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Item 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.Item Embargo COUNTERING BACTERIAL RESISTANCE VIA INHIBITION OF TWO-COMPONENT SYSTEMS(East Carolina University, May 2025) Hondros, Alexander DavisThe 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.Item Embargo A Grounded Theory Study of Birthing People’s Decision-Making on Labor Pain Management(East Carolina University, May 2025) Guard, KaitlinUsing a Constructivist Grounded Theory (CGT) approach, this study addressed the following research questions: 1) What sociocultural factors contribute to decision-making about labor pain management among nulliparous birthing people? and 2) How does the pain management decision develop or change from antepartum to delivery among nulliparous birthing people? Data to address the research questions was collected through semi-structured interviews. Seventeen nulliparous participants, at least 37 weeks pregnant, partook in initial interviews following their antepartum outpatient visit. Nine participants were retained for follow-up interviews that took place either at the hospital or over the phone between one day and four months postpartum. The overarching theme, Navigating Decision-making amid Uncertainty, was supported by two theoretical categories: 1) Shaping decisions, which involved receiving advice from social networks, engaging with social media, and awaiting provider-led education, and 2) Shifting decisions, which involved responding to labor realities and considering support system guidance. Of the follow-up participants, seven initially intended to avoid an epidural. However, only two of the seven did so. This shift was often a result of unanticipated pain intensity exacerbated by medical interventions such as the foley balloon and IV Pitocin. Policy and practice changes that promote person-centered care and informed decision-making should be considered. These findings offer a novel perspective on decision-making among nulliparous birthing people. They can serve as a step toward evaluating the potential for an intervention to promote informed decision-making about pain management during labor.Item 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.Item Embargo EVALUATING VELOPHARYNGEAL FUNCTION DURING PHONATION IN CHILDREN UTILIZING STATIC MRI(East Carolina University, May 2025) Snodgrass, Taylor DanielleMany children born with cleft palate go on to present with velopharyngeal insufficiency (VPI) characterized by speech that is hypernasal and/or has nasal air emissions. Magnetic resonance imaging (MRI) is the only imaging technique that allows for direct visualization of velopharyngeal musculature to plan for surgical correction of VPI. Normative data for MRI are available at rest and these data indicate age, race, and sex are important variables that may need to be considered when determining what is typical for velopharyngeal anatomy. A current limitation of these normative data are that they do not include speech production. This study used structural MRI at rest, during sustained phonation of /i/, and sustained production of /s/ to investigate differences in velopharyngeal anatomy between speech production and rest in five and six-year-old children who were either Black or White. The study had two aims. Aim I determined if racial differences were present in velar and pharyngeal structures and resulting physiology. Aim II determined if racial differences were present in the LVP muscle (LVP), which is the most important muscle for velopharyngeal closure, Aim I determined there are significant differences in velar structure size during speech production between Black and White Children, with Black children presenting with thicker velums and longer effective velums. These anatomical differences result in Black children needing less velar stretch than White children to achieve velopharyngeal closure. Aim II determined there are no differences in LVP length and LVP shortening between Black and White children.Item Embargo The Relationally-Centered Integrated Behavioral Health Model: Exploring Holistic Patient Health Outcomes(East Carolina University, May 2025) Hellstern, RylanIntegrated Primary Care (IPC) is a system that relies on the biopsychosocial (BPS) framework to provide holistic care to patients by organizing collaborative teams of biomedical and psychosocial health services. Due to the diversity of patient needs, many IPC models have been created with varying methods of organizational structure and foci. This dissertation explores the physical and mental health outcomes of the Relationally-Centered Integrated Behavioral Health Model (RCIBH), an IPC model that strives to provide all patients and their families with behavioral health services, regardless of the presenting problem. The following document is divided into six manuscripts: (a) an introduction to the dissertation and a narrative history of IPC models from around the globe, (b) a systematic review of the holistic outcomes of IPC models, (c) an introduction to the RCIBH model and its comparison to other prominent models, (d) a chapter outlining the methodology for the original research study, (e) an empirical research study utilizing machine learning techniques to explore the factors contributing to biological and psychological outcomes of the RCIBH model, and (e) a perspective piece calling for Medical Family Therapists (MedFTs) to engage with Electronic Health Record (EHR) data and further establish their role in the healthcare system.Item Embargo Kininase I Mediated Signaling Evidenced in Hypertension and Target Organ Damage(East Carolina University, May 2025) White, Acacia Mahogany NecoleCarboxypeptidase N (CPN) and Carboxypeptidase M (CPM) are isoforms of kininase I, with CPN restricted to the plasma and CPM located on the plasma membrane. Kininase I is a critical pleiotropic regulator of inflammation, as it cleaves several inflammatory mediators such as anaphylatoxins, creatine kinase MM, stromal cell-derived factor-1alpha and kinins. Kinins are important vasoactive peptides which activate the kinin B2 receptor (B2R), and cause vasodilation. However, kininase I converts these kinins into agonists for the kinin B1 receptor (B1R), a receptor associated with vasoconstriction and hypertension. However, the role of kininase I in hypertension has not been identified. Male and female carboxypeptidase N1 (Cpn1) whole-body gene knockout (CPNKO) mice were utilized to investigate the role of CPN in the development of angiotensin II (Ang II)-induced hypertension and target organ damage. CPNKO and wild-type (WT) mice were implanted with radiotelemetry probes to measure blood pressure, and later with mini osmotic pumps to infuse mice with either saline or angiotensin II (Ang II; 600/ng/kg/min -1) for 4 weeks. In a separate experiment, we used tail cuff plethysmography to measure blood pressure in WT male and female mice infused with either saline or a combined dosage of the non-selective kininase I pharmacological inhibitor (Mergetpa; 10 mg/day) and Ang II (600/ng/kg/min -1) over 4 weeks. Aortic segments isolated from these mice were subjected to wire myography, to assess the effect of CPN gene deletion on vascular function. The activity and expression of kininase I and B1R were analyzed using various protein detection techniques in the brain, heart, and aorta. Proximity ligation assays were utilized to identify alterations in protein-protein interactions between B1R and other protein partners, such as CPM and aminopeptidase N (CD13), in the hypothalamic paraventricular nucleus (PVN) of WT and CPNKO mice. CPN activity assays revealed that overstimulation with Ang II in WT mice promoted CPN activity in plasma. Interestingly, Ang II infusions did not cause a significant modulation in CPM expression specifically in the PVN, heart, and aorta of CPNKO mice, compared to saline treated CPNKO and WT mice. Alternatively, CPM expression was robustly increased following Ang II infusions in WT mouse PVN, heart and aortic tissues. Mean arterial pressure (MAP), measured using radiotelemetry, was not significantly different in saline treated CPNKO and WT mice in both sexes. However, MAP was significantly elevated in WT mice infused with Ang II, but this effect was attenuated in Ang II infused CPNKO mice. Furthermore, the expression of B1R in the brain, heart and aorta was significantly enhanced by Ang II in WT mice compared to CPNKO counterparts. Based on the extent of hypertrophy and picrosirius red staining observed in Ang II infused WT and CPNKO mice, the genetic deletion of CPN protects against cardiac hypertrophy and fibrosis caused by Ang II in both sexes. Interestingly, markers associated with inflammation (IL-1β, IL-6, MCP-1/CCL2, and TNF) and oxidative stress (superoxide, 3-nitrotyrosine) were also found elevated in the heart, aorta, and PVN of Ang II infused WT mice, and these outcomes were mitigated in Ang II infused CPNKO counterparts. Furthermore, the expression CD13 in the PVN was enhanced following Ang II infusion in WT mice, while CPNKO exhibited significantly lower levels of CD13 expression in the PVN. Astonishingly, proximity ligation assays revealed that protein-protein interactions between both CPM and B1R, as well as CD13 and B1R were heightened in WT mice infused with Ang II but diminished in CPN deficient mice. Mergetpa, the non-selective kininase I inhibitor, was associated with a reduction in MAP and the expression of CPM and B1R in the PVN of WT mice, compared to WT mice infused with Ang II alone. Lastly, vascular function was assessed using wire myography revealed that male Ang II infused CPNKO and Mergetpa treated mice have preserved endothelial dependent relaxation mechanisms, compared to WT mice overstimulated with Ang II. Based on our results, kininase I blockade prevented Ang II induced hypertension and targeted end-organ damage by mitigating B1R activation. B1R mediates inflammation and oxidation and CPM/B1R or CD13/B1R protein-protein interactions may promote Ang II-induced hypertension. Specifically, the pro-inflammatory effects of kininase I initiate B1R activation, leading to the generation of reactive oxygen species (ROS) and subsequent immune cell infiltration/activation and endothelial dysfunction. Alternatively, we suggest that B1R activation mediated by CD13 signaling may contribute to Ang II-induced inflammation and fibrosis, to a lesser extent. Within the context of this study, we did not identify any clear potential sex differences associated with kininase I blockade, in WT and CPNKO mice. We have evidenced for the first time that kininase I mediated B1R activation is correlated with pathogenesis of Ang II and may serve as a potential therapeutic target for the treatment of an overactive RAAS in hypertension.Item Embargo The Impact of Virtual Simulation on Clinical Judgment Development in Undergraduate Nursing Students: A Pilot Study(East Carolina University, May 2025) Cherry, Brittany BakerBackground: Healthcare today is more complex than ever, with patients arriving at hospitals severely ill, often with multiple comorbidities. As patient care acuity rises, so does the need for nurses who can make safe and effective clinical judgments while ensuring quality care. Clinical judgment involves recognizing important aspects of a patient's condition, interpreting them, and responding appropriately. For years, clinical judgment and decision-making have been critical in prelicensure education, and recent research has focused on measuring these skills with psychometric precision using case studies and innovative item types on the licensure exam determining whether new graduate nurses are prepared for the complexity of patient care. Method: This study utilized David Kolb’s Experiential Learning Theory and Christine Tanner’s Clinical Judgment Model as the theoretical framework, emphasizing the development of thought processes that enhance learning. A quasi-experimental quantitative research design was employed to assess whether virtual simulation with instructor feedback improved clinical judgment among undergraduate nursing students. A pre- and post-case study assessment using Next Generation NCLEX (NGN) questions, based on the Modified Lasater Clinical Judgment Rubric, was developed to measure clinical judgment before and after virtual simulation. The case study results were graded, and clinical judgment development was evaluated using the Modified Lasater Clinical Judgment Rubric by Christina Zamaripa. Results: The results indicated that the clinical judgment scores on the pre-case study assessment were higher than the post-case study assessment scores, following the virtual simulation with instructor feedback. Despite the decrease in scores, on average, the level of clinical judgment remained the same at the developing. Regarding individual components, skillful response within the responding phase showed significant improvement from the pre- to post-case study assessment score following the virtual simulation, however, the moderate negative effect size indicates that the change was not very large. Conclusion: Findings from this study indicate that while students reported positive perceptions and felt the virtual simulation enhanced their learning, clinical judgment scores declined post-intervention. Despite student engagement, the simulation did not lead to measurable improvements, likely due to factors such as class length, content complexity, and gaps in foundational knowledge. When examining individual components and scoring, students showed some variation in their clinical judgment development. For certain components, their scores declined, while in others, their clinical judgment levels remained unchanged on average. For responding, both the scores and clinical judgment level declined for confident tone. As for well-planned intervention and flexibility, although their scores declined, students remained at the developing level of clinical judgment. When it came to clear communication, the score improved, but their clinical judgment level remained at developing. As for skillful response, the score improved significantly, yet the clinical judgment level stayed at developing. In the reflecting phase, both evaluation/self-analysis and commitment to improvement scores declined, but the clinical judgment level remained at developing. During the noticing phase, scores for focused observation declined, but the level remained unchanged at developing. For recognizing deviations from expected patterns and information seeking, there was a significant decline in both the scores and clinical judgment level. Lastly, in the interpreting phase, there was a notable decline in both scores and clinical judgment level for prioritizing data and making sense of data. These results suggest that the success of virtual simulation in developing clinical judgment is influenced by the students' existing knowledge base. Several factors highlighted in student feedback may have contributed to the decline in post-assessment scores. Despite its limitations, this study offers an innovative teaching approach and a valuable tool for assessing students' knowledge and clinical judgment skills.Item Embargo Assessing association between PFAS exposure, social determinants of health, and risk outcomes in adolescents and adults in the United States(East Carolina University, May 2025) Sharpe, BrendaPer- and poly-fluoroalkyl substances (PFAS), aka forever chemicals, are known for their eco-persistence, resistance to degradation, and longer half-life. Emerging studies have linked PFAS exposure to various adverse health effects, including cancer, liver damage, immune system disruption, reproductive issues, and endocrine disruption. By evaluating PFAS blood serum concentrations (n=4,444) using complex logistic regression and the USEPA deterministic risk assessment framework, this research aimed at elucidating the role of social determinants on PFAS exposure and estimate potential health risks among adolescents and adults from drinking contaminated water. Males showed higher odds of detecting pefluorodecanoic acid (PFDA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and branched PFOA than females. PFDA and 2-(N-Methyl-perfluorooctane sulfonamido) acetic acid (Me-PFOSA-AcOH) and Perfluoroundecanoic acid (PFUA) detection in blood was significantly associated with adolescents (12-15 and 16-20 years) and adults (21-40 and 41–60 years). Adults aged 21-40 years had 33% lower odds of detecting PFNA in blood compared to adults aged 61 years and older. Detection of PFAS in blood was chemical-specific for Race/Ethnicity. Non-Hispanic Asians had significantly higher odds of blood PFDA (p=.007, OR=2.39) and PFUA (p<.001, OR=2.482) compared to Other Race; whereas Mexican Americans, Other Hispanics, and Non-Hispanic Asians had 32%, 44%, and 46% lower odds of blood Me-PFOSA-AcOH detection compared to Mixed or Other Race, respectively. Individuals with higher income-to-poverty ratios had significantly higher odds of blood Me-PFOSA-AcOH (p<.001, OR=1.705). Furthermore, estimated non-cancer risks based on drinking water PFAS concentrations were highest for PFHxS, PFNA, PFUA and PFOA across various age groups and exposure scenarios. Cancer risk for PFOA was highest in the high exposure scenario [12 to 15 age group: 4.0 x 10-6, 16 to 20 age group: 3.0 x 10-6, 21 to 40 age group: 3.0 x 10-6, 41 to 60 age group: 3.0 x 10-6, 61 years and older: 2.0 x 10-6] with younger individuals being more at risk than older adults. These findings support the hypotheses that social determinants (gender, age, race/ethnicity, and income) are associated with higher PFAS exposure and that adolescents may face a higher risk of adverse non-cancer health effects from PFAS in drinking water compared to adults. The findings of this study stress the importance of continued monitoring, additional regulatory action to safeguard public health, targeted public health interventions to limit exposure (particularly in younger populations), and call for further research to identify additional sources of exposure and reassess risk estimates.Item Embargo Health Risk Assessment of Arsenic and Manganese Contamination in West Virginia's Public Water Systems(East Carolina University, May 2025) Sharpe, Linda DThis study examines arsenic (As) and manganese (Mn) contamination in West Virginia's public water systems, focusing on Community Water Systems (CWS) and Non-Transient Non-Community Water Systems (NTNCWS). The research addresses the critical public health issue of heavy metal exposure through drinking water, which can lead to various adverse health effects. Water sampling data from 2011 to 2023 were obtained from the West Virginia Department of Health and Human Resources and analyzed for As (n=305) and Mn (n=112) across the state. This study utilized the U.S. Environmental Protection Agency's risk assessment framework, calculating Average Daily Dose (ADD), Lifetime Average Daily Dose (LADD), Hazard Quotients (HQ), and Cancer Risk (CR) for various age groups under multiple (default, low and high) exposure scenarios. Socioeconomic factors were evaluated using county-level data and examined using univariate analysis. The dependent variables were log-transformed As and Mn concentrations, while the independent variables included median income, county districts, collection year, and water system type. The findings revealed significant geographic variations in As and Mn contamination across counties. Arsenic levels remained relatively stable from 2011 to 2022 (1.6-6.7 µg/L) but spiked dramatically in 2023 (60.17 µg/L). Manganese levels increased significantly from 2017, peaking at 889 µg/L in 2019. Several samples exceeded regulatory limits: 19 As samples surpassed the maximum contaminant level (MCL) of 10 µg/L, and 67 Mn samples exceeded the secondary maximum contaminant level (SMCL) of 50 µg/L. Arsenic levels were significantly influenced by the year of collection and its interaction with the geographic district. Manganese contamination was influenced by geographic district and its interaction with collection year. The study found no significant correlation between As or Mn concentration and median income (p = 0.704, p = 0.283, respectively), suggesting that environmental factors may have a stronger influence on contamination levels than socioeconomic factors. All exposure scenarios for As in both the community water system (CWS) and non-transient non-community water system (NTNCWS) showed hazard quotient (HQ) values greater than 1, indicating considerable non-cancer risks across all age groups. Manganese exposure presented considerable non-cancer risks, particularly for infants and young children. Cancer risk characterization revealed elevated lifetime cancer risks for As exposure, exceeding the threshold of 1 × 10⁻⁶ in most scenarios. The study confirms significant variations in As and Mn contamination between counties and reveals considerable cancer and non-cancer risks from exposure to these metals in drinking water among West Virginia residents. These findings highlight the urgent need for targeted interventions to improve water quality and reduce exposure to these contaminants, particularly in high-risk areas. Recommendations include implementing targeted interventions in high-risk counties, enhancing water quality monitoring and remediation efforts, conducting public awareness campaigns, considering stricter regulations on industrial activities, and investing in water treatment infrastructure and distribution systems.Item Embargo CULTIVATING BELOVED SCHOOL COMMUNITIES: HOW SCHOOL LEADERS LEARN AND LEAD FOR EQUITY(East Carolina University, May 2025) Steiner, Jennifer MIn the participatory action and activist research (PAAR) study, I explored how equity-centered school leaders who cultivate beloved communities can interrupt systemic inequities in education. Grounded in psychological safety, collective responsibility, and equity-centered leadership principles, nine school leaders as co-practitioner researchers (CPRs) and I engaged in three cycles of inquiry. Using the PAAR methodology and through Community Learning Exchange (CLE) protocols, 1:1 consultations, and reflective practices, participants examined their dispositions and actions. As a result, the CPR members analyzed their leadership practices collaboratively and implemented strategies to foster beloved communities where students, families, and educators feel seen, heard, valued, and supported. Two findings from the study demonstrate that leaders need to experience collaborative, authentic holding spaces—brave spaces—with equity-centered colleagues to become a beloved community (Arao & Clemens, 2013; Drago-Severson et al., 2023; Khalifa et al., 2016). As leaders engaged in a beloved community, their concept of leadership evolved, leading to a shift in their leadership practices. Leaders navigated systemic challenges—including fiscal instability, leadership turnover, and political pressures—by coming together in brave spaces and grounding their leadership in shared frameworks. A key insight from the research was the shift from a model of shared accountability to collective responsibility, emphasizing interconnectedness and mutual care over hierarchical enforcement. This shift aligns with the Kwanzaa principle of ujima (collective work and responsibility) and underscores the importance of co-constructing school environments that are healing-centered and justice-oriented. The study highlights the necessity of affinity groups and networks that support leaders in processing the emotional and strategic complexities of their roles. These spaces enabled leaders to practice vulnerability, engage in productive conflict, and develop sustainable self-care strategies, mitigating the burnout often associated with equity-centered leadership. The research is important for district leaders, policymakers, and scholars seeking to cultivate beloved school communities that prioritize healing, transformation, and racial justice. By centering the experiences of school leaders and fostering a culture of relational trust, this study contributes to the growing body of scholarship on transformative leadership and equity in education.Item Embargo LEADING IN NEPANTLA: THE COMPLEXITY OF SERVING AS A TEACHER AND LEADER IN A TEACHER-LED SCHOOL(East Carolina University, May 2025) De Alba, AdrianTeachers traditionally work in a classroom and have little to no say outside those walls. I engaged a group of four co-practitioner researchers (CPR) from an alternative teacher-led school in a participatory action and activist research (PAAR) project and study. As teachers and leaders, they occupied nepantla – a liminal space between teaching and leading and between their values and district constraints. I analyzed how teachers as leaders developed and implemented a project-based instructional program in an alternative school and the assets and challenges they encountered. In the final data cycle, I conducted an ethnographic study of two teacher leaders as they shared their successes and challenges in operating a teacher-led school focused on shifting the power structure to include students. Using the Community Learning Exchange axioms and protocols (Guajardo et al., 2016), the team crossed boundaries to develop their individual and collective capacity as school leaders. At the intersection between teaching and leading and between district demands and their belief in the power of student-generated curriculum, the teachers as leaders collaborated to leverage student voices amid challenges. The team challenged the traditional hierarchy, elevating teachers as leaders and allowing them to exist in a complex environment in which they experienced freedom and conflict. The findings emerged as a cautionary tale: (1) Teacher leaders in an alternative school without a principal did not rely on a traditional hierarchy for decision-making, simultaneously existing in a position of decision-making dissonance and empowerment, and (2) teacher leaders in an alternative setting focused on project-based learning were empowered to authentically use student voices to develop curriculum and school structures, causing internal conflict due to longstanding educational traditions and limited resources. While teachers felt liberated beyond the classroom to lead, they simultaneously felt overwhelmed by the demands of teaching and leading. They recognized that they needed decision-making and conflict-resolution structures to more effectively navigate the liminal space of teacher and leader. The implications for those engaging in democratic structures for schools need processes and protocols for decision-making and conflict resolution.Item Embargo CULTIVATING SUPPORTIVE AND CARING IEP PROCESSES: ADDRESSING THE UNDERLYING CONDITIONS FOR SPECIAL EDUCATORS(East Carolina University, May 2025) Lund, AlyssaStudents with disabilities and their families have a right to informed consent in a supportive and caring IEP process. To do so, understanding the conditions that impact special educator professional capacity and effective collaboration between special educators and the general education teachers and administrators is necessary. In a participatory activist and action research (PAAR) study at an urban charter high school in Oakland, California, I facilitated a project and study as a practitioner-researcher and worked closely with a co-practitioner researcher (CPR) team of site-based special educators. Using Community Learning Exchange (CLE) axioms and practices (Guajardo et al., 2016) to examine current practices, I determined two finding related to the underlying conditions that are necessary for effective IEP processes: (1) To cultivate the necessary emotional resilience that special educators need for their work, the special educators need structured time for personal and professional reflection, and (2) special educators and general education teachers must engage in conversations about shared values and the school processes necessary for effective collaboration before they can co-plan inclusive practices and co-develop supportive IEP processes for students and families. To address the underlying conditions necessary for effective IEP design and implementation, schools must mitigate the impact of burnout and compassion fatigue by tending to the emotional resilience of special educators and providing space and time for individual and collective reflection and self-care. In addition, school leaders must design systemic structures that provide time and space for collaborating, building relational trust, and centering decisions on a shared values system that encourage collaborative practices to address the needs of students with disabilities. Broader implications include improving the preparation of administrators and special educators for the responsibilities related to serving students with disabilities, providing coaching and mentoring to administrators and special educators early in their careers, and expanding opportunities for building relational trust and deeper understanding of the hopes and dreams of our students and families.Item Embargo NECESSARY PRECONDITIONS FOR RE-IMAGINING CASE MANAGEMENT: CULTIVATING A SECURE MINDSET IN HIGH SCHOOL CASE MANAGERS(East Carolina University, May 2025) Thomas, SuniquaTo combat the disproportionate outcomes of Black and Brown students who transfer to continuation schools, students need a case management system to support them to stay on track for graduation at a comprehensive high school. In this participatory action research (PAR) study, a group of co-practitioner researchers (CPR) and I examined how to address a change in the case management system. As a result, we learned that the staff who support our most vulnerable students need parallel support to be case managers and to re-imagine case management. Using CLE protocols for discussion and engagement and qualitative research methods to analyze iterative data from three cycles of inquiry over 14 months, we cultivated relational trust and participated in open discussions about student and staff needs. Two findings pointed us to the need to fully engage adults in dialogue and self-care before they can effectively change the case management system for the students in our school. First, adults who engage with students as case managers need supportive parallel spaces in which to build trust, reflect, and sustain their resilience and agency. Secondly, to re-imagine a case management system, staff need support to develop secure mindsets and fully commit to caring for students. The team identified compassion fatigue as a primary factor in staff inability to take on full responsibility or re-imaging a more effective case management system. This study brings attention to the compassion fatigue that staff experience and their need for supportive structures before they can make systemic changes. The findings have implications for educational leaders trying to guide teachers and staff in reform efforts; adult caregivers in schools need concurrent emotional support so they can support students. To collaborate, leaders must work to ensure the preconditions for effective change efforts -- secure mindsets and practice using tools and protocols that support them to engage in productive change processes.Item Embargo IMPROVING INSTRUCTIONAL PRACTICES IN LITERACY: SUPPORTING THE GROWTH AND DEVELOPMENT OF INTERVENTION TEACHERS(East Carolina University, May 2025) Guzman, Monica MarieThe goal of the participatory action research study was to examine how intervention teachers contribute to the improvement of literacy instructional practices in elementary schools. A team of nine elementary intervention teachers volunteered to participate and engage in three cycles of inquiry throughout the 14-month study. Collaborating more frequently with a smaller group of intervention teachers as three co-practitioner researchers, we identified experiences and practices to support the growth and development of the team of intervention teachers in their work to increase literacy in schools. To develop relational trust and identify the supports they needed to be effective in their work, the team engaged in Community Learning Exchanges to share effective instructional practices and strategies as they collaboratively addressed literacy needs at their schools. The study describes how participants navigated fragmented centralized reform efforts and the impact made on literacy intervention instruction and outcomes. The findings of the study reveal the need for established systems of support for the team of intervention teachers. In addition, by clarifying their complex roles and providing relevant professional learning, the challenges the intervention teachers faced were minimized. These findings support and add to the research literature in the areas of supporting the development of the capacities and practices of teachers in specialized roles.Item Embargo BECOMING CHEECHAKOS: DEVELOPING CAPACITY FOR IMPLEMENTING AN INTERNATIONAL BACCALAUREATE (IB) PRIMARY YEARS PROGRAMME (PYP) AT AN ELEMENTARY SCHOOL.(East Carolina University, May 2025) Sosa, Arthur LeeCheechako originally was a term used to describe a person who newly arrived in the mining districts of Alaska. Currently, cheechako is used describe newly arrived individuals to the state of Alaska. For this dissertation, I used the term to describe the new arrival of the IB PYP program to the state and how I, as the principal, was a cheechako new to the International Baccalaureate (IB) Primary Years Programme (PYP). This participatory action research (PAR) study aimed to understand how one elementary school understood and planned for implementation of an IB program. PAR is used as a qualitative data collection and analysis processes (Herr & Anderson, 2014; Saldaña, 2016). Specifically, this research uses iterative cycles of inquiry to increase the collective understanding of the with particular focus on the IB Learner Profile (LP) attributes. In the project and study, we utilized the Community Learning Exchange (CLE) protocols to engages the IB PYP learning community to examine elements of the IB PYP implementation. This study underscores the importance of shared leadership among administrators, teachers, and parents in enhancing the implementation of IB PYP initiatives. Additionally, co-developing a common understanding of innovations among practitioners ensures success. These elements foster a system that promotes adaptability and continuous improvement. The research also presents potential policy changes for the school district and state, using evidence from the Harbor Bay International Elementary School learning community to inform curriculum decisions aligned with IB principles. This insight can guide the district in determining the viability of alternative and IB school models, ultimately influencing best practices that could improve outcomes across schools.Item Embargo DEEP ROOTS: EMPOWERING TEACHERS OF ELEMENTARY GIFTED STUDENTS IN INCLUSIVE CLASSROOMS(East Carolina University, May 2025) Lindner, Matthew D.Elementary students bring many assets with them to school each day. Throughout their formative years, elementary students show leadership, collaboratively solve problems, and persevere through challenges. However, structural and policy issues, including traditional methods of instruction, pose challenges for educators attempting to identify and build upon student strengths. Leaders and teachers often gloss over the need to focus on giftedness as supporting students with learning difficulties often takes priority. Yet, gifted students need our support and attention. The purpose of the participatory action research (PAR) project and study was to engage a team of co-practitioner researcher (CPR) team members in three iterative cycles of inquiry to understand how elementary teachers analyzed equitable and inclusive giftedness and how they planned for equitable and inclusive teaching strategies to support gifted students. The primary methodology was participatory action research (Herr & Anderson, 2014) using qualitative analysis processes in the three cycles of inquiry to determine emergent themes and findings (Saldaña, 2016). I relied on the Community Learning Exchange (CLE) axioms to engage eight educators in authentic dialogue to examine their conceptions of giftedness and how they address the learning needs of gifted learners within general education classrooms. We engaged in root cause analysis to refine a definition of giftedness, explore multiple processes for identifying giftedness, re-examine teacher responses to gifted instruction, and investigate instructional practices to support gifted students in inclusive classrooms. The findings are: (1) intentionally cultivating relational trust by co-generating a space of inclusion and attending to educator preferences is a necessary condition for professional learning; and (2) to address the needs of gifted students in public elementary classrooms, educators shifted their definition, mindsets, and giftedness-related instructional practices. As a result, the processes we used in the study to create holding spaces for adults (Drago-Severson, 2012) are replicable and useful for adult professional learning. Teachers and district leaders can collaborate to identify equitable and inclusive practices that support gifted students in elementary classrooms, depending on the identified needs of learners in the local context.Item Embargo ELEVATING THE VOICES AND ROLES OF PARAPROFESSIONALS IN INCLUSIVE MIDDLE SCHOOL CLASSROOMS(East Carolina University, May 2025) Sweeney, Lena HeatherThe purpose of the participatory action and activist research (PAAR) project was to engage in iterative cycles of inquiry to understand how a school-based team could shift the role of instructional assistants (IAs) to provide students with disabilities support for independence within inclusive classroom environments. The primary methodology for the study was participatory action and activist research (PAAR) (hunter et al., 2013) using qualitative analysis processes in three cycles of inquiry (Saldaña, 2016). We relied on Community Learning Exchange (CLE) axioms and processes to engage two teams – a co-practitioner researcher (CPR) team and an instructional assistant professional learning group (PLG) -- in authentic dialogue to examine practices related to instructional assistants shifting from a 1:1 role to a classroom assistant (CA) role. In sharing personal stories and fostering relationships, we cultivated a dedication to equity and inclusion for IAs. In consultation and collaboration with instructional assistants, we analyzed and supported their experiences, skills, and needs as primary factors in changing our approach to inclusion and changing how paraprofessionals fully engage with teachers and leaders to support students in inclusive classrooms. Actively listening to instructional assistants enhanced their sense of belonging within the adult school community as evidenced by their participation in the professional learning. Secondly, the people who became anchors of the work and acted as bridges accelerated the change effort. As a result, the instructional assistant role shifted from isolated to integral and included collaboration time, resources, and access to information to facilitate inclusion and empowerment. Key individuals emerged as pivotal leaders in this change process, bridging the initiatives of the IA PLG and CPR group and advancing a systemic approach to integrating instructional assistants in the school community and in classrooms to better provide support for students. The findings of the study offer a roadmap for other schools and districts to rethink the roles of paraprofessionals. The power of the collaborative work and the processes are replicable in other contexts.Item Embargo Investigating the Molecular Defects Contributing to Skin Fragility in Two Ectodermal Dysplasias(East Carolina University, May 2025) Salois, Maddison N.Ankyloblepharon ectodermal dysplasia and cleft lip/palate (AEC) and ectrodactyly-ectodermal dysplasia cleft lip/palate syndrome (EEC) are rare genetic skin disorders caused by missense mutations in TP63. AEC is caused by mutations to the sterile alpha motif domain of TP63, while EEC is caused by mutations in the DNA binding domain. TP63 is an essential component in epidermal and epidermal appendage development and maintenance. Mutations to TP63 cause a wide variety of ectodermal defects, including abnormal development to the skin, hair, nails, and limbs. The most severe manifestations are extensive skin erosions that can be life-threatening and decrease the patient's quality of life. Additionally, these erosions have no therapeutic options. To develop novel therapies, we need to first understand how TP63 mutations lead to skin erosions. Our laboratory aims to uncover the pathological mechanisms that contribute to the devastating skin fragility in AEC and EEC. To investigate this, we have generated complementary in vitro model systems. First, we have generated patient-derived induced pluripotent stem cells (iPSC) which can be differentiated into keratinocytes (iPSC-K), one of the cell types affected in AEC and EEC. Additionally, with CRISPR/Cas we have corrected the disease-causing mutation, generating conisogenic pairs of keratinocytes. This is an ideal model as they have the same genetic background as the patients and only differ in the presence or absence of the single point mutation. Additionally, using the commercially available immortalized keratinocyte line, NTERT, and lentiviral constructs harboring AEC- or EEC-mutant TP63, we generated an additional disease-relevant model (AEC-NTERT and EEC-NTERT). RNA sequencing was performed with subsequent in silico pathway analysis on the conisogenic AEC iPSC-K and EEC iPSC-K pairs. A comparison of AEC iPSC-K to their respective gene-corrected (GC) iPSC-K showed “integrin cell surface receptors” and “cell adhesion genes” as abnormally expressed. However, EEC iPSC-K, compared to their GC iPSC-K, had “epidermal differentiation” as a significant defect, suggesting different pathological mechanisms. AEC iPSC-K RNA and protein analyses show reduced expression of several cell-cell and cell-extracellular matrix (ECM) adhesion structures, including the desmosome, hemidesmosome, and focal adhesion. Additionally, AEC-NTERT keratinocytes revealed significantly reduced adhesion and migration on several ECMs. The inability to adhere and migrate over ECMs could explain long-lasting wounds in AEC patients, suggesting wound healing might be impaired. AEC patient skin samples validated focal downregulation of several cell adhesion structures. However, EEC iPSC-K did not show abnormalities to hemidesmsosmal or focal adhesion genes. Instead, we observed inappropriate expression of genes associated with epidermal terminal differentiation in keratinocytes cultured under proliferating conditions. Further, 3D epidermal equivalents generated from EEC-NTERT showed increased differentiation programs which was validated in EEC patient skin. Altogether, our data suggests that skin fragility in AEC and EEC is due to different molecular mechanisms. AEC epidermal defects can be at least in part attributed to the cooperative downregulation of cell-cell and cell-ECM components while EEC epidermal defects show accelerated terminal differentiation. Further understanding these molecular mechanisms may eventually lead to the development of novel therapies for these patient populations and improve patient quality of life.Item Embargo Exploring the Relationship Between Health Insurance Literacy and Health Equity Among College Students: A Pilot Study(East Carolina University, May 2025) Wynn, JaelynHealth insurance literacy is a critical factor that should be addressed in the college student population. This population has been found to have lower health insurance literacy levels, which can impact health outcomes. This study examines health insurance literacy and health equity among undergraduate college students at a Minority-Serving Institution (MSI) and a Predominantly White Institution (PWI) and examines demographic factors in health insurance literacy. A total of 276 students participated in a survey examining perceptions of health equity and health insurance literacy levels using items from the Kaiser Family Foundation (KFF). Bivariate associations were assessed using Mann-Whitney U tests, Chi-Square tests (or Fisher-Freeman-Halton tests as appropriate), and Spearman correlations. Multiple linear regression models were used to control for covariates in comparisons. Results found that students at the PWI had significantly higher mean health insurance literacy scores than students at the MSI (PWI Mean = 4.33 vs. MSI Mean =3.14, p=0.027) after controlling for student demographics. Perceptions of health equity between the two institutions were similar. Prior knowledge of health insurance was found to be positively associated with health insurance literacy levels (p<0.001). The number of barriers that students reported, and experiences of unfair treatment was not significant between the two institutions. Additionally, it was found that age (p<0.001), race (p=0.003), and academic standing (p=0.005) were significantly associated with health insurance literacy levels among all undergraduate students in this study, regardless of institution. Additional research should be conducted to determine if educational programs should be implemented to improve health insurance literacy levels among the college student population. Age, race, and academic standing were found to be disparities that contribute to health insurance literacy in this study and should be explored in future studies. Addressing gaps in health insurance literacy will improve college students’ overall understanding of health insurance and improve health equity.