Full text for dissertations and theses included in this collection dates back to 2011. For older dissertations, check the library’s catalog CatalogUSMAI or Dissertations and Theses.

Recent Submissions

  • Smoking Cessation Among People With Severe Mental Illness

    Alghzawi, Hamzah Mohammad; Storr, Carla L. (2019)
    Introduction: People living with mental illnesses have a high rate of smoking and make up over half of those dependent on nicotine. A considerable body of research has shown that social support, stressful life events (SLE), receiving help for tobacco/nicotine use, intention to quit, and smoking use-related factors are associated with smoking cessation in the general population. Yet, little is known about these factors among people with severe mental illness (SMI). Purpose: This study aims to: 1) examine gender differences in the interrelations among social support, SLEs, and smoking cessation, 2) estimate the probability of remission from NUD by type of help/services received for tobacco/nicotine use (pharmacological, non-pharmacological, and both), and 3) estimate gender and racial/ethnic differences in the probability of smoking cessation among those with a history of intention to quit. Methods: A sample of 4610 people with SMI and a history of tobacco/nicotine use were identified in a public limited dataset of the 2012-2013 National Epidemiologic Survey on Alcohol and Related Conditions (NESARC-III). Four mediation and moderated mediation models were used in the first manuscript, whereas survival analyses were used in the second and third manuscripts. All analyses took into account the complex sampling design and controlled for possible confounders (i.e. sociodemographic characteristics) and covariates (i.e. comorbidity with another mental illness). Results: Total, appraisal, and tangible support in females exerted indirect effects on improving smoking cessation via decreased SLEs (total=.0094, appraisal=.0229, tangible=.0298; p<.05). The probability of remission from NUD was higher among those who received non-pharmacological services (28.5%, HR=1.95, p<.05) or those who received both services (19.6%, HR=1.52, p<.05) compared to those who only had pharmacological services (17.6%). Among those with a history of intention to quit, 31.7% had stopped. The probability of smoking cessation was highest for Hispanic females (HR=2.07, p<.05), non-Hispanic other females (HR=1.59, p<.05), non-Hispanic other males (HR=1.45, p<.05), Hispanic males (HR=1.40, p<.05), and non-Hispanic Black females (HR=1.35, p<.05) compared to non-Hispanic Black males. Conclusion: A greater understanding of subgroup differences and the correlates of smoking cessation among tobacco/nicotine users with SMI can enhance efforts to design and implement smoking cessation programs for people with SMI.
  • Impact of Host and Parasite Factors on Gametocyte Production in Plasmodium falciparum

    Vareta, Jimmy Amakwa; Laufer, Miriam K.; Takala-Harrison, Shannon (2023)
    Stalled progress in reducing the malaria burden over the past few years suggests the need to develop new interventions to augment existing ones, including interventions aimed at interrupting gametocyte transmission from humans to the mosquito vector. To develop and effectively apply interventions that target gametocytes, there is a need to understand patterns of gametocytemia observed between individuals. Gametocytemia varies by host age, season, symptom status, antimalarial drugs use, and complexity of infection; however, the underlying mechanisms of this variation are not fully understood. Complexity of infection may modulate gametocytemia in P. falciparum; however, mechanisms of how clone composition influences gametocyte production are not clear. Addressing this gap requires a genotyping assay that can detect and estimate relative clone frequency of gametocytes and asexual parasites in infections. The dissertation aims were two-fold: 1) To develop an amplicon sequencing assay to genotype P. falciparum mature gametocytes; and 2) To evaluate the impact of host and parasite factors and gametocyte production in P. falciparum infections. We identified a polymorphic region of the pfs230 gene as a marker to distinguish P. falciparum mature gametocyte clones. When evaluating the impact of host and parasite factors on gametocyte production and gametocytemia, we found that more complex and high parasite density infections were more likely to produce and harbor gametocytes. The proportion of infections that produced gametocytes were similar between age-groups and between symptomatic and asymptomatic individuals, but children and asymptomatic individuals were more likely to harbor gametocytes than adults and symptomatic individuals, respectively. These findings suggest that complexity of infection and parasite density may increase gametocyte production, but additional factors such as host immunity and duration of infection may contribute to the presence or absence of gametocytes after initiation of gametocyte production. Coupled with the development of the gametocyte genotyping assay which will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal infections, understanding the impact of host and parasite factors on gametocyte production and gametocytemia will help explain variation in gametocytemia observed between individuals. This knowledge could inform development and effective deployment of transmission interrupting interventions.
  • Attitudes Toward Medical Aid in Dying in a National Sample of Hospice Clinicians

    Becker, Todd D.; Becker, Todd; Cagle, John G. (2023)
    As of this writing, medical aid in dying (MAID) is available in 11 U.S. states and the District of Columbia, with further expansion projected. Legal protections for conscientious objection foreground clinician attitudes as substantial barriers or facilitators to MAID access for interested patients. Although upward of roughly three quarters of patients who use MAID are enrolled in hospice care, little is known about hospice clinicians’ attitudes toward MAID. The purpose of this three-paper dissertation was to examine attitudes toward MAID in a national sample of hospice clinicians. Participants were recruited from national hospice and palliative care membership associations representing the four core disciplines of the hospice interdisciplinary group (i.e., medicine, nursing, social work, chaplaincy) to complete a one-time, self-administered survey. Paper 1 examined the preliminary psychometric properties of a modified version of the only empirically evaluated scale on attitudes toward MAID. Confirmatory factor analysis results indicated that the Attitudes Toward Medical Aid in Dying Scale demonstrated factorial validity. Construct validity was established through correlation analyses targeting convergent validity (vis-à-vis a researcher-constructed measure of attitudes toward MAID) and discriminant validity (vis-à-vis a researcher-constructed measure of attitudes toward euthanasia and a scale assessing religiosity). High congeneric reliability estimates supported internal consistency reliability. Despite the favorability of these statistical results, conceptual mismatches between scale items and the U.S. practice context as defined by state laws caution against wider scale use. Further psychometric development is warranted. Paper 2 explored institutional factors tied to the hospice context of care as correlates of MAID attitudes. Using a 3-point version of Paper 1’s ordinal convergent validity item, results of a partial proportional odds model indicated that professional experience working in a state where MAID was legal and increased orientation toward patient-centeredness were both significantly associated with higher odds of more supportive MAID attitudes across each threshold of the dependent variable. Increased commitment to the hospice philosophy of care also was significantly associated with higher odds of more supportive MAID attitudes. Accounting for differing slopes across dependent variable thresholds, however, this association reached statistical significance only when estimating the odds of being in a category above the midpoint response option (neither support nor oppose). Findings support the assessment of ecological factors that drive hospice ethos and functioning when exploring attitudes toward MAID. Paper 3 explored attitudes toward being physically present throughout MAID in a hypothetical patient scenario governed by certain safeguards. The 74% of participants who indicated willingness to be present did so based on feelings of personally derived support for MAID, definitions of quality clinical care, and values from their professional training. This broad support, however, was conditioned by boundary setting though which participants described specific conditions required for their participation. In contrast, 15% of participants were unwilling to be present. These attitudes were attributed to objections to the concept of MAID, objections to participation in MAID, and perceptions that MAID is misaligned with health care. Merely 11% of participants were unsure, relating their hesitation to feelings of ambivalence and a lack of experience with MAID. The tensions that participants across samples reported experiencing with themselves, their profession, and broader society reflect a need for greater professional guidance on the safe and effective provision of MAID.
  • An Insight to Further Malaria Vaccine Development: PfSPZ Vaccine Correlates of Protection Appear to be Cross-Reactive Antibodies to Immunodominant Low-Complexity Epitopes

    Berry, Andrea; Takala-Harrison, Shannon (2023)
    Plasmodium falciparum circumsporozoite protein (PfCSP) coats the sporozoite surface and is the target of multiple malaria vaccines in development. Discovery of additional vaccine candidate antigens beyond PfCSP may lead to improved vaccines. To identify new antigens, we used peptide microarrays to map antibody responses to P. falciparum proteins in adults who received a whole organism sporozoite vaccine, PfSPZ Vaccine, and were protected or unprotected after controlled human malaria infection. We discovered antibody responses that correlate with protection, but further examination, including of two monoclonal antibodies derived from protected PfSPZ Vaccine recipients, suggests that some antibodies elicited by PfCSP cross-react with peptides representing non-CSP proteins, a demonstration of inter-protein cross-reactivity. This work provides evidence that PfSPZ Vaccine elicits inter-protein cross-reactivity, provides amino acid-specific detail of putative epitopes, and introduces opportunities for further exploration that may help to elucidate underexplored immunological mechanisms and inform development of next-generation malaria vaccines.
  • Nitric Oxide in Mucormycosis Pathogenesis

    Soare, Alexandra; Bruno, Vincent, Ph.D. (2023)
    Mucormycosis is classified by NIAID as an emerging disease and is caused by Mucorales fungi. The recent surge of mucormycosis cases among COVID-19 patients has thrust the disease and lack of available treatments into the spotlight. Clinical data suggests a lack of inflammatory responses during mucormycosis despite severe fungal angioinvasion and tissue necrosis. In this dissertation, I sought to characterize immune evasion mechanisms by Mucorales, focusing on the interaction between fungi and macrophages. Macrophages infected with Mucorales fungi block the production of nitric oxide, a free radical molecule with strong antimicrobial properties and an important signaling role in immunity. Despite the increased expression of Nos2 mRNA and inducible nitric oxide synthase (iNOS) protein in Mucorales-infected macrophages, these macrophages are unable to produce nitric oxide, even when stimulated with nitric oxide-producing stimuli (LPS and IFN-γ). My results suggest that Mucorales fungi prevent the accumulation of nitric oxide through at least 2 mechanisms: (1) removal of nitric oxide from the surrounding environment, and (2) depletion of nutrients required to make nitric oxide. Additionally, a potent nitric oxide-donor (DETA-NONOate) inhibits in vitro growth of Mucorales fungi indicating that nitric oxide may be have antifungal activity against Mucorales. At lower concentrations of DETA-NONOate that are unable to inhibit growth of Mucorales, I observed downregulation of mRNAs encoding Mucorales virulence proteins including Mucoricin, a ricin-like toxin that is critical for Mucorales pathogenesis. By downregulating these genes, nitric oxide could be attenuating the virulence potential of the fungus, rendering it less pathogenic. My research describes a new immune evasion mechanism by Mucorales fungi and presents nitric oxide as a potential therapeutic for mucormycosis.
  • Role of Galectin-3 in Airway Epithelial Barrier Integrity During Influenza A Viral Infection

    Iqbal, Muddassar; Vasta, Gerardo R. (2023)
    Influenza A virus (IAV) infects the airway and alveolar epithelia in humans, and causes seasonal influenza annually which is a major global health concern, along with COVID-19, besides exhibiting pandemic potential. In severe cases IAV infection causes acute respiratory distress syndrome (ARDS) resulting from increased alveolar permeability due to the disruption of cell-cell tight junctions. The detailed mechanisms involved, however, remain to be fully elucidated. Galectins are β-galactose-binding lectins implicated in diverse cellular functions as well as in pathogen infections, including those from respiratory viruses. In previous studies from our lab on a murine model, IAV infection enhanced galectin-3 (Gal-3) secretion in the bronchoalveolar fluid. In the present study, I investigated in vitro the potential role of the secreted Gal-3 in airway epithelial barrier function during IAV infection. The results thus acquired using the human airway epithelial A549 cells indicate that IAV infection leads to a significant desialylation of the cell surface, exposing the sub-terminal β-galactose ligands, with a concomitant increase in the binding of recombinant galectin-3 (rGal3). I detected potential Gal-3 receptors CD147, integrin-β1 and MUC1 on the surface of A549 cells, and observed an increase in the secretion of matrix metalloproteinases MMP2 and MMP9 after exposure of these cells to rGal3. Furthermore, I observed disruption of the A549 cell surface distribution of tight junction proteins occludin and ZO-1 upon both IAV infection and Gal-3 exposure, as well as significant increase in the monolayer permeability. Using rGal-3, I demonstrated its direct interaction with the A549 cell CD147 as well as integrin-β1 which possibly mediated the above-mentioned cellular effects and validated some of the key findings in A549 cells from this study to the primary small airway epithelial cells (SAECs). In the end by searching for the sequence variants in the Gal-3 encoding genes LGALS3 in two public genetic databases, I found three rare variants that might alter protein function and one that was associated with the phenotypes indicative of a role for Gal-3 in influenza outcome: including influenza vaccine, flu treatment, acute sinusitis, and emphysema.
  • Microtubules as a therapeutic target in Duchenne muscular dystrophy

    Vanegas, Camilo; Ward, Christopher, Ph.D. (2023)
    Microtubules (MT) are dynamic polymers of tubulin protein whose post-translational modifications regulate MT interactions with other proteins, including intermediate filaments and actin. This integrated cytoskeletal network performs a vast array of cellular functions, including cell movement, transport of cellular cargo, defining cell shape and stiffness, and transmitting mechanical information to proteins that generate biological signals (mechanotransduction). My work is in Duchenne muscular dystrophy, where the absence of dystrophin leads to the proliferation of microtubules marked by an increase in posttranslational (PTM) modifications to their tubulin including detyrosination (deTyr-tubulin) and acetylation (acetyl-tubulin). The consequences of this change are increased cytoskeletal mechanics (i.e., stiffness) of the muscle fiber and increased mechanotransduction through NADPH Oxidase 2 (Nox2) dependent reactive oxygen species (ROS) and calcium signals during contraction that together have been implicated in the contraction injury that drives the pathology. My work addressed two open questions related to the pathobiology of these microtubule changes independent of dystrophic disease (Aim 1; Chapter 2) and the potential for targeting their reduction as a therapeutic option for halting or slowing disease progression (Aim 2; Chapter 3).
  • Biological, psychological, and sociocultural contributions to pain processing in the central nervous system: A whole-person approach to understanding chronic pain

    Cundiff-O'Sullivan, Rachel; Colloca, Luana (2023)
    Introduction: Chronic pain affects approximately 20% of the global population, yet effective treatments remain elusive. This study employs a biopsychosocial model, recognizing the importance of all facets of life, to investigate the complex interactions of biological, psychological, and sociocultural factors influencing pain outcomes and pain processing in the central nervous system. Methods: This project utilized functional magnetic resonance imaging to assess pain severity, interference, and endogenous pain modulation via placebo analgesia in a sample of participants with temporomandibular disorder. Brain-age difference was estimated via a pre-trained Gaussian Process Model using cortical thickness as a predictor of pain outcomes. Structural equation modeling and high dimensional multivariate mediation were employed to examine the influence of pain catastrophizing (PC) on pain outcomes. Differences in pain outcomes between religious/spiritual (R/S) and atheist participants were also explored. Results: TMD participants exhibited accelerated brain aging which was associated with pain severity and interference but not placebo analgesia. Structural equation modeling revealed that PC did not mediate pain outcomes or influence placebo analgesia. However, resting-state functional connectivity between the hippocampus and cuneal cortex mediated the relationship between PC and pain interference. Although there were no significant differences in behavioral outcomes between R/S and atheist participants, differential connectivity underlying these outcomes was found, such that atheist participants exhibited greater reliance on low-level sensory input compared to R/S participants who utilized higher cognitive regions for better emotional regulation of pain. Conclusion: This work provides valuable insights into the neurobiological mechanisms underlying how brain age, PC, and R/S factors influence chronic pain and placebo-induced pain reductions. By unraveling the complexities of pain processing using a whole-person approach, this research promotes a more holistic understanding of chronic pain and contributes to the development of more effective interventions for individuals suffering from chronic pain.
  • Immunomodulatory Nanoparticles as a Multimodal Approach to Attenuate Immune Dysregulation in Severe Inflammation and Sepsis

    Truong, Nhu; Pearson, Ryan M. (2023)
    Sepsis, a life-threatening condition triggered by an uncontrolled immune response to infection, currently lacks an FDA-approved therapeutic intervention to enhance patient survival. Severe inflammatory conditions can disrupt the balance of histone acetyltransferase (HAT)/histone deacetylase (HDAC) activity, leading to global cellular hypoacetylation. Histone deacetylase inhibitors (HDACi) restore acetylation profiles and reverse transcriptional silencing. Suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, was modified by para-hydroxymethylation (termed SAHA-OH), which resulted in a favourable reduction in SAHA-associated toxicity under inflammatory lipopolysaccharide (LPS) challenge. SAHA-OH was incorporated into immunomodulatory nanoparticles (iNPs), previously developed by our lab, to form iNP-SAHA using a prodrug approach through the covalent modification with poly(lactic-co-glycolic acid) (PLGA). iNP-SAHA treatment significantly reduced proinflammatory cytokines in vitro and in vivo, improved the viability of LPS-stimulated primary macrophages, and enhanced survival of mice in an LPS-induced endotoxemia model. iNP-SAHA treatment did not significantly improved mice survival compared to the iNP treatment alone; however, the synergistic anti-inflammatory properties of iNP-SAHA are potentially promising for future exploration in alternative models of inflammatory disease. We evaluated the efficacy and cellular mechanism of iNP activity using a clinically relevant cecal ligation and puncture (CLP) murine model of polymicrobial sepsis. iNPs, when administered as an adjuvant to antibiotics, significantly improved survival compared to antibiotics alone. Interestingly, iNP treatment marginally affected local and systemic cytokine profiles, despite mitigating organ dysregulation. Minimal effects on immune cell proportions at local sites were observed, but iNP treatment normalized monocyte levels in peripheral blood and alveolar macrophages in lung tissues. Further studies enumerated that iNPs modulated cellular adhesion and migration surface marker expression as well as apoptotic levels on immune cells. These findings highlight the potential of iNPs as an adjunctive therapy for sepsis, particularly when combined with antibiotics, suggesting promising prospects for future clinical translation. Lastly, a high-throughput microfluidic approach for iNP formulation to enable facile scale-up was developed. We optimized the microfluidic method and the impact of polymer and surfactant concentrations, surfactant chemistry, flow rate ratio (FRR), and anti-inflammatory activity. This work demonstrated a controlled and reproducible microfluidic method for iNP formulation, showcasing their inherent anti-inflammatory properties and offering a promising avenue for inflammation management.
  • Conversion of Small-Molecule Inhibitors into Heterobifunctional Compounds in the Discovery of Novel Chemotherapeutics

    Chan, Alexandria; Fletcher, Steven (2023)
    Heterobifunctional polypharmacologic agents are compounds that have individual pharmacophores for at least two separate biological targets. Our work spans two distinct sets of heterobifunctional molecules: 1. Polypharmacologic agents that inhibit two proteins known to contribute to the disease state, and 2. Protein degraders: Proteolysis targeting chimeras (PROTACs) and molecular glues. Both types of protein degraders function through recruiting an E3 ligase to the protein of interest, resulting in a hijacking of the ubiquitin-proteasome system, and the subsequent destruction of the target protein. The use of type 1 compounds is rapidly growing as such polypharmacologic agents are postulated to exhibit distinct advantages over the monovalent, parent drug compounds from which they are constructed, including the ability to increase therapeutic effect, lower effective dosage, and circumvent treatment resistance. Type 2 compounds – the protein degraders – can eliminate a target of interest, requiring the cell to resynthesize the protein to regain its cellular function. These compounds may have a catalytic mechanism of action wherein the compounds are recycled after mediating the degradation of the target protein, thereby requiring non-stoichiometric amounts of drug while also directly countering resistance that manifests through target protein upregulation. Moreover, such degraders retain activity with resistant proteins where traditional, non-covalent small-molecule drugs fail. Due to these advantages, there is increasing enthusiasm that targeted protein degraders may herald a new class of anti-cancer therapeutics. Herein, our efforts towards the discovery of heterobifunctional pharmaceuticals for the treatment of drug-resistant hematological malignancies are described.
  • Novel Bioactive Low-Shrinkage-Stress Composite with Antibacterial and Remineralization Properties

    ALHUSSEIN, ABDULLAH; Xu, Huakun H. (2023)
    Methacrylate-based resin composites are frequently employed in dentistry for their aesthetic qualities, durability, and adhesive properties. Nevertheless, these restorations generally exhibit a lifespan of 5 to 10 years, with recurrent caries and tooth fractures being primary failure factors. Marginal integrity and the absence of bioactivity at the tooth-restoration junction contribute to recurrent caries development. Consequently, this dissertation endeavors to introduce a novel bioactive low-shrinkage-stress nanocomposite, featuring dimethylaminododecyl methacrylate (DMADDM) as an antibacterial agent, as well as remineralization nanoparticles of calcium fluoride (nCaF2) and nanoparticles of amorphous calcium phosphate (NACP), with the potential of increase the longevity of dental restoration and protect tooth structure. All novel formulations of low-shrinkage-stress composite were subjected to a series of mechanical, antibacterial, cytocompatibility, and ion release assessments. First, we investigated the optimum concentration of DMADDM that can be incorporated with a low-shrinkage-stress composite without compromising mechanical properties. We found that incorporation of up to 5% DMADDM into a low-shrinkage stress composite efficiently inhibited Streptococcus mutans (S. mutans) biofilm commonly associated with secondary caries. This potent antibacterial effect is achieved while maintaining excellent mechanical properties and minimizing polymerization shrinkage stress, potentially improving the long-term success of dental restorations. Next, we investigated the antibacterial and cytocompatibility of the incorporation of 3% DMADDM with 20% nCaF2 or 20% NCAP into a low-shrinkage-stress nanocomposite. We found that incorporating DMADDM with either nCaF2 or NACP into a low-shrinkage-stress nanocomposite provides a potent antibacterial effect against S. mutans biofilm while maintaining excellent mechanical properties. In addition, the novel formulations demonstrated excellent biocompatibility against human gingival fibroblasts and dental pulp stem cells. Lastly, we investigated the ions release and antibacterial properties against a salivary biofilm for our innovative formulations. The innovative mixture of DMADDM, NACP, and nCaF2 demonstrated strong antibiofilm effects on salivary biofilm, while concomitantly releasing a significant amount of remineralizing ions. This nanocomposite is a promising dental material with antibiofilm and remineralization capacities, with the potential to reduce polymerization-related microleakage and recurrent caries.
  • Utilizing Pharmacometrics to Facilitate Generic Drug Development of Orally Inhaled Products and Optimize Pharmacotherapy of Antifibrinolytics

    Li, Shuhui; Gobburu, Jogarao (2023)
    This thesis has two parts. The first part is related to the pharmacokinetic (PK) batch-to-batch variability of orally inhaled products, which may pose challenges for generic product development. I applied the techniques of pharmacometrics to propose and evaluate alternative PK bioequivalence (BE) study designs using Advair Diskus as an example product, aiming to facilitate generic development. First, population PK models for Advair Diskus were developed and qualified to simulate PK BE study. Next, the effect of batch-to-batch variability on the establishment of BE was evaluated using the developed models. Batch-to-batch variability substantially elevates the probability of reaching a false conclusion in a PK BE study for equivalent and inequivalent comparisons. Therefore, ignoring batch-to-batch variability when presenting will increase the risk of either patients being treated with an inequivalent formulation or pharmaceutical companies not obtaining approval for an equivalent formulation. This calls for alternative PK BE approaches to account for the batch-to-batch variability. I proposed and evaluated a two-phase study framework that uses a pilot study to select reference and test batches for the pivotal BE study. A parallel design with ≥ 12 patients per sequence or a crossover design with ≥ 6 patients per sequence is recommended for the pilot study design. The proposed criteria for selecting batches based on the pilot study results include (1) 0.9 ≤ T/R ≤ 1.11 and (2) higher conditional power. The two-phase study design offers the flexibility to select batches in a PK study to minimize the impact of batch-to-batch variability on the generics development. The two-phase framework might be applied to other products with similar characteristics and high batch-to-batch variability in the reference products. The second part of this thesis used pharmacometrics to optimize the pharmacotherapy of an anti-fibrinolytic, tranexamic acid (TXA), in special patient populations. The PK and pharmacodynamics (PD) of TXA in special populations are understudied; therefore, the PK/PD-driven optimal doses for them are unknown. First, I characterized the PK and PD of TXA in pregnancy and found that pregnant women have up to 30% higher clearance and volume of distribution than the general non-pregnant population. A dose of 650 mg maintains both PK and PD targets for > 1 hour in most patients, which is recommended as the postpartum prophylactic dose for future confirmatory clinical studies. In addition, I evaluated a current dosing regimen for cardiac surgery patients who use cardiopulmonary bypass (CPB) during their surgeries from a PK perspective. This dosing regimen consists of a long infusion of TXA at 100 mg/hr for 5 hours before CPB initiation, a 1 g bolus of TXA at CPB initiation, and another 1 g bolus at the end of CPB. While kidney function affects the clearance of TXA, and the CPB procedure increases the volume of distribution of TXA, the current dosing regimen was confirmed to provide sufficient TXA exposure (15 mg/L) from CPB initiation till 3 hours post-CPB, achieving the therapeutic goal. Both studies contribute to understanding how TXA dosing can be optimized in special patient populations.
  • Role of Social Determinants of Health on HIV Testing and Treatment Cascade

    Mohanty, Kareshma; Stafford, Kristen Alyce (2023)
    Introduction: The Joint United Nations Programme on HIV and AIDS proposed that to achieve epidemic control of HIV by 2025; 95% of all people living with HIV are aware of their status, 95% of people diagnosed with HIV receive sustained antiretroviral therapy (ART), and 95% of all people on ART are virally suppressed (VLS). The 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS) found that in Nigeria, while only 47% knew their status, 96% had received ART, and 81% had achieved VLS. Social determinants of health (SDH), like wealth index (WI), have been shown to play a significant role in HIV in western countries, but the evidence has been limited and mixed in Nigeria. Identifying political, social, cultural, demographic, economic, and behavioral indicators of SDH, can better explain and address the disparities in the HIV epidemic, especially in the testing and treatment cascade, that are preventing the UNAIDS targets from being met in Nigeria. Objective: Examine the role of singular and composite indicators of SDH on the 95-95-95 targets: HIV testing, receipts of ART, and VLS in people living with HIV in Nigeria. Additionally, examine if wealth modifies the relationship between SDH indicators and the three 95 targets. Methods: Using the World Health Organization-SDH framework and Factor Analysis, I constructed composite indicators of SDH for Nigeria from various population-level survey data sources. Scores from the sub-indices and Global Terrorism Index were categorized as low, medium, and high, and individual or states were assigned one of these categories. Subsequently, I examined the association of the composite and singular SDH factors with HIV testing, receipt of ART, and achievement of VLS through survey-weighted multivariable logistic regression. Additionally, I examined the significance of the SDH indicators with the testing and treatment outcomes, by each of the wealth quintiles. Results: Out of the seven sub-indices constructed, only Access to Public Services, Crime & Conflict, Government Corruption, and Government Performance met the internal reliability criterion (Cronbach alpha > 0.7). Global Terrorism Index was constructed based on the prescribed methodology. When examining HIV testing, the first target in the 95-95-95 UNAIDS strategy, medium levels of Government Corruption, lower/medium Government Performance, and high Terrorism was associated with lower testing. Unemployment, living in rural areas, and married before 18 years of age were significantly associated with lower odds of HIV testing. For receipt of ART, second 95-95-95 target, low/medium treatment coverage was associated with lower odds of being on treatment. Younger age, male sex, being single, and living in rural areas were the singular factors associated with lower receipt of ART. Finally, for the third 95-95-95 target, only singular SDH, like lack of condom usage during sex, CD4 count (<500), and ethnic languages were associated with lower VLS. Wealth modified the relationship between the social determinants and HIV testing and treatment, but the role was weak. Wealth may increase the gap between the lowest and highest wealth index strata; HIV-related disparities experienced might be more pronounced between the two ends. Conclusion: Understanding and addressing structural determinants like political stability, terrorism, gender equality, accessibility to public services, and treatment facility coverage, rather than individual-level behavioral factors, could help Nigeria achieve the 95-95-95 targets.
  • Regulation of Epidermal Skin Immunity by a Tick Bite

    Marnin, Liron; Pedra, Joao H. F. (2023)
    Hard ticks are hematophagous arthropods of public health and veterinary importance. Following a tick bite, these arthropods take prolonged, continuous bloodmeals that facilitate pathogen transmission. Successful bloodmeals are attributed to components of the tick saliva that alter inflammation, inhibit hemostasis, and block pain and itch responses in the mammalian skin. Recent studies have reported that extracellular vesicles (EVs) in the saliva enable tick feeding and redirect skin immunity. Notably, the skin epidermis, which interfaces with the external environment, has been mostly neglected when studying the relationship between ticks and their mammalian hosts. Here, we report that Ixodes scapularis EVs enable tick feeding by affecting epidermal γδ T cells frequency and co-receptor expression at the skin site. Epidermal γδ T cells have a critical role in wound healing and interact with keratinocytes, which comprise 95% of the epidermal layer. We further coupled entomological approaches with flow cytometry, single cell RNA sequencing, and animal strains devoid of epidermal γδ T cells to demonstrate that tick EVs disrupt networks involved in keratinocyte proliferation, suggesting an effect on epithelium wound repair. Collectively, this work broadens our knowledge of ectoparasitology, vector-host interactions, and principles of immunology.
  • Characterization of Plasmodium vivax transcriptome by Single Cell RNA-sequencing in a non-human primate model

    Hazzard, Brittany; Serre, David (2023)
    Malaria is a significant cause of morbidity and mortality worldwide, responsible for 241 million clinical cases and 627,000 deaths in 2020 according to the World Health Organization. Plasmodium vivax infections, while less deadly than the more widely studied Plasmodium falciparum, is a significant risk to many people in SE Asia and South America. P. vivax infections often consist of heterogenous populations of parasites at different developmental stages and with distinct transcriptional profiles, which complicates gene expression analyses. The advent of single cell RNA sequencing (scRNA-seq) enabled disentangling this complexity and has provided robust and stage-specific characterization of Plasmodium gene expression. However, scRNA-seq information is typically derived from the end of each mRNA molecule (usually the 3’-end) and therefore fails to capture the diversity in transcript isoforms documented in bulk RNA-seq data. P. vivax infections also often contain multiple, genetically-distinct, parasites but the consequences of this polyclonality on the regulation of asexual parasites, their sexual differentiation, and the transmission to a new host remain unknown. Here, we describe the sequencing of P. vivax scRNA-seq libraries obtained from a non-human primate model using traditional Illumina sequencing and Pacific Biosciences (PacBio) chemistry to characterize full-length Plasmodium vivax transcripts from single cell parasites, as well as experimental infections with the NIH-1993-F3 and the Chesson strains of P. vivax. Our results show that many P. vivax genes are transcribed into multiple isoforms, primarily through variations in untranslated region (UTR) length or splicing, and that the expression of many isoforms is developmentally regulated. Our experimental coinfection experiments revealed limited polyclonality when the strains were inoculated one after the other, but yielded robust polyclonal infections by simultaneous infection. In contrast to our hypothesis, this polyclonality did not seem to modify the regulation of individual parasites, or their sexual commitment. Overall, this work yielded unique insights on the mechanisms regulating the establishment of polyclonal P. vivax infections, and their consequences for disease transmission, and provided a validated framework to further study of Plasmodium polyclonal infections.
  • Nocifensive Behavior and Transcriptomic Biomarkers in a Translational Pig Model of Spared Nerve Injury

    Weiss, Miriam; Nahm, Eun-Shim (2023)
    Neuropathic pain is a prevalent condition that significantly impacts quality of life. Translational animal models are needed to understand the mechanisms and develop novel treatments. The spared nerve injury (SNI) model is a well-validated neuropathic pain model that causes allodynia and hyperalgesia. While the SNI was originally developed in rats, rodent models have translational limitations. Pigs, by contrast, share many characteristics with humans, making them uniquely suitable for modeling painful conditions. Objective: The goal of this study was to test the feasibility and efficacy of a translational pig model of SNI. Methods: The left common peroneal nerve of SNI pigs was ligated, while the nerve remained untouched in sham pigs. Mechanical and dynamic allodynia, thermal hyperalgesia, pressure, and gait alterations were assessed over four weeks. Video footage was recorded during testing and analyzed for a pig grimace scale. Blood and spinal cord tissue were harvested and sequenced to determine the presence of differentially expressed genes and associated enriched pathways. Results: Surgical ligation of the common peroneal nerve was feasible and safe. However, there were significant floor and ceiling effects for mechanical and dynamic allodynia and thermal hyperalgesia testing. Females showed lower pressure tolerance than males over time at the medial left (p=0.003), medial right (p=0.003), lateral left (p=0.041), and lateral right (p=0.003) testing locations. The left side had a lower threshold tolerance than the right side for both medial (p<0.001) and lateral (p=0.03) locations, although this difference was not significant over time. There was no significant difference in threshold tolerance between the sham and SNI groups. There were no significant changes in gait. The grimace scale showed substantial interrater reliability for ear position (kappa=0.73) and moderate interrater reliability for snout tension (kappa=0.43). Several pathways related to inflammation and wound healing were differentially expressed between the SNI versus sham groups and left versus right sides of the SNI animals. Conclusion: While the pig model of SNI is feasible, special considerations are needed when acclimating animals and calibrating study equipment to avoid floor and ceiling effects. Relevant enriched pathways may be targets of interest for future neuropathic pain research.
  • The Role of Pathogenic Mimicry Response Leading to Mitochondrial Dysfunction in Ovarian Cancer

    Stojanovic, Lora; Rassool, Feyruz V. (2023)
    Approximately 10-15% of epithelial ovarian cancer (EOC) patients that have BRCA mutations and homologous recombination deficiency (HRD) are successfully treated with FDA-approved poly (ADP-ribose) polymerase (PARP) inhibitors resulting in synthetic lethality and antitumor responses. While some patients with BRCA-wildtype EOC have shown a response to single agent PARP inhibitor (PARPi) therapy, the majority do not respond. This suggests the need for the development of novel combination therapies for EOC. DNA methyltransferase inhibitors (DNMTis) activate transcriptionally silenced genes and repeat sequences, including endogenous retroviruses (ERVs), that increase dsRNA in the cytosol, leading to interferon (IFN) signaling and antitumor immune responses, in a mechanism known as viral mimicry. Combining PARPi with DNA methyltransferase inhibitors (DNMTis) have been shown to not only induce synergistic cytotoxicity in multiple cancers, including EOC with wildtype BRCA, but also increase both cytosolic dsDNA dsRNA, leading to STING-mediated interferon (IFN) and inflammasome (NFKB-TNFα) signaling, resulting in generation of HRD. Recent research shows that mitochondria (mt) are a gateway to IFN/inflammasome signaling and that release of mtDNA into the cytosol can activate innate immune signaling pathways, including STING. Importantly, recent reports suggest that the little studied ds RNA/DNA sensor ZNFX1, through interaction with mt proteins, is an important regulator of the IFN response in viral infection. Here, I show that in EOC, DNMTis and PARPi treatment increases expression of ZNFX1 and colocalization with mitochondrial antiviral protein MAVs in the mt outer membrane. This combination drug treatment also induces mt reactive oxygen species (ROS) and fragmented mtDNA release into the cytosol, resulting in STING-dependent inflammasome signaling. ZNFX1 knockout attenuates these dynamics, thus defining ZNFX1 as essential for interferon /inflammasome signaling induced by mtDNA damage. Overall, we suggest that ZNFX1 represents a novel master regulator of mt-mediated STING-dependent IFN and inflammasome signaling in OC and other solid malignancies.
  • Exploring the Design and Delivery of Sexual Health Education for US-based Doctor of Physical Therapy Students

    Felter, Cara; Gordes, Karen L. (2023)
    Curricular guidelines to prepare United States-based Doctor of Physical Therapy (DPT) students to address sexual health do not exist. The literature about this topic is scant and does not describe how DPT faculty design and deliver educational content related to sexual health. The available literature does not link curricular design and delivery to learning theories. This qualitative, phenomenological study explored how DPT educators (n=15) who teach sexual health determine the curricula for this topic and how they design learning experiences for their students. Participants reported a variety of content, design and delivery strategies, and resources that they use to guide curricula. Most participants did not use learning theories to guide their content, but they did advocate strongly for content inclusion in both the didactic and clinical components of their respective DPT programs. Student feedback was a major influence on content design and delivery. While research is still warranted to further understand the experiences of students learning this content, and the opinions of the patients who ultimately participate in physical therapy, this study provides a deeper understanding of the sexual health content that is currently taught in DPT programs.
  • Age-Related Deficits in Natural Killer Cell-Derived Interferon-Gamma Production Contribute to Severe Bordetella pertussis in Infant Mice”.

    Mitchell, Ashley Elizabeth; Carbonetti, Nicholas H. (2023)
    Many respiratory infections are injurious in infants; however, the etiology of age-associated susceptibility is unknown. One such bacterial pathogen is Bordetella pertussis. In healthy adults, B. pertussis infection typically occurs within the lungs; however, systemic dissemination in infants can cause severe disease. In mouse models, NK cell- and IFN-?-deficient adult mice suffer disseminated lethal infection resembling the infant condition. Accordingly, we hypothesized that infants exhibit age-related deficits in NK cell frequency, maturation, and responsiveness to B. pertussis, associated with low IFN-? levels. To evaluate this hypothesis, we characterized an age-dependent mouse model of pertussis and further delineated the mechanisms underlying age-dependent susceptibility. Analogous to human disease, increasing age in mice is associated with declining susceptibility. Infant mice develop the most severe aspects of B. pertussis infection, including bacterial loads, disseminated infection, leukocytosis, and lethality. Moreover, this increase in protection directly correlated with an increase in innate NK cell-derived IFN-γ production and type 1 cytokines during infection. Postnatal day 7 infant mice had fewer pulmonary NK cells than adult mice during infection. Unlike NK cells from adult mice, Infant NK cells express high levels of CD27, which suggests an immature state with limited effector functions, most notably an inability to produce IFN-?. In addition, the infant's lung showed no up-regulation of IFN-?-inducing cytokine IL-12p70. Adoptive transfer of adult NK cells into infants, or treatment with exogenous IFN-?, significantly reduced bacterial dissemination. These data indicate that the lack of NK cell-produced IFN-? significantly contributes to infant fulminant pertussis. Future studies can explore how age impacts the capacity to produce IL-12/IFN-? during development and how intrinsic age-related differences in NK cell responses could impact early protection during B. pertussis infection.
  • Development of Mass Spectrometric Methods for Analysis of Sphingolipids and Oligonucleotides

    Tran, Anh Quan; Jones, Jace W., 1978- (2023)
    Mass Spectrometry (MS) is a powerful method for analysis of biomolecules due to its selective and sensitive analytical benchmarks, providing information about their structures and abundance, further enabling their functions to be studied. This thesis focuses on the analysis of sphingolipids and oligonucleotide therapeutics due to knowledge gaps in their MS analysis. Sphingolipids (SPs) are pivotal membrane lipids with very diverse structures, setting the stage for challenging analysis. To enhance analytical performance, lithium was incorporated into the MS workflow, consolidating adducts and simplifying the mass spectrum and proving informative fragmentation patterns. An extraction protocol was developed that integrated a base hydrolysis step for SP enrichment using lithium hydroxide, which effectively hydrolyzed esterified lipids with the added benefit of lithium adduct consolidation. A high throughput screening method was developed with lithium adduct consolidation, resulting in detection enhancement of low abundant SPs. A multidimensional analytical platform was developed to provide higher structural quality of SPs, utilizing off-line liquid chromatograph (LC), ion mobility and high-resolution tandem MS. The off-line LC provided separation and allowed lithium adduction for further analysis while ion mobility and elevated energy tandem MS are used to structurally characterize and resolve SP isomers. Data processing, analysis and visualizations techniques were also developed, tailored to the specific needs of the workflow. A MS imaging method for spatial localization of SPs was developed with lithium adduction and on tissue hydrolysis to enhance SP analysis of intact tissue. OGN (oligonucleotide) therapeutics are becoming increasingly more popular for complex diseases. Despite this, rigorous analytical techniques to monitor biomanufacturing processes and the final formulation product are lacking. A high-throughput screening method was developed to verify the molecular weight and to scan for non-isomeric impurities while minimizing alkali salt contamination that notoriously adduct to OGNs during ionization. LC methods were developed for both analytical and preparative separation while tandem MS was used to confirm their sequence. For isomeric impurities, ion mobility was utilized to interrogate and compare the extremely complicated diastereomeric composition of various OGN drug products.

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