Biomedical Sciences

Permanent URI for this collectionhttp://hdl.handle.net/10342/3934

Browse

Now showing 1 - 20 of 44

Embargo
The Effects of the Proton-Sensing G-Protein Coupled Receptors GPR4 and GPR68 on B16F10 Mouse Melanoma Growth and Responses to Immune Checkpoint Inhibitors.
(East Carolina University, May 2025) Swyers, Madison Marie
This research delves into the study of proton-sensing G-protein coupled receptors (GPCRs) GPR4 and GPR68 and their relation to the modulation of the tumor microenvironment in metastatic melanoma. Melanoma especially at a higher stage often are harder to treat and have a greater likelihood of recurrence. The main treatment for early-stage melanomas is surgery. Though due to melanomas being an immunogenic tumor, immune checkpoint inhibitors (ICI) and other forms of immunotherapies have been approved for use in treatment of this disease. However, with ICI treatments they are not as effective as they could be, and some patients are removed from treatment due to immune related adverse events (IRAEs). Our aims were three-fold in terms of these GPCRs and tumor treatment. First, we aimed to assess how the absence of these GPCRs in a host body would affect tumor growth and modulate the tumor microenvironment. GPR4 and GPR68 are linked to both inflammatory diseases and cancer, and have been shown to reduce tumor growth when inhibited or knocked-out in a host. With GPR4 it plays a key role in the expression of several key adhesion molecules and chemokines on vascular endothelial cells which are key for immune cell migration and infiltration into the tumor and other tissues. GPR4 is also linked to the promotion of angiogenesis. GPR68 plays a role in immune cell suppression. So when there is a lack of these receptors in the host the B16F10 melanoma lacks key communication targets to stimulate pro-tumoral responses. We observed a significant reduction in tumor growth in the GPR4 KO/GPR68 KO mice alongside a significance both in an increase in CD8+ T-Lymphocyte infiltration into the TME and a reduction in blood vessels in the TME. Next with the ICI treatments we investigated how the tumor growth and IRAEs were affected in the GPR4 knock-out (KO) mice when treated with ICI therapy as compared to wild-type (WT) mice. There was no significant reduction in the efficacy of the ICI treatment on inhibiting tumor growth in the GPR4 KO mice as compared to WT mice. In the GPR4 KO mice there was a reduction in immune cell infiltration into the normal tissues of the mice. Based on these findings, though more work still needs to be done, we can see that there are many possibilities for future antagonists targeting both GPR4 and GPR68, alongside also GPR4 antagonists when paired with ICI treatment.
Embargo
The Role of Response Regulators in Gram-Negative Bacterial Resistance
(East Carolina University, May 2025) Jaimes, Felicia E.
Since their beginning, antibiotics have been hailed as a miracle of modern medicine. And while their discovery brought about exciting advances across a multitude of industries, the ubiquity of their use has paved the path leading towards the current resistance crisis and the dawn of a post-antibiotic era. As resistant bacteria have emerged steadily over time, updated methods of treating infections have developed. From double treatments using two antibiotics or recycling older antibiotics, these changes have come about in hopes to delay resistance and prolong the lifespan of the antibiotics currently in use. Included in the older classes of antibiotics is polymyxins, their usage gone to wayside due to their innate nephro and neuro-toxic characteristics. The use of polymyxins has now been reconsidered to overcome highly resistant pathogens typically found in healthcare settings. And while the side effects of polymyxin treatment can be deleterious it is a more preferable outcome than succumbing to a fatal infection. Nosocomial infection have become more and more prevalent, threatening patients within ICUs and under long term care. Some of the major contributors of the most concerning nosocomial infections are the E.S.K.A.P.E. pathogens, these are (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Named for their ability to escape treatment, they are notorious for causing dangerous infections and rapidly developing resistance characteristics. Globally recognized institutions such as the World Health Organization have declared a state of emergency and have issued a call to action to academic institutions, pharmaceutical developers, and government agencies to come together to prevent the reality of a world overrun with untreatable infections. In response, there has been extensive work done to understand the variety and nuance of resistance mechanisms. Additionally, strides have been made towards newer methods of ameliorating resistance through the use of small molecule antibiotic adjuvants. Adjuvants work in tandem with drugs to improve their efficacy. The following body of work explores the mechanisms and application of two such adjuvants, 2-aminoimidazole compounds and several salicylanilide kinase inhibitors. Both of these compounds target separate components of systems found in most bacteria and are responsible for regulating the expression of resistance inducing genes. Two-component systems (TCSs) are present in most bacteria, playing a crucial role in how they sense and respond to environmental signals. TCSs have been an attractive drug target since their roles in a wide variety of cellular processes, such as nutrient uptake, motility and virulence, have been elucidated. Conveniently they appear to have no mammalian homolog, making then an ideal candidate for intervention. As their name would suggest, TCSs have two distinct components, a membrane-bound sensor kinase and a response regulator that is responsible for directly binding DNA and altering expression levels. Working as a unit, the histidine kinase responds to environmental signals by autophosphorylating and transferring the phosphoryl group to the response regulator to activate its regulatory function. While not every TCS works in this way, this is a typical example and is the most common mechanism. The TCS responsible for polymyxin resistance in species such as K. pneumoniae, A. baumannii, E. coli and numerous others is the PmrAB system. The histidine kinase, PmrB, and its cognate response regulator, PmrA, have been implicated as the mechanism responsible for inducing changes to the outer membrane of the aforementioned bacteria, these changes alter the normal charge of the outer membrane. Polymyxins are cationic and in susceptible populations of bacteria have an affinity for the negatively charged outer membrane. However, due to the PmrAB system resistant strains are able to decorate lipid-A molecules located on the outer membrane with functional groups that change the charge of the membrane as whole. The cross talk between these two molecules is subtle but complex. We have found through investigations of point mutations in highly resistant strains of A. baumannii that a single amino acid substitution can alter this signaling cascade in profound ways to increase the bacteria’s ability to survive treatments. These alterations were shown to affect PmrA’s ability to accept the phosphoryl group from PmrB in one of two ways, either by altering regions necessary for kinase recognition or perturbing the binding pocket. And while we originally hypothesized the point mutations would exert a noticeable effect of DNA-binding affinity we found that the mutants were slower to activate, lessening the energy exerted by the cell and prolonging the lifetime of the activation signal. The goal of developing small molecule adjuvants to target the PmrAB system is to interrupt the system on both sides. IMD-0354 has shown an ability to bind and inhibit the proper functioning of sensor kinase, PmrB, while 2-aminoimidazole compounds bind and inhibit the response regulator in a similar fashion. Both compounds have a minimum inhibitory concentration (MIC) lowering effect, however because polymyxins are highly toxic our goal evolved into bringing down the necessary concentration of polymyxin even lower. We performed minimum inhibitory concentration experiments to test if these two compounds can be used congruently with colistin to break the resistance mechanisms and make bacteria susceptible to treatment once again. Our endeavor was ultimately successful, bringing down the colistin MIC in resistant A. baumannii strain AB4106 to 10 µg/mL and under, a 200-fold decrease. Our goal is to apply this method to other TCSs and displace other organisms from their status as resistant pathogens.
Embargo
COUNTERING BACTERIAL RESISTANCE VIA INHIBITION OF TWO-COMPONENT SYSTEMS
(East Carolina University, May 2025) Hondros, Alexander Davis
The emergence of bacterial resistance is a current and rapidly growing threat to human health worldwide. Each year, pathogenic bacteria exhibit new or increased resistance to a limited arsenal of existing antibiotics at a rate that greatly outpaces new antimicrobial discovery. In several species the reality of pan-resistant bacterial infections with no means of treatment has already arrived. To counter this, the production of creative, unconventional therapeutics with specific concern for eliminating or reducing resistance development is imperative. To achieve this goal, the development and application of antibiotic adjuvants - a therapeutic that enhances the effect of another treatment - offers a promising solution. By nature, successful application of an adjuvant would allow for restored efficacy of existing antimicrobials and offset the massive time and monetary investment required for developing new antibiotics. Two-component systems (TCSs) serve as ubiquitous communication modules that enable bacteria to detect and respond to various environmental stimuli by regulating cellular processes such as growth, viability, and most notably, antimicrobial resistance. Classical TCSs consist of two proteins: an initial membrane-bound sensor histidine kinase and a DNA-binding response regulator that induces the appropriate response within the cell, namely the upregulation of genes that elicit bacterial defense mechanisms. TCSs are undeniably one of the most effective therapeutic targets against bacterial resistance. Given their function, the inhibition of the TCS would remove the bacteria’s ability to sense and respond to changes in its environment, i.e. the presence of antimicrobials. In laymen’s terms, these treatments effectively ‘blind’ the bacteria to the threat of the therapeutic/antibiotic, removing the ability to activate resistance mechanisms. Further, due to these therapeutics ability to render bacteria oblivious to the threat of the antibiotics, there is no increase in selective pressure. As a result, the bacteria do not develop resistance to the adjuvant compounds or increased resistance to the antibiotic. As the initiators of the signaling pathways that elicit resistance, the histidine kinases present as the ideal target within the TCS for developing antibiotic adjuvant drugs. Despite this, due to the membrane-bound nature of histidine kinases, in vitro investigations for TCSs have been predominantly limited to response regulators. This includes the development of targeted therapeutics. In this work, we counter this limitation by producing recombinant truncation mutants of the cytosolic portion of HKs that retain ATP-binding, autophosphorylation, and phosphotransfer functions. This method was initially used in A. baumanii’s PmrAB system to make a truncation of PmrB (Polymyxin resistance protein B), dubbed PmrBc. The PmrAB system is the main mechanism of resistance to colistin (polymyxin E) in Acinetobacter baumannii. This truncation mutant allowed for in vitro evaluation of potential salicylanilide histidine kinase inhibitors, previously shown to eliminate resistance in vivo. Experimental findings from kinase assays, limited proteolysis, and hydrogen-deuterium exchange (HDX) mass spectrometry enabled us to determine these compounds’ mechanism of action as well as the likely binding site on the ATP-lid of the histidine kinase’s catalytic domain. Following successful production of PmrBc, other functional cytosolic truncations were produced across multiple species for several resistance mechanisms, including Klebsiella pneumonia. While K. pneumonia also utilizes the PmrAB system to produce colistin resistance, it contains a second mechanism, PhoPQ, that is capable of upregulating resistance genes outside those of the PmrAB system. With the goal of addressing both systems, truncations of K. pneumonia PhoQ and PmrB were produced for screening and evaluation of the salicylanilide inhibitors. Notably, these results revealed variations in specificity/selectivity of the compounds between kinases, suggesting solution of structural data for the kinases will allow for structure-activity-relationship (SAR) based drug design and enable targeting of specific HKs. Beyond applications for drug development, truncation mutants also enabled expanded in vitro investigation of TCSs. The cytosolic construct of A. baumannii PmrB, was utilized to assess the effect of resistance inducing point mutations in its cognate response regulator. Results of phosphorylation assays identified discrepancies in the phosphorylation rates between point mutants, distinguishing altered phosphotransfer activity as the means of increased Polymyxin resistance within these clinical isolates. Finally, previous efforts have also been successful in targeting the response regulators within the TCS. A class of compounds called 2-aminoimidizoles (2-AIs), developed from a natural product, have shown the ability to target response regulators. Early work gave clues to their mechanism of action and binding site but were never confirmed. Our recent assessment of the effects of two novel 2-AIs on the PhoPQ system support this previously proposed mechanism of action and indicate how these compounds affect transcriptional regulation. In whole, the work presented here cohesively establishes the efficacy, binding site, and mechanism of action for 2-AI compounds in inhibiting response regulators within the PhoPQ system. In summation, this work utilizes a range of biochemical techniques to evaluate compounds aimed at inhibiting the histidine kinase and response regulator of TCSs to ameliorate antibiotic resistance. We also develop methods for producing functional cytosolic truncation mutants that counter current limitations in research of two-component systems. These works offer promising avenues in countering antibiotic resistance that simultaneously limit the potential for bacteria to develop resistance to the adjuvant compounds or increased resistance to antibiotics.
Embargo
Phytochemicals Confer Neuroprotection in Models of Contaminant-induced Neurodevelopmental Disorders
(East Carolina University, May 2025) Bessetti, Riley N.
Phytochemicals that target antioxidant and anti-inflammatory pathways have gained attention as promising interventions for mitigating oxidative stress and neuroinflammation in neurodevelopmental and neurodegenerative disorders. Many neurodevelopmental disorders, such as autism spectrum disorder (ASD), are linked to oxidative stress-driven disruptions in neural development. Among the many contributing factors, fetal exposure to environmental insults including maternal infection, pollution, and pharmaceuticals increase oxidative stress and the likelihood of ASD development. The response to cellular oxidative stress is regulated in part by the transcription factor Nuclear Factor Erythroid 2-related Factor 2 (NRF2), which promotes phase II enzyme and antioxidant gene expression. The work described in this dissertation investigates the neuroprotective potential of two phytochemicals, sulforaphane and cannabidiol (CBD), focusing on their ability to activate the NRF2 pathway. Using human induced pluripotent stem cell (iPSC) technology, we modeled the susceptibility of early brain development to the toxicant valproic acid (VPA) in vitro with parallel assessment in primary mouse cultures. Sulforaphane robustly activates NRF2-mediated antioxidant responses, mitigating VPA-induced oxidative stress and synaptic alterations. In contrast, CBD recruits NRF2 to the nucleus without increasing transcription of antioxidant genes, but may promote neuroprotection through the suppression of neuroinflammatory pathways. Our continuing work, influenced by the growing recognition of the role of neuroinflammation in neurodevelopmental disorders, explores how phytochemicals modulate inflammatory responses. Ultimately, these findings contribute to our understanding of how NRF2 activators and the regulation of oxidative stress and inflammation can be harnessed as potential preventative strategies to combat environmental exposures that increase the risk of neurodevelopmental disorders.
Embargo
The Effects of Increased Oligomannose N-Glycan Expression on Neuroblastoma Progression and Epidermal Growth Factor Receptor Signaling
(East Carolina University, December 2024) Burch, Adam Patrick
N-glycosylation is an essential post-translational modification with known roles in cancer, including the pediatric cancer neuroblastoma (NB). The folding, stability, regulation, and trafficking of proteins are all dependent on proper N-glycosylation. There are three general classes of N-glycans: oligomannose, hybrid, and complex ranging from the least processed to the most respectively. Changes in metabolism, endoplasmic reticulum (ER) stress, and other cell signaling events can and do exert influence on the N-glycosylation processing pathway to ensure all glycoproteins are processed to fit the current needs of the cell. Pathological diseases are known to exert influence on the types of N-glycans produced. Regarding cancer biology, β1,6 complex N-glycans have often been attributed to the malignant transformation of cells. This work seeks to define the types of N-glycans (oligomannose, hybrid, or complex) that contribute to the progression of neuroblastoma. The link between N-glycosylation and the malignant transformation of cells has often been centered around more processed N-glycans (i.e., complex N-glycans). However, in the body of this work, we find that oligomannose N-glycans are responsible for aggressive neuroblastoma phenotypes, mainly increased invasiveness. By CRISPR/Cas9 knockout of MGAT1, MGAT2, or MGAT3 in human and rat neuroblastoma cell lines, we were able to generate cells with varying levels of N-glycan processing of proteins. MGAT1 encodes N-acetylglucosaminyltransferase-I (GnT-I), a critical glycosyltransferase that converts oligomannose N-glycans to hybrid N-glycans. By loss of GnT-I, cells have reduced synthesis of hybrid N-glycans and therefore also reduced production of complex N-glycans, resulting in cells with increases in oligomannose N-glycan content. MGAT2 encodes GnT-II, which converts hybrid N-glycans to complex N-glycans. Loss of GnT-II prevents the synthesis of complex N-glycans resulting in cells enriched with hybrid N-glycan structures. MGAT3 encodes GnT-III, which produces bisecting hybrid and complex N-glycans and is responsible for terminating N-glycan processing. By knockout of GnT-III, hybrid and complex N-glycans should be more susceptible to additional modifications (e.g., fucosylation, sialylation, galactosylation, etc.). With these various cell lines, we were able to explore how the reduced amounts of various N-glycan modifications influenced neuroblastoma growth, invasion, and cell-cell adhesion; ultimately revealing that increased expression of oligomannose N-glycans leads to neuroblastoma cells that are highly invasive but have decreased proliferation. Furthermore, we examined unmodified human neuroblastoma cells derived from a SK-N-BE(2) cells: BE(2)-C and BE(2)-M17. We found that BE(2)-M17 cells expressed more oligomannose N-glycans and were significantly more invasive but less proliferative relative to BE(2)-C cells, furthering the support that oligomannose N-glycans drive neuroblastoma invasiveness. These studies were instrumental in designating oligomannose N-glycans as perpetrators of aggressive neuroblastoma phenotypes. A primary focus of this work was to better understand how oligomannose N-glycans impacted neuroblastoma progression. Neuroblastoma cells expressing high amounts of oligomannose N-glycans exhibit increased invasiveness but decreased proliferation leading us to examine intracellular signaling, specifically the oncogenic receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Knockout of MGAT1 is a powerful approach to enrich all N-glycosylated proteins of cells with oligomannose N-glycans, including EGFR. This investigation builds upon the towering literature surrounding N-glycosylation and EGFR, in particular how N-glycosylation modification of EGFR impacts the receptor’s ability to initiate downstream signaling. The 12-13 N-glycans of EGFR have been characterized as being mostly complex N-glycans and have been shown to be essential to both EGFR’s function and regulation; however, very few have attempted to examine how EGFR functions when decorated primarily with oligomannose N-glycans. Here we show BE(2)-C(-MGAT1) cells produce oligomannosylated EGFR (EGFR decorated with oligomannose N-glycans). Further characterization of oligomannosylated EGFR revealed that ligand independent phosphorylation and EGF stimulated phosphorylation are significantly increased in BE(2)-C(-MGAT1) cells. Furthermore, when observing proliferation in response to EGF stimulation in 3D conditions, BE(2)-C cells do not have a major proliferate response whereas BE(2)-C(-MGAT1) cells proliferate robustly likely due to the heightened EGFR phosphorylation of oligomannosylated EGFR. The increased autophosphorylation and sensitization of BE(2)-C to EGF stimulation when expressing oligomannosylated EGFR, due to loss of MGAT1, is novel and further details how changes in N-glycosylation, due to a pathogenic state, could alter a cellular phenotype without genetic mutation in EGFR. Here using both rat and human neuroblastoma cells with selective defects in the N-glycan processing of proteins we further define the role of N-glycosylation in the progression of neuroblastoma. By examining various N-glycosylation mutations as well as unmodified neuroblastoma cell lines we were able to establish that oligomannose N-glycans contribute to neuroblastoma progression through heightened invasiveness. Utilizing newly generated BE(2)-C(-MGAT1) cells we further explored the role of oligomannose N-glycans by examining EGFR signaling in BE(2)-C and BE(2)-C(-MGAT1) cells. Neuroblastoma cells bearing oligomannosylated EGFR respond intensely to EGF stimulation, leading to heightened autophosphorylation and EGF stimulated proliferation. Ultimately, we conclude that neuroblastoma cells with increased oligomannose N-glycan content is consistent with a highly invasive cancer that is readily able to undergo EGF stimulated proliferation.
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 Adler
Chronic 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.
Open Access
Kinin B1 Receptor Mediates Bidirectional Interaction between Neuroinflammation and Oxidative Stress
(East Carolina University, 2023-05-02) Theobald, Drew
Hypertension 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.
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, Youngyong
Conserved 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.
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, Sydney
Restless 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.
Restricted
The Circadian Rhythm and its Role in the Dynamic Dopamine Neuron Phenotype
(East Carolina University, 2021-05-04) Barker, Samantha
The 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.
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.
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, Ryan
The 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.
Restricted
Matrix Metalloproteinase 12 is Critical for Granuloma Formation in the Murine Model of Granulomatous disease
(East Carolina University, 2019-08-16) Neequaye, Nicole N
Matrix 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.
Open Access
DEVELOPMENT OF SMALL-MODELCULE INHIGITORS OF THE INTIATING PROTEASES, C1r AND C1s, OF THE CLASSICAL COMPLEMENT PATHWAY
(2019-12) Rohlik, Denise
Complement 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.
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 disclosed
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 L
Sarcoidosis 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.
Open Access
30-Day Immunotoxicity Study of PFMOAA in C57BL/6 Mice
(East Carolina University, 2019-07-18) Vance, Samuel
Within 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.
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 G
Poxviruses, 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.
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.
Restricted
Macrophage Regulation in the Murine Inflammatory Response
(East Carolina University, 2018-08-30) Shine, Sherri M
The 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.

Browse

Recent Submissions