controls at 20- and 60-days post-instillation with MWCNT+ESAT-6. Further, TH17 associated transcription factor ROR[gamma]t and immune regulatory marker PD1 were elevated in PPAR[gamma]-KO mice treated with MWCNT+ESAT-6 compared to sham mice at both time points, with elevation of multiple other TH17 associated genes at 60 days post instillation. Carbon nanotubes were observed in the mediastinal lymph nodes of PPAR[gamma]-KO and wild-type mice post-instillation with MWCNT and MWCNT+ESAT-6, and flow cytometric analyses revealed that CD4+ T cells were significantly elevated in PPAR[gamma]KO mice receiving MWCNT+ESAT-6. These findings suggest that instillation of PPAR[gamma]-KO mice with MWCNT+ESAT-6 elicits an exacerbated granulomatous and T lymphocyte mediated response.Item Open Access 30-Day Immunotoxicity Study of PFMOAA in C57BL/6 Mice(East Carolina University, 2019-07-18) Vance, SamuelWithin the past five years, two classes of per- and polyfluoroalkyl substances (PFAS) were phased out of production in the U.S., which led to the development and production of PFAS to replace these two major classes. One family of these PFAS are perfluoro-ether carboxylic acids (PFECA), which have emerged in the public and scientific arenas due to their presence in drinking water systems across the U.S., including Wilmington, NC. Although manufacturers have touted them as having more favorable environmental and toxicological properties very little is known about the toxicity and environmental fate these emerging PFECA. One compound, perfluoro-2-methoxyacetic acid (PFMOAA), was identified as the dominant PFECA in the Cape Fear River, in concentrations as high as 35,000 ng/L. There is very little mention of PFMOAA in the publicly available scientific literature and to our knowledge, we are the first to investigate its potential for toxic effects. In this 30-day study, we orally administered 25,000, 2,500,000, or 250,000,000 ng/L of PFMOAA in water to male and female C57BL/6 mice and investigated immune and liver alterations following exposure. Mice given PFMOAA showed no signs of overt toxicity during the study and no evident changes were observed in liver mass or peroxisomal enzyme activity. While mild alterations in splenic and thymic lymphocyte sub-populations were observed in males, these results do not point to any definitive alterations in immune function. Ultimately, we concluded that the doses administered were too low to achieve an internal dose sufficient to induce changes to immune endpoints, likely due to rapid excretion of PFMOAA in mice. Further investigation into serum and organ concentrations of PFMOAA as well its effects on antibody production will be more conclusive of immunotoxic effects.Item Open Access Investigating the Role of Poxvirus Virulence Genes A35 and O1L in the Virus Life Cycle(East Carolina University, 2019-07-22) Hayes, Alexandra GPoxviruses, some of the largest viruses in existence, have a great impact on the human and animal world due to their ability to infect a broad assortment of organisms and cause significant disease. Today, poxvirus infections remain a danger to human health, as natural and potential bioterrorism threats. Vaccinia virus (VACV), the species of poxvirus used in smallpox vaccines, is the most studied poxvirus, but there is still much to learn in regards to its virulence factors and their role in the virus life cycle. Our laboratory has identified two VACV genes/proteins that play an important role in virulence, A35 and O1L, which we hypothesized were immunoregulatory, so their effects on host immune responses were assessed. We found that the A35 protein inhibits anti-viral antibody production and cytokine responses by T lymphocytes in vivo. However, there was no evidence to suggest that A35 inhibits recall antigen presentation by infected BMDC in vitro. There were also no A35 effects observed on VACV cell killing, replication, or integrin expression for bone marrow dendritic cells (BMDC), which were used as antigen presenting cells (APC). When looking at the function of O1L, we did not find an effect of O1L on anti-viral antibody production or T cell response, so the O1L effects on replication and spread, cell killing, integrin expression, cytokine production, and innate immunity were also measured. In each of these cases, the O1L deletion mutant (O1LDel) had a similar phenotype to the wild type virus. We did observe that plaques formed by the O1LDel virus appeared smaller in some cases compared to wild type plaques, which was due to reduced cell clearance in the center of the O1LDel plaques. However, the biological relevance of this finding is unclear at this time. The fact that the VACV O1L gene encodes a large protein that is conserved in mammalian tropic poxviruses with 92-100% homology supports that the gene performs an important function in the poxvirus life cycle. Our laboratory has shown that both A35 and O1L deletion viral mutants make safer vaccine alternatives against poxviruses. Understanding how poxviruses turn off immune responses will aid in our understanding of viral pathogenesis and support anti-viral drug design, improve vaccines, and may allow us to mimic poxvirus immunosuppression to control autoimmune diseases.Item Open Access THERAPEUTIC AND TOXICOLOGICAL EVALUATION OF BROAD-SPECTRUM ANTIMICROBIAL TRICLOSAN AS A CHEMOTHERAPEUTIC AGENT(East Carolina University, 2019-05-07) Alfhili, Mohammad A.Triclosan (TCS) is a phenolic antimicrobial incorporated in personal care products and medical devices. Interest in the antiproliferative properties of TCS has recently grown owing to its antilipogenic effects. Through the studies presented here, we provide an appraisal of TCS as a chemotherapeutic agent by investigating its influence on the growth and survival of lymphoma cells. We also examine the contribution of TCS to the development of anemia; a major side effect of chemotherapy with a prevalence as high as 90% in cancer patients. Finally, we identify nonionic detergents, often used as excipients in drug formulations, as potent inhibitors of TCS in vivo.Item Restricted Macrophage Regulation in the Murine Inflammatory Response(East Carolina University, 2018-08-30) Shine, Sherri MThe development of acute and chronic liver disease is a complex condition involving the dysregulation of hepatic metabolism and the accumulation of critical immune cells; specifically, the recruitment and activation of macrophages. The relationship between lipid accumulation, cytokine expression, and hepatic macrophages remains an area of further study. The purpose of these studies was to assess the role of the macrophage in the context of liver disease. Various mechanistic studies involving genetic mouse models of liver disease including alcoholic (ALD), nonalcoholic (NAFLD) and toxin-induced hepatitis were used to replicate the multiple variables associated with a hepatic immune response. Further, cellular based studies using bone marrow derived macrophages (BMDMs) and isolated Kupffer cells (KCs) were used to verify whole animal data in areas of immune cell regulation. Fatty acid binding proteins (FABPs) are becoming recognized as key regulators of both the inflammatory response and lipid metabolism. Therefore, the effect FABP1 and FABP5 deletion on the early signs of liver injury associated with ethanol exposure in mice were first investigated. These studies demonstrated that FABP1, but not FABP5, regulates hepatic lipid accumulation and inflammation in the context on ALD. On the other hand, FABP5 is highly expressed in macrophages and may play an important role in the hepatic inflammatory response after endotoxin (LPS) exposure in mice. Specifically, these findings demonstrate that loss of FABP5 promotes a more anti-inflammatory response in the macrophage. Lastly, we focused on the hypothesis that Dicer regulates the development of a unique macrophage population that facilitates the resolution of hepatic fibrosis. We found that loss of Dicer in the macrophage delays hepatic fibrosis repair. In summary, this dissertation discusses the mechanisms of innate immune cell activation and the regulation of macrophage function in relation to acute and chronic liver injury. Further, these studies demonstrate that macrophages are an integral component of the immune system which delicately regulate hepatic metabolism and inflammatory and anti-inflammatory processes in the context of liver disease.Item Open Access Identification of Biomarkers in Non-Small Cell Lung Cancer Patients Treated with PD-1 Monoclonal Antibody Immunotherapy(East Carolina University, 2018-08-22) Atwell, Druid CarlisleCancer immunotherapy works by taking a patient's existing immune system and priming it to recognize cancer cells in order for immune cells to mount an effective response to the disease. This is a less invasive means of treating cancer for the patient. However current immunotherapy does come with its own unique side effects such as auto immune disorders that manifest in the patients' treatment due to the blocking of essential immune regulatory checkpoints. In this study, patients are treated with drugs nivolumab and pembrolizumab, both of which are PD-1 (Programmed Death Receptor 1) monoclonal antibodies. These antibodies bind to PD-1 and prevent ligand interaction with PD-L1. PD-1 is a receptor expressed on the surface of activated B-cells, macrophages and T-cells. When PD-1 is activated by PD-L1 a signal propagates from the receptor to inside the cell that results in the apoptosis of the cell that expresses PD-1. The activation of PD-1 on activated T-cells ultimately results in a reduction of T-cell proliferation and IFN-[gamma] secretion. An apoptotic signal occurs through the inhibition of the cell survival signal that is propagated through the PI3K pathway. While there is knowledge on how the expression and activation of PD-1 on immune cells regulates the progression of cancer, there is a lack of evidence to suggest biomarkers in non-small cell lung cancer patients for optimizing immunotherapy. This study serves to identify biomarkers in non-small cell lung cancer patients undergoing PD-1 monoclonal antibody immunotherapy. To accomplish this, blood samples were collected from non-small cell lung cancer patients undergoing the immunotherapy treatment and the cell counts were taken. Cell types of interest include cytotoxic T-cells, helper T-cells, B-cells, and granulocytes. Cytotoxic T-cells were identified by CD8 expression, a known marker of cytotoxic T-cells. Helper T-cells were identified by CD4 expression and B-cells were identified by CD19 expression, both of which are known markers of helper T-cells and B-cells, respectively. Secondly, this study investigated the expression levels of known immune regulatory genes and how these changed over the course of the immunotherapy treatment. Known immune regulatory genes included PD-L1, PD-1, CTLA4, CD28, A2A, CD80 and CD86. The expression levels of the proton sensing family of G-protein coupled receptors (G2A, GPR4, OGR1 and TDAG8) were also investigated. Thirdly, we investigated how tumor cell expression of PD-1 and PD-L1 was altered when introduced into an acidic environment. Due to the tumor microenvironment being characteristically acidic this would provide insight on how anti PD-1 and anti PD-L1 immunotherapies could potentially be used in various cancers and may also lead to the development of potential future combination therapies. Our study shows that approximately 90% of patients exhibited an increase in cytotoxic T-cell counts with 50% of patients achieving healthy donor cytotoxic T-cell counts after receiving immunotherapy. Additionally 2 patients out of the total 16 patients achieved and sustained cytotoxic T-cell counts above that of healthy donors. There was an observable trend that indicated a possible correlation that PD-1 levels at baseline could predict patient response to the PD-1 monoclonal antibody immunotherapy. In addition to our research into the clinical aspects of PD-1 monoclonal antibody immunotherapy, we also investigated the change in expression of PD-1 and PD-L1 mRNA in several cancer cell lines. We observed that there was a variation in how cancer cells responded to acidosis. PD-1 and PD-L1 mRNA expression was shown to be regulated through several variables such as the acidity of the media, duration of exposure to acidic conditions and cancer cell type. It was also observed that there was PD-1 and PD-L1 expression in these cancer cell lines, at 5 hour and 24 hour treatment times, with a prominent level of PD-L1 mRNA expression in most of these cancer cells.Item Restricted TGF-β Signaling, Via Smad3, Mediates Notch Pathway-Induced Stemness and Epithelial to Mesenchymal Transition in Colon Cancer Cells(East Carolina University, 2017-08-21) Clark, Alexander G.Late stage colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways in CRC contribute to these phenomena, including the Notch and Transforming Growth Factor-Beta (TGF-[beta]) pathways. These pathways integrate external signals by cross-talking with one another to fine-tune cellular responses. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-[beta] signaling pathway. Therefore, we hypothesized that the TGF-[beta] pathway, via Smad3, mediates the Notch-induced stemness and EMT observed in these cells. To test our hypothesis, we used the human colorectal carcinoma cell line HCT-116 and derivative cell lines GFP-v (a control cell line transfected with a retrovirus containing the green fluorescent protein), and ICN1 (a cell line transfected with a retrovirus containing both the green fluorescent protein and constitutively active intracytoplasmic Notch1). These cells were treated with different combinations of TGF-[beta]1 (a TGF-[beta] receptor ligand), DAPT (a [gamma]-secretase inhibitor), or SIS3 (a novel Smad3 inhibitor). Western blot procedures and statistical analysis were performed to determine the cross-talk between the Notch and TGF-[beta] pathways and to assess the effects of Smad3 stimulation and inhibition on Notch and its downstream targets. The role of Smad3 on colosphere formation was also determined using the aforementioned cell lines. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression. Additionally, SIS3 effectively inhibited key stemness and EMT markers such as CD44, Slug, Snail, and Hes1. Colosphere forming ability was also considerably reduced in cells treated with SIS3. These results indicate a key role of TGF-[beta] signaling in Notch1-induced tumorigenesis, and they also suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.Item Open Access Targeting Kremen1 Downregulation with RVG-9R/siRNA Complexes in the Triple-Transgenic Mouse Model of Alzheimer’s Disease(East Carolina University, 2017-07-19) Baker, Kelly E.Alzheimer's disease (AD) is a progressive disease characterized by cognitive decline and memory loss. Memory loss observed in AD results from the loss of neurons and synapses which may be caused by the disruption of the canonical Wnt signaling pathway by Dickkopf-1 (Dkk-1). Under normal conditions, the canonical Wnt signaling pathway is responsible for normal neuronal development, synaptic plasticity, and overall normal brain function. Amyloid-[beta] (A[beta]) plaques and neurofibrillary tangles are two characteristic morphological changes observed in AD. An increased level of A[beta] has been associated with increased expression of Dkk-1, which may be linked to synaptic loss seen in AD. Kremen1 (Krm1) is a receptor for Dkk-1. Published and unpublished observations from our laboratory showed that silencing Krm1 with miR-431 can promote regenerative axon growth and prevent synaptic loss in a cell culture model. This study focused on downregulating Krm1 the triple-transgenic mouse model of AD (3xTg-AD). It was hypothesized that application of siRNA-431 in vivo would downregulate Krm1 thereby preventing synaptic loss and memory deficits in the 3xTg-AD mouse model of AD. Tail vein injections of RVG-9R/siRNA complexes and control injections were administered to 3xTg-AD mice and wild-type (WT) mice at 4, 6, or 12 months of age. Within each age cohort there were three different groups: 3xTg-AD mice injected with RVG/siRNA, 3xTg-AD mice injected with control peptide/siRNA, and WT mice injected with saline. Each group of mice was approximately half male and half female. Following the injections, the Barnes Maze was administered to each mouse in order to assess memory function. Data gathered from the Barnes Maze shows 3xTg-AD mice have a longer primary latency in the probe phase compared to WT mice. Of mice tested, fewer 3xTg-AD mice have been successful in finding the target hole during probe phase compared to WT mice. After completion of the Barnes Maze, mice were sacrificed and brains were collected for analysis. The brains were analyzed for Krm1 downregulation at the protein and mRNA levels via Western blot and qPCR, respectively. In 4 month old mice, WT mice showed the lowest levels of Krm1 protein and mRNA expression levels and 3xTg-AD CPep/siRNA treated mice showed the highest. The 4 month old 3xTg-AD RVG-9R/siRNA treated mice had Krm1 protein and mRNA expression levels that fell between the other two groups. Immunofluorescence was performed on coronal brain sections to analyze number of synapses. Six month old 3xTg-AD CPep/siRNA treated mice had significantly fewer synapses than both the WT and 3xTg-AD RVG-9R/siRNA treated groups. In conclusion, IF, qPCR, and Western blot data reveal the potential for RVG-9R/siRNA treatment to target and downregulate Kremen1 in vivo and provide protection from synaptic loss. However, further studies are need to confirm the ability of RVG-9R/siRNA treatment to downregulate Kremen1.Item Restricted Notch 3 Affects Chemoresistance in Colorectal Cancer via DNA Base Excision Repair Enzymes(East Carolina University, 2017-07-28) Khan, AzeemApproximately 1.2 million cases of colorectal cancer (CRC) arise each year, and 40-50% of CRC patients will reach metastasis. The Notch pathway is known to be dysregulated in CRC, and its relationship with DNA repair mechanisms, which contribute to drug resistance, is currently being established. Previously, we observed a decrease in DNA base excision repair (BER) enzymes and drug resistance upon Notch 1 targeting. We have also observed that Notch 1 signaling is associated with promoting cancer stemness and epithelial to mesenchymal transition in CRC via upregulation of the Notch 3 receptor. Thus, we hypothesized that targeting Notch 3 will increase drug sensitivity in CRC via signaling effects on proteins associated with the DNA BER mechanism. Methods: In order to assess our hypothesis, the colon cancer cell line HCT 116 was transduced with a small hairpin messenger RNA construct that effectively knocked down the Notch 3 receptor, creating the Sh-N3 cell line. Culturing and Western blot analyses were conducted using standard methodology. Drug resistance was analyzed by treating cells with cisplatin or cytarabine, potent DNA damaging agents, and cytotoxicity was assessed. Microscopy was used to confirm effects of the DNA damaging agents. Proteins from the Notch 3 receptor targeted cell line treated with the DNA damaging agents were also studied. Results: Notch 3 targeted (Sh-N3) cells resulted in 1.5-fold lower plating efficiency compared to their counterpart controls (p<0.01). Western blot analysis showed that Notch 3 targeting led to a decrease in poly (ADP-ribose) polymerase (PARP1) expression by 34% in comparison to the parental cell line control (p<0.05), while apurinic/apyrmidinic endonuclease (APE1) expression was decreased by 47% (p<0.05). Sh-N3 cells treated with 20 [micro]g/mL of cisplatin for 48 hours showed a 2-fold increase in cell death compared to the controls (p<0.001). Additionally, cytotoxicity in Notch 3 null cells treated with 0.64 [micro]g/mL of cytarabine at 48 hours displayed a 1.7-fold increase compared to the controls (p<0.001). Microscopic observations confirmed these cytotoxicity results. Conclusions: This study further reinforces the importance of Notch signaling in drug resistance, and highlights the potential use of Notch 3 inhibition in conjunction with DNA BER protein inhibitors to effectively target chemoresistant CRC cells.Item Open Access Structural and functional analysis of the Vaccinia virus O1 virulence protein(East Carolina University, 2017-07-26) Weeks, Anastasia C.Poxviruses are double-stranded DNA viruses capable of causing disfiguring and deadly disease in a wide range of hosts, from insects to mammals. Orthopoxviruses (OPXV) encode many proteins that are not essential for viral replication, but are responsible for vast differences in pathogenesis. Of the>200 proteins in the prototypical OPXV vaccinia virus (VACV), many remain functionally cryptic. The objective of these studies was to understand how the VACV O1 protein functions by investigating cell-specific effects that may contribute to virulence. The O1L gene is expressed early as the O1 protein, a 78 kDa protein that lacked N-linked glycosylation. These data are the first to demonstrate the reduced ability of an O1 deletion mutant ([delta]O1) to induce cell migration compared to the parental VACV Western Reserve strain (VACV-WR). [delta]O1-infected cell monolayers also exhibited reduced plaque diameter and clearance in plaque foci. These observations indicated that O1 is a significant contributor to VACV cytopathic effects (CPE) in vitro, in agreement with published reports. The results reported herein are the first to describe an altered immunological response with [delta]O1, as levels of anti-VACV immunoglobulin significantly increased with [delta]O1 infection at a time point (seven days post-infection) when VACV-WR induced VACV-specific antibody levels were comparable to sera from mock-infected mice. [delta]O1 was more immunogenic in an ex vivo antigen presentation assay, although mitogen-induced CD4+ T cell activation during [delta]O1 infection was equivalent to VACV-WR infection. Surprisingly, of all the immune cell types tested, [delta]O1 significantly differed from VACV-WR infection in the metabolic readout of only one cell type - RAW 264.7 macrophages. VACV-WR infected RAW 264.7 macrophages were more metabolically active than [delta]O1-infected cells at higher infectious doses, which may be indicative of a specialized niche for O1 function. Taken together, these data may provide clues into the mechanism of O1 virulence.