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    Establishment and Improvement of Preclinical Murine Models of Severe Scrub Typhus to Define Immune Signatures and Pathogenic Mechanisms
    (August 2023) Thiriot, Joseph David 1989-; Soong, Lynn; Sun, Keer; Walker, David; Comer, Jason; Riley, Sean
    Scrub typhus is a leading source of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Scrub typhus causes 150,000 deaths per year, with an estimated 1 million cases, and a further 1 billion people at risk. No vaccine is available for this reemerging and severely neglected infection. Frustratingly, scrub typhus can result from infection due to a variety of Ot strains with limited cross protection. However, little is understood about strain-specific virulence factors or host immune response. While there are many established murine models of scrub typhus, differences in route of infection, host resistance, and clinically unrelated pathologic outcomes diminish the usefulness of model studies. Therefore, there is a great need for the development of small animal models that reproducibly mimic human diseases for immunologic investigation and future vaccine studies. Our recent studies have established a new inbred murine model of hematogenous spread. To expand these models, we have established an outbred murine model of hematogenous transmission that mimics tissue tropism and pathologic lesions of scrub typhus patients. Interestingly, we find this outbred model exhibits the same pro-inflammatory/Th1 skewed immune response as seen in our previously established inbred model. We further developed our outbred and inbred models by using two clinically prevalent O. tsutsugamushi strains, Karp and Gilliam, and reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. We find that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice are susceptible to Karp, but resistant to Gilliam (with self-limiting infection). Our outbred preclinical model is particularly useful for future translational and vaccine studies for severe scrub typhus. This body of work provides the first evidence of differential tissue cellular responses in an inbred model against Karp vs. Gilliam infection, thus offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis.
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    HCV Core Protein affects Lipid Metabolism in a Genotype-Dependent Manner
    (2020-08-01T05:00:00.000Z) Hull, Madeleine E
    Chronic HCV infection is the leading cause of steatosis (fatty liver disease) and hepatocellular carcinoma (HCC). The virus establishes a chronic infection in 70% of patients and infects approximately 71 million people worldwide. Genotypes (gt) 1 and 3 are the most prevalent, with gt3 HCV being associated with more severe disease. It is known that core protein plays a role in the development of steatosis, but the precise mechanism is not yet understood. In this study, we investigate the role of genotypes in core-mediated differential regulation of lipid metabolism. Prior studies described the lipid induction by HCV infection or ectopic expression of core derived from gt1a and 3a HCV qualitatively. The goal of this thesis is to define the gt1a and 3a HCV core-mediated lipid regulation in a quantitative manner. Using FACS analysis, and immunofluorescence analysis by using confocal microscope, we found that gt3a core protein induces larger lipid droplet formation, although the quantity of lipids remains similar to that induced by gt1a core protein. We then attempted to determine the difference in fatty liver-associated gene expression levels induced by gt1a and gt3a core proteins. To do this, we utilized microarray to analyze the gene expression in cells transfected with different HCV core proteins. The analysis showed that both gt1a and gt3a core upregulated SOCS3; this upregulation has been shown to influence the liver response to previously used antiviral therapies, as well as the induction of insulin resistance. Additionally, genes that were downregulated by gt1a and gt3a core are largely associated with insulin resistance, which plays a role in the development of diabetes. Interestingly, more genes appear to be downregulated by gt3a than gt1a core protein, which could suggest that gt3a core protein induces insulin resistance and steatosis in a different manner, or to a more severe degree. Overall, these results present a potential mechanism for the relationship between insulin resistance and lipid metabolism deregulation induced by gt1a and gt3a core proteins.
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    Proviral Roles of Ebola virus VP35 Ubiquitination
    (2022-05-01T05:00:00.000Z) Van Tol, Sarah Rose
    Ebola virus’ structural protein VP35 is polyfunctional and plays vital roles in Ebola’s life cycle from antagonizing the host’s type I interferon pathway to acting as the polymerase co-factor. The mechanisms that regulate which function any given VP35 molecule engages remains unknown. Previously, we observed the host E3 ubiquitin ligase TRIM6 conjugates ubiquitin onto VP35 at lysine (K) 309. This post-translational modification was found to be proviral, but we did not know which VP35 role(s) ubiquitination regulated. We generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant’s suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This dysregulation of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. Blocking VP35/K309 ubiquitination enhanced interaction with the viral nucleoprotein and may trigger premature nucleocapsid packaging. Prior work also showed that at least one lysine residue other than K309 is ubiquitinated and that VP35 non-covalently interacts with ubiquitin. We observed that multiple residues in VP35’s N-terminus can receive covalent ubiquitin, including K119, 126, and 141. Substitution of K119-, 126-, and/or 141-to-R significantly attenuates VP35’s polymerase co-factor activity, but their mutation does not alter interactions with the viral polymerase or nucleoprotein. When looking into VP35’s non-covalent ubiquitin interaction, we found that VP35 specifically binds K63-linked ubiquitin chains via its C-terminus. The specific cleavage of unanchored ubiquitin chains, using the deubiquitinase isopeptidase T, impedes VP35’s non-covalent binding to ubiquitin and stunts EBOV’s polymerase activity. Finally, we also found that TRIM25, TRIM6’s relative, facilitates ubiquitin ligation onto VP35 and enhances VP35’s non-covalent interaction with ubiquitin. EBOV replication is attenuated 100-10,000 fold in cells lacking TRIM25. Overall, our data support that TRIM6- and TRIM25-mediated VP35 ubiquitination and VP35’s non-covalent interaction with unanchored ubiquitin is proviral.
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    Small Molecule Allosteric Modulation of G Protein-Coupled Receptors and Applications in the Pharmacological Targeting of 5-HT2 Receptors
    (2021-05-01T05:00:00.000Z) Wold, Eric A.
    The discovery of G protein-coupled receptors (GPCRs) and the extensive second messenger systems associated with their activation has ushered in an enormously productive period in drug discovery. The extent to which around one-third of FDA-approved medications target GPCRs. Predictably, small molecule allosteric modulation has emerged in recent years as a new means to control GPCR function with numerous examples nearing FDA approval. This body of work begins with a substantive review of drug discovery efforts in the development of Class A GPCR allosteric modulators as a means to compile successful strategies and take note of the distinct challenges in the field as our group approaches allosteric modulator discovery for the serotonin 5-HT2C receptor (5-HT2CR). From the time of its characterization, the 5-HT2CR has been marked by unique, untapped potential as a drug discovery target for numerous diseases and disorders of the central nervous system. Thus, the second chapter in this work provides a clear rationale for 5-HT2CR allosteric modulator discovery in context of the neurobiological framework wherein the 5-HT2CR plays an integral role in reward-related behaviors and cortical executive functions. With the rationale established, the following sections report our efforts in the discovery of novel 5-HT2CR positive allosteric modulators (PAMs) from their design and chemical synthesis to the in vitro and in vivo characterization of these molecules. Additionally, further work describing 5-HT2CR PAM pharmacokinetic properties as suitable for rodent behavioral assays and the structural determinants of 5-HT2CR PAM binding via molecular modeling are discussed. Having discovered allosteric modulators with functionality across the 5-HT2R subfamily and benefiting from the wealth of available structural data for these targets, the final chapter delves into a theoretical mechanism underpinning allosteric modulation of 5-HT2Rs. The enhanced activation state (EAS) is thus coined for the first time herein to describe 5-HT2R PAM functionality and is treated with a rigorous theoretical framing comprised of the observed pharmacological, structural, and computational studies that shape our understanding of GPCR dynamics, specifically the activation dynamics of 5-HT2Rs. The resultant body of work provides the reader a comprehensive understanding of allosteric modulation and its pharmacological utility in targeting 5-HT2Rs.
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    Opioid Prescribing and Opioid-Related Health Outcomes Among Cancer Survivors
    (2023-05-01T05:00:00.000Z) Gibson, Derrick C
    The US population of older long-term cancer survivors—Americans who are free of cancer 5-years post-cancer diagnosis and not receiving cancer treatment—is growing. The prevalence of pain among cancer survivors after curative treatment is approximately 40% and opioids are frequently prescribed to manage the pain. The purpose of this dissertation is to explore long-term opioid therapy and opioid-related harms in cancer survivors using Surveillance Epidemiology and End Results – Medicare linked datasets. First, we explored the temporal and geographical variation in long-term opioid therapy among cancer survivors in the United States. We found that long-term opioid therapy rates were highest in the south and lowest in the northeast and that long-term opioid therapy rates peaked in 2012 but declined until 2016. Second, we assessed if patient level pain conditions and provider specialties seen at outpatient visits by cancer were associated with long-term opioid therapy. We found that cancer survivors who had been diagnosed with chronic pain or noncancer pain conditions and who were treated by noncancer specialists were more likely to receive long-term opioid therapy. Third, we assessed if cancer survivors were more likely than noncancer controls—matched on age, gender, race, pain conditions, previous opioid use—to experience an opioid-related emergency department visit or hospitalization. We found that the incidence of opioid-related adverse events were five times higher among cancer survivors who used opioids previously than opioid naïve cancer survivors. We found cancer survivors were as likely as persons without cancer to experience an opioid-related emergency department visit or hospitalization. In conclusion, we found high prevalence rates of long-term opioid therapy that differed by time and US geographical region and the risk of an opioid-related emergency department visit and hospitalization is comparable between cancer survivors and persons without a history of cancer. Our findings support the idea that policies and guidelines should continue to promote and incentivize the use of nonpharmacological and nonopioid interventions for managing pain among older adults.
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    Regulation of signaling and function of the voltage-gated sodium channel complex by protein:protein interactions
    (2020-05-01T05:00:00.000Z) Wadsworth, Paul A
    As fundamental determinants of neuronal function, voltage-gated Na+ (Nav) channels are important targets for therapeutic development against a wide range of health conditions. Dysfunction of Nav channels in the CNS is associated with disorders ranging from neurological (i.e., epilepsy, neurodegeneration) to psychiatric (i.e., major depression disorder, schizophrenia). Unfortunately, commercially available drugs targeting Nav channels are directed against highly conserved domains across Nav isoforms, giving rise to severe side effects such as cardiotoxicity and movement disorders. Thus, there is an unmet need for discovering new probes and pathways that regulate Nav channels that could potentially help designing new medications. Recent evidence suggests that protein:protein interactions (PPI) between Nav channels and their accessory proteins play a key role in regulating neuronal firing, and that minimal disturbances to these tightly controlled PPI can lead to persistent maladaptive plasticity. These PPI interfaces are highly specific and provide ideal targets for drug development, especially in the CNS where selectivity and specificity are vital for limiting side effects. However, for the most part how these protein:channel interactions are regulated in the cell is still poorly understood, and methods for assessing these interactions are lacking. Therefore, the goal of the present study was to develop robust assays to reconstitute the Nav channel complex in cells and identify cellular pathways and small molecules regulating PPI interfaces with the Nav channel complex. Specifically, we focused on the PPI between Nav1.6 and its regulatory protein, fibroblast growth factor 14 (FGF14). Using a newly developed assay we screen cellular pathways followed by biophysical validation, we discovered a mechanism by which the JAK2 tyrosine kinase might directly influence neuronal firing through phosphorylation of FGF14. Furthermore, we conducted a high-throughput screening of ~45,000 small molecules and identified potent modulators of the FGF14:Nav1.6 complex that are functionally active and predicted to be permeable to the blood-brain barrier. While providing a robust in-cell screening platform that can be adapted to search for any channelopathy-associated regulatory protein, these results lay the potential groundwork for a new class of drugs targeting Nav channels with a broad range of applicability for CNS disorders.
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    A Naturalistic Inquiry of Nigerian Immigrant Nursing Students’ Experiences in United States (U.S.) Baccalaureate Nursing Programs
    (2020-05-01T05:00:00.000Z) King, Shatoi T
    Nigerian immigrant nursing students have been identified in the literature as having difficulty in making a transition to the teaching methods used in nursing programs in the U.S. Among the problems they experience are understanding the vocabulary and terminology of the Western culture. Previous research suggests Nigerian students remain silent in seeking help regarding the challenges of their learning in part due to the isolation they face from peers and faculty (Sanner, Wilson and Samson, 2002). There is a paucity of research associated with Nigerian immigrant nursing students and how they acclimate to U.S. nursing programs. This study utilized Naturalistic Inquiry (Erlandson et al., 1993; Lincoln & Guba, 1985) to explore the perceptions and experiences of ten Nigerian immigrant nursing students. Study participants were Nigerian immigrant nursing students and the researcher recruited students enrolled in baccalaureate nursing programs in Texas. Interviews, face-to-face or by telephone, for the ten participants were conducted in a private, agreed upon place. Data were collected until reaching saturation, indicating data redundancy, and with no new themes evolving. Analyzed data used procedures described by Lincoln and Guba (1985) and Erlandson et al. (1993) to seek emerging patterns. Guided by Lincoln and Guba (1985) procedures, the five emergent themes included Stepping into America, Navigating the Rough Waters, Unexpected Changes, Searching for Consistency, and Hopes of Finding Clarity. Lincoln and Guba’s (1985) criteria were used to evaluate trustworthiness of the data. Study findings revealed that Nigerian immigrant nursing students experience positive and negative encounters with faculty and classmates, challenges and obstacles related to the American English language, their Nigerian accent, family expectations, and work obligations. The study findings may aid U.S. nursing schools and faculty to incorporate strategies in the learning environment to assist Nigerian immigrants and other international nursing students, to better acclimate to Western programs to achieve success.
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    Employee Assistance Program and Occupational Health Providers’Perceptions and Experiences with Intimate Partner Violence in the Workplace: A Grounded Theory Study
    (2020-05-01T05:00:00.000Z) Adams, Carin Dale
    Abstract:Title: Employee Assistance Program and Occupational Health Providers’ Experiences and Perceptions of Intimate Partner Violence in the WorkplacePurpose/Significance: Employee Assistance Program/Occupational Health Providers(“providers”) are often the first point of contact for employees, both victims and perpetrators, affected by intimate partner violence (IPV). This Classical Grounded Theory (CGT) study explored providers’ experiences and perceptions of working with employees in IPV relationships. This study advances the mission of the Southern Nursing Research Society by communicating research findings related to a persistent social and health issue: IPV.Methods: CGT (Glaser, 1978) guided the study. Semi-structured interviews were conducted with ten providers from across the United States. The interviews were analyzed using constant comparison and open coding to identify categories and concepts within transcripts to ultimately reveal a substantive theory grounded in the study data. The study and resulting substantive theory adhered to Glaser’s (1978) criteria for trustworthiness: Fit, work, relevance and modifiability.
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    The Roles of Estate Planning and Social Support in Racial/Ethnic Disparities in Advance Care Planning and End-of-Life Care
    (2020-05-01T05:00:00.000Z) Moran, Jacob
    Advance Care Planning (ACP)– completing advance directives, discussing end of life care preferences, and assigning a durable power of attorney for healthcare – may be associated with improvements in quality of end of life care and more specifically with receiving care congruent with one’s wishes. Despite this, stark differences in completion rates by Non-Hispanic Blacks and Hispanics compared to Non-Hispanic Whites are observed. Much of the research on ACP has focused on describing associations between sociodemographic factors and planning completion or planning completion and health care received at the end of life (End-of-Life). However, few studies have proposed testable hypotheses and investigated causal relationships for ACP completion, particularly among Hispanic subgroups, and effects on end of life care received. This study uses nationally representative data from the Health and Retirement Study to investigate explanatory causal pathways in ACP completion and its effects on End-of-Life healthcare with a focus on Hispanics. Results from this study will help providers better understand sociodemographic factors that predispose patients to high risk for failing to plan for the end of life, health systems identify target areas for system change, and policy makers understand the role socioeconomic disparities play in end of life planning.
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    The Relationship Between Gastrointestinal Disease and Post-traumatic Stress Disorder in United States Military Veterans
    (2020-05-01T05:00:00.000Z) Kent, Kelsey
    This study examined relationship between gastrointestinal disease (GI Disease) and post-traumatic stress disorder (PTSD) in United States Military Veterans. Based upon literature and clinical practice data sources from the US Veterans Administration, GI disease and PTSD were hypothesized to be positively correlated in Veterans. The specific aims of the study were to determine the frequency with which: 1) GI Disease and PTSD are diagnosed co-morbidities; 2) a diagnosis of GI Disease accompanies a diagnosis of PTSD; and, 3) a diagnosis of PTSD accompanies a diagnosis of a GI Disease. The methodology was a retrospective, correlational design using data collect from the Department of Veteran’s Affairs Patient Database. Findings were that PTSD is bi-directionally correlated with the GI diseases of gastroesophageal reflux disease (GERD), peptic ulcer disease, functional dyspepsia, Crohn’s disease, diverticular disease, and irritable bowel syndrome (IBS), and the symptoms of constipation and nausea/vomiting within Veterans who served during wartime periods. The study also found that PTSD is not correlated with ulcerative colitis in Veterans.
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    Investigating the Potential Role of α-Synuclein in Tau Aggregation and Toxicity
    (2020-05-01T05:00:00.000Z) Sengupta, Urmi
    A group of neurodegenerative diseases that are pathologically characterized by the presence of intracellular abnormal aggregation of α-Synuclein (α-Syn) in Lewy bodies (LBs) and Lewy neurites (LNs), are collectively known as synucleinopathies. Even so, tau protein pathology is abundantly found in these diseases. Apart from Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), LBs and LNs have been reported in Alzheimer’s disease (AD) patients as well. Both α-Syn and tau can exist as polymorphic aggregates, and this phenomenon has been widely studied, mostly in their fibrillar assemblies. Growing evidence suggests that intermediate metastable oligomeric assemblies of several amyloidogenic proteins, including α-Syn and tau are actual neurotoxic species. However, little is known about the structural and functional heterogeneity among α-Syn oligomers occurring in different diseases. Moreover, the functional crosstalk between these toxic oligomers has not been scrupulously studied. Here, by using biochemical, biophysical and cell-based techniques, I have studied the structural and functional diversity of distinct recombinant α-Syn oligomers, prepared by modifying the protein with two physiological inducers, dopamine (DA) and docosahexaenoic acid (DHA). The two recombinant α-Syn oligomers differed in aggregate size, conformation, sensitivity to proteinase K digestion, tryptic digestion and toxicity, suggesting them as distinct α-Syn oligomeric strains. I have also analyzed brain-derived α-Syn oligomers (BDSOs) from AD, DLB and PD brain tissues. I observed that disease associated BDSOs were diverse in their functional properties. Notably, they can be uptaken via gap junction protein Cx50 in the primary neurons, thus suggesting a unique mechanism that might be involved in the oligomers mediated toxicity. Additionally, both recombinant and brain-derived α-Syn oligomeric strains effectively cross-seeded tau aggregates with diverse biochemical, biophysical and biological properties. Interestingly, BDSOs cross-seeded tau aggregates were more potent seeds causing cellular tau aggregation than the ones cross-seeded with recombinant α-Syn oligomeric strains. The findings here represent a significant step to elucidate the toxic interplay between α-Syn oligomeric strains and tau, altering the aggregation profiles and nature of the amyloid deposits. This will lay the groundwork for more successful therapeutic interventions by targeting multiple candidate molecules, such as α-Syn and tau in diseases.
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    Efficient Identification and Comprehension of Molecular Pathways Associated with Irradiation Induced Hepatic Carcinogenesis
    (2020-05-01T05:00:00.000Z) Nia, Anna
    As human exploration into deep space continues to expand in the future, risk prediction for irradiation-induced diseases will become an increasingly important task. It will be critical to identify the biological effects of high-charge, high energy (HZE) and low energy 137Cs γ rays, which are the major components of space irradiation on the human body during longer stay in deep space, including a mission to Mars. It has been shown that there is a significant increase in incidence of Hepatocellular carcinoma (HCC), after exposure to low dose HZE. There is, however, limited knowledge of the effects of low dose irradiation on the formation of HCC. To address this gap in knowledge, RNA-Seq and MALDI-MSI were used to assess the effects of space irradiation on the pathogenesis of HCC. In particular, RNA-seq was used to determine transcriptional changes, and MALDI-MSI to determine lipid changes, in the hepatic microenvironment of ion irradiated compared to non-irradiated controls, in two different strains of mice, at five different time points post-irradiation. For our RNA-Seq datasets, we first present a novel pipeline to perform gene co-expression network analysis, and use this to show that mitochondrial pathways are dysregulated in response to 56Fe irradiation compared to non-irradiated control, in the wildtype mouse strain (C57BL/6NCrl.) Next we performed a comparative transcriptomic analysis in a mouse model for irradiation-induced HCC (C3H/HeNCrl), in order to assess the carcinogenic effects of 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy), compared to non-irradiated control. Our data demonstrated a clear difference in the effects of these HZE ions, particularly immunological, suggesting different molecular mechanisms of tumorigenesis for each ion. Additionally, we observed novel, functionally unannotated transcripts that were significantly affected by HZE. The biological functions of these transcripts were investigated using Self-Organizing Maps (SOMs). Finally, we used MALDI-MSI to identify lipid changes 12 months post-exposure to low dose 28Si and 137Cs γ rays’ irradiation. We identified a number of lipid species; in particular, we have confirmed the identity of GSL; which is of special interest because its up-regulation have been reported in patients with HCC.
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    An Exploration of School Nurses' Perspectives in Caring for Homeless Children
    (2020-05-01T05:00:00.000Z) Wilson, Michele Atteberry
    Naturalistic inquiry methodology was incorporated into a qualitative study designed to explore the experiences and perceptions of school nurses working with homeless, elementary school-aged children. The researcher employed purposive sampling to recruit participants from members of the National Association of School Nurses. Recruiting was supplemented by snowball sampling. Eligibility criteria were nurses with a minimum of one school year prior experience and current employment as school nurses providing direct care to elementary, school-aged children. Thirteen school nurses participated in semi-structured telephone interviews. Data were collected from demographic questionnaires and interview guides. Interview data were analyzed using Lincoln and Guba’s (1985) processes of unitizing data, emergent category designation, negative case analysis; and bridging, extending, and surfacing the data. Analysis of study findings resulted in the identification of three categories describing school nurses encounters with the health and social needs of homeless, elementary school-aged children: (a) school nurse education; (b) school nurse practice; and (c) policy impacts. Additional insights into school nurses’ practice issues related to nursing care of homeless, elementary school-aged children were discovered. Implications of the study findings provided a basis to recommend further research into this important but understudied aspect of school nurse practice.
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    Description of interactions of Ebola virus with a putative reservoir species using next-generation sequencing
    (2020-05-01T05:00:00.000Z) Ronk, Adam James Dean
    Ebola virus (EBOV) causes a severe, often fatal disease in humans and nonhuman primates. Recently, EBOV has caused two very large outbreaks, one of which is ongoing in the Democratic Republic of the Congo. Bats are the likely reservoir of EBOV, but little is known of their relationship with the virus. Next-generation sequencing has become an extremely powerful and flexible tool in virology over the past decade as new library preparation techniques have been developed that permit the selective sequencing of small RNAs, and the characterization of entire viral populations at incredible levels of detail. For this work, I exploited this technology to explore two aspects of the bat/virus nexus; namely the small RNA profile of infection, and the evolution of the virus in bat cells. The biology of the virus in human cells was used for comparison. Here I describe a new class of small noncoding RNAs produced by EBOV during infection of bat and human cells that resemble microRNAs, but are not associated with the microRNA machinery, and lack any discernable RNAi function. I also describe the evolution of EBOV in an experimental passage series in bat and human cells. This work led to the discovery of a potential role for host RNA editing enzymes in the evolution of EBOV in bats, and identified loci within the viral genome that appear to be associated with adaptation to human cells.
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    Vector Competence of Aedes aegypti for Zika Virus and Effects of Colonization
    (2020-05-01T05:00:00.000Z) Roundy, Christopher
    The following dissertation aims to determine how vector colonization of influences the vector competence of Aedes aegypti for Zika virus (ZIKV) as well as the microbiome as a correlating factor. Ae. aegypti is the vector of multiple arthropod-borne viruses including dengue, yellow fever, and Zika virus, making it one of the most globally significant disease vectors and is studied in laboratories world-wide with significant research focus on vector competence studies. Many of these studies, however, utilize strains of Ae. aegypti that have been colonized in insectaries for laboratory use and may not reflect the phenotype of wild mosquitoes. While studies have shown differences lab adaptation of mosquitoes resulting in an altered phenotype compared to field mosquitoes, a comprehensive study examining the process of adaptation and effects on vector competence has not been conducted. I hypothesize that the colonization of Ae. aegypti results in an increase in vector competence for ZIKV, correlated with a change in microbiome diversity and composition. First, the vector competence of multiple species of mosquitoes (Ae. aegypti, Ae. albopictus, and Culex quinquefasciatus) was determined for ZIKV, using various strains of both virus and each vector species. A field-collected population of Ae. aegypti was then colonized and experimentally examined for vector competence for ZIKV and microbiome over the course of ten generations. I found that the vector competence of this population did increase over the course of the study and that this change occurred abruptly after multiple generations, resulting in two distinct groups of low and high competence. I then identified a number of bacteria that exhibited different levels of abundance between the low and high competence groups, many of which remain uncharacterized in the mosquito microbiome. Further studies to elucidate the role of these bacteria in determining vector competence as well as the development methods to minimize the effects of colonization could lead to better standardization across vector competence studies and increased relevance to field mosquitoes. These findings are incorporated into the existing literature with recommendations on the design of vector competence studies.
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    Profiling the Serotonin2 Receptor System Functional Capacity: Towards Identification of a Cocaine Use Disorder Biosignature
    (2020-05-01T05:00:00.000Z) Land, Michelle Ann
    Vulnerability to developing a cocaine use disorder (CUD) starts with a background of genetics and environment and results in changes in neuroplasticity that leads to a greater drive to take cocaine. The intricate interplay between these variables differs for each individual, presenting a barrier to understanding the origin of the disorder as well as the development of treatments. Characterization of a CUD biosignature through the use of defining biological markers will greatly improve our ability to predict patient response, disease risk, accurate CUD diagnosis and potentially even identify novel targets for medications development, all the while moving CUD treatment towards an age of precision medicine. The cycling progressive nature of CUD stymies efforts to stay abstinent with vulnerability to abuse and relapse during abstinence often precipitated by impulsive behavior. The loss of impulse control has been particularly noted in cocaine-dependent subjects who also express high reactivity to cocaine-associated cues (“cue reactivity”) suggesting that impulsivity and cue reactivity are interlocked contributors to relapse, a cardinal facet of addiction. Serotonin (5-HT) neurotransmission through the 5-HT2C receptor (5-HT2CR) and 5-HT2A receptor (5-HT2AR) within the central nervous system is a critical driver of the cognitive and/or behavioral dimensions underlying impulsivity and cue reactivity. Characterization of how these receptors are regulated or altered by genetic and epigenetic means will lead to the identification of a 5-HT2R-mediate biosignature for CUD. To bridge this gap in knowledge, we first investigated the functional effects of a single nucleotide polymorphism (SNP) of the 5-HT2CR that converts a cysteine (Cys) to a serine (Ser) at amino acid codon 23 in the N-terminal extracellular domain (Cys23Ser; rs6318). We established that the Cys23Ser SNP dramatically reduces efficacy of 5-HT at the 5-HT2CR with a decrease in potency as a result of reduced plasma membrane expression from altered localization through the secretory and recycling pathway. We also demonstrate in vivo, that overexpressing the human Ser23 5-HT2CR in the medial prefrontal cortex, a key region implicated in relapse-like behaviors, of rodents exhibit dampened cocaine-seeking behavior accompanied by greater plasma membrane expression of the 5-HT2CR versus rodents expressing the Cys23 5-HT2CR. Finally, we characterized methylation patterns of the HTR2A, 5-HT2AR human gene, promoter that correlated with relapse-related behavior in cocaine-dependent participants. Taken together, we have identified genetic and epigenetic makers of the 5-HT2R system with great potential to define a high-risk relapse biosignature of CUD.
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    Deletions in SARS-CoV-2 nsp6 enhance antagonism of type-I interferon signaling
    (2023-05-01T06:00:00.000Z) Bills, Cody Jay 1993-; Routh, Andrew (; Shi, Pei-Yong (; Leiman, Petr (; Ward, Michelle (; Wilusz, Jeffrey (
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and adapt long after it first emerged in 2019. As the causative agent of the coronavirus disease 2019 (COVID-19), a tremendous effort has been made to understand the molecular pathogenesis of SARS-CoV-2. Recent research has identified nonstructural protein 6 (nsp6) as a major contributor to SARS-CoV-2 replication through the formation of replication organelles, antagonism of interferon type I (IFN-I) responses, and NLRP3 inflammasome activation, a major factor of severe COVID-19. Here, I review the most recent published findings regarding the multiple roles of nsp6 in promoting SARS-CoV-2 replication and investigate further the effect of variant nsp6 mutations in molecular pathogenesis of SARS-CoV-2, specifically the antagonism of IFN-I pathways. I demonstrate that a mutant SARS-CoV-2 USA/WA1-2020 (WA1) containing a nsp6 mutation (ΔSGF-WA1) seen in the Alpha (B.1.1.7) and Omicron sublineages (BA.2, BA.4, BA.5) is less susceptible to IFN-α treatment in African green monkey kidney epithelial cells expressing the human co-factor TMPRSS2 (Vero E6-TMPRSS2) compared to full-length WA1. Nsp6 mutations ΔSGF and ΔLSG, a similar deletion found in BA.1 nsp6, augment the ability of nsp6 to block phosphorylation of STAT1 and STAT2 in vitro compared to WA1 nsp6, thereby suppressing the IFN-I signaling pathway. Furthermore, ΔSGF-WA1 infection of primary airway cultures secretes similar levels of infectious virus and viral RNA than WA1-infected cells but produces higher levels of intracellular viral RNA than WA1 and outcompetes parental WA1 in a competition experiment. Lastly, ΔSGF-WA1 infected mice have higher levels of viral RNA than WA1-infected mice and experience lower survival rates with a longer disease period. These data suggest that variants containing ΔSGF or ΔLSG mutations are more virulent and may cause more severe disease in COVID-19 patients.
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    Commercial Sexual Exploitation of Children and School Nursing: A Non-Experimental Quantitative Study
    (2023-05-01T05:00:00.000Z) Steele, Megan D. 1985-; Verklan, Terese; O'Keefe, Mary; Campo-Englestein, Lisa; Lorenzo, Elizabeth; Nguyen, Hoang; Fraley, Hannah
    Underlying the healthy learning environments of our nation’s schools are students who are unwillingly entangled in the darkness of a form of human trafficking known as commercial sexual exploitation of children (CSEC). There is substantial and compelling evidence that CSEC is a serious problem in the United States with immediate and long-term adverse consequences for children and adolescents. These victims are attending school and school nurses are uniquely positioned in a frontline role to identify and intervene with victims of CSEC. Therefore, the overall objective of this study was to investigate the awareness, attitudes, and perceptions that Kansas school nurses have regarding CSEC. To attain the objective, a non-experimental quantitative study was conducted using the SNAPS survey. Quantitative data was analyzed and the theoretical framework, School Nurses ‘Seeing’ Youth Vulnerability to Trafficking, was applied. The study revealed that Kansas school nurses significantly lack awareness of CSEC and have negative attitudes regarding CSEC. Surprisingly, Kansas school nurses have positive role perceptions regarding CSEC and their student populations.
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    Differences between Men and Women in Risk Profile of commonly used metrics in Screening for Obstructive Sleep Apnea using NHANES data.
    (2023-05-01T05:00:00.000Z) Leary, Paul Edward 1992-; Weller, Susan (; Yu, Xiaoying (; Sultana, Rizwana (
    Obstructive Sleep Apnea (OSA) is a common chronic condition that is often underdiagnosed, especially in women due to differences in symptomology. OSA is diagnosed using polysomnography which is time intensive and impracticable as a screening method. Alternate methods for screening based on symptoms exist with varying sensitivities. This study used the National Health and Nutrition Examination Survey (NHANES) based on a nationally representative US sample (n=20,497) to test for associations between self-reported sleep apnea and risk factors, such as snoring, fatigue, hypertension, and obesity. Data was taken from the sleep questionnaire 2005-2008, along with BMI, blood pressure, and demographic data for 12,600 subjects at least 16 years old. After excluding those with missing data on the set of risk factors, the final sample was 8373. A main objective of the study was to see if there was a difference in risk profile between men and women. Multivariable model (using logistic regression) compared [snoring, observed apnea, somnolence, age, gender, hypertension, BMI, history of smoking, race/ethnicity, education, and annual household income] to self-reported sleep apnea. Results indicated that observed apnea had the highest risk of OSA (OR:7.435, 95% CI:5.698, 9.745) followed by obesity (OR:4.524, 95% CI:3.523, 5.849). Snoring, age, and annual household income had statistically differing risks between men and women.
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    Spike Processing and Protease Usage Effects on SARS-CoV-2 Pathogenesis
    (2023-05-01T05:00:00.000Z) Vu, Michelle Nguyen 1995-; Menachery, Vineet D.
    The emergence of SARS-CoV-2 and its subsequent variants have ignited a multitude of studies on the spike protein and its domains. While most studies focus on the impact of receptor binding domain changes, mutations in the C-terminus of S1 (CTS1) have largely been overlooked. The SARS-CoV-2 CTS1 contains various features that are unusual to sarbecoviruses – the furin cleavage site (FCS) and the QTQTN motif. In this dissertation, we demonstrate that the CTS1 is more complex than previously thought. We have established the aspects of cleavage site efficiency, differential protease usage, loop length, and post translational modifications that all contribute to SARS-CoV-2 pathogenesis. Using our reverse genetics system, we generated several infectious clones to examine the various features of the CTS1: ΔQTQTN demonstrated the importance of loop length and glycosylation. PQQA established that the integrity of the FCS is necessary. Omicron CTS1 mutants YKH (H655Y, N679K, and P681H) and N679K demonstrated the interplay between mutations with different contributions and expanded on the impact of post translational modifications. These studies show that alterations to any of these aspects greatly affect the pathogenicity of the virus. Together, we demonstrate that the mutations in the CTS1 are key determinants of coronavirus pathogenicity and should be especially considered in surveillance for the next coronavirus outbreak.