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 database.

Recent Submissions

  • 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.
  • 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.
  • 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.
  • 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.
  • Complement Component C1q is an Immunological Rheostat That Regulates Fc:FcγR Interactions

    So, Edward; Strome, Scott E.; Flajnik, M. F. (Martin F.) (2023)
    The complement system appears to play a paradoxical role in the pathogenesis of autoimmune diseases. Although the activation of complement contributes to tissue injury and inflammation, individuals with a genetic deficiency in C1q, the first component of the classical complement pathway, have shown to be highly susceptible for developing systemic lupus erythematosus (SLE), which is characterized by the generation of self-reactive antibodies (Abs) and aberrant lymphocyte activation. Research indicates that the development of SLE can be traced back to a breakdown in the clearance of apoptotic cells and the subsequent deposition of immune complexes (ICs). To investigate the role of ICs in autoimmune pathology, circulating immune complexes were physiologically replicated in vitro through the utilization of recombinant immunoglobulin G1 (IgG1) Fc multimers (GL-2045). IC-mediated engagement of FcγRs on NK cells resulted in the upregulation of the cosignaling molecule, 4-1BB (CD137), demonstrating a comparable response to immobilized intravenous immunoglobulin (IVIG), but not to soluble IVIG or recombinant IgG1 Fc monomers (G001). Since the binding sites for C1q and FcγRs on IgG molecules overlap, the extent to which C1q decoration of immune complexes (ICs) influences their ability to engage FcγRs remains unknown. Experimental data demonstrate that C1q engagement of ICs directly and transiently inhibits their interactions with FcγRIII (CD16) on NK cells. This inhibition occurs by C1q engagement alone as well as in concert with other serum factors. Furthermore, the inhibition of CD16 engagement mediated by avid binding of C1q to ICs is directly associated with IC size and dependent on the concentrations of both C1q and Fc multimers present. Functionally, C1q-mediated Fc blockade limits the ability of NK cells to induce CD137 upregulation, and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Although C1q is traditionally viewed as a soluble effector molecule, we demonstrate that C1q may also take on the role of an “immunologic rheostat,” buffering FcγR-mediated activation of immune cells by circulating ICs. Thus, the unchecked IC-mediated immune activation may be a functional consequence attributed to a genetic deficiency in C1q, leading to the etiopathogenesis of SLE. These data define a novel role for C1q as a regulator of immune homeostasis and add to our growing understanding that complement factors mediate pleiotropic effects.
  • Postsynaptic Control of Trans-synaptic Nanostructure

    Dharmasri, Poorna; Blanpied, Thomas A. (2023)
    Many synaptic proteins form subsynaptic nanoclusters to execute their vital functions in information transmission in the central nervous system. Nanoclusters are dynamic, with properties that change over multiple timescales and in response to specific stimuli, supporting a wide range of functional states in the synapse. We still lack a complete accounting of the factors shaping properties of synaptic nanostructure, especially the trans-synaptic alignment of nanoclusters, which strengthens communication between neurons. I tested how the nanostructure of the synapse overall is influenced by the postsynaptic compartment. First, I tested if postsynaptic cell identity results in distinct organization of core, ubiquitous excitatory synapse proteins by comparing excitatory synapses forming onto principal cells with those forming onto parvalbumin expressing interneurons (PV-INs). PV-INs contained larger synapses with larger, denser nanoclusters of postsynaptic scaffold protein PSD-95, which were also fewer in number when accounting for synapse size. Postsynaptic cell identity also impacted presynaptic organization, as Munc13-1, vital for neurotransmitter vesicle docking and priming, formed larger but less dense and more numerous nanoclusters at excitatory synapses forming onto PV-INs. Trans-synaptic alignment also differed at PV-INs, occurring further away and over a wider distance than at excitatory synapses forming onto principal cells. These results demonstrate how postsynaptic cellular contexts yield distinct trans-synaptic protein organization. Second, I tested if postsynaptic ionotropic glutamate receptors (iGluRs) influence synaptic nanostructure. Knockout of GluN1, the NMDAR obligate subunit, resulted in profound pre- and postsynaptic reorganization. The resulting synapses were larger and contained denser, more numerous nanoclusters of PSD-95 and Munc13-1. Intriguingly, more Munc13-1 nanoclusters were enriched with PSD-95, suggesting enhanced trans-synaptic alignment. Prolonged NMDAR activity block did not fully mimic the changes to Munc13-1, suggesting that NMDAR presence regulates presynaptic nanostructure. Furthermore, mutations to the GluA2 AMPAR subunit differentially altered PSD-95 nanocluster number and size. These data demonstrate that iGluRs have the underappreciated capacity to shape the organization of other synaptic proteins. Overall, my findings show both cis- and trans-synaptic nanostructure is influenced by postsynaptic factors. Specific protein expression profiles of postsynaptic cells, including variability in glutamate receptor subtypes, may provide a basis for circuit-specific diversity of excitatory synapse function and organization.
  • The Role of Bardet-Biedl Syndrome Protein 1 in the Pancreatic Beta Cell

    Bryan, Hadley; Rizzo, Megan A. (2023)
    Type 2 diabetes (T2DM) affects over 400 million people worldwide, a majority of whom are overweight or obese. In the past few decades, the prevalence of T2DM has dramatically increased, putting millions of people at risk for comorbidities and premature death. With approximately 2 billion adults overweight, understanding why some individuals who are overweight progress to T2DM while others do not is of critical importance. However, even with increasing sample sizes and genetic data thanks to genome wide association studies and meta-analyses, only a small portion of the genetic risk for T2DM has been explained. Modeling common complex diseases via monogenic causes of disease allows for the removal of many confounding factors. The obesity-related ciliopathies Alström (AS) and Bardet-Biedl (BBS) Syndromes provide a unique model for understanding why some individuals develop T2DM under very similar conditions. Both ciliopathies are characterized by early onset truncal obesity, but only AS presents with childhood T2DM. Here we endeavor to understand the relative lack of T2DM seen in individuals with BBS despite similar rates and types of obesity. To accomplish this, we first analyzed glucose and insulin data from carriers of the most common disease-causing variant of BBS finding that carriers maintained lower blood glucose levels than matched controls. Next, to elucidate whether this enhanced glycemic control was due to changes in beta cell function, we assessed various measures of glucose metabolism and insulin secretion in beta cells depleted of BBS1. Here we found that although BBS1 does not have a clear role in GCK activity or intracellular calcium level in response to glucose, there is increased beta cell response to incretin stimulation in cells lacking BBS1. Finally, we investigate pathways that are not linked to the primary cilium to uncover potential extraciliary roles of BBS1 in the beta cell. Together this work provides evidence of additional sources of variability in glucose homeostasis in both rare disease and the general population.
  • Rab27 in Tick Extracellular Biogenesis and Infection

    Butler, Lillian; Pedra, Joao H.F. (2023)
    The blacklegged tick, Ixodes scapularis, is the most medically relevant arthropod vector of infectious disease in the United States. It transmits over six pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum, but remains critically understudied. I. scapularis releases a variety of anti-inflammatory, anesthetic, and anti-coagulant molecules through its saliva during a blood meal. Recently, it was found that extracellular vesicles (EVs) are secreted in its saliva and may impact microbial transmission to the mammalian host. EV biogenesis and function have been studied in mammalian systems, but their role in arthropods of public health relevance remains elusive. In mammals, the large Rab GTPase family is known to be closely associated with EV biogenesis and many of these proteins appear in the I. scapularis genome. We report that tick Rab27 is critical for tick EV biogenesis and infection. Through an in vivo model, we have shown that silencing Rab27 impacts tick fitness. Furthermore, the tick acquires less A. phagocytophilum after Rab27 silencing. In vitro experiments show that silencing of Rab27 causes a size increase of tick EVs which indicates that Rab27 is needed to regulate vesicle biogenesis. Overall, we have observed that Rab27 plays an important role in tick EV biogenesis and the tripartite interactions between vectors, hosts, and microbes.
  • Mast Cell Modulation of the Developing Hippocampus

    Blanchard, Alexa; McCarthy, Margaret M., 1958- (2023)
    Brain development requires orchestration of overlapping critical periods defined by responsiveness to endogenous or exogenous stimuli (i.e. hormones, light, touch). Simultaneously, sensitive periods represent heightened risk to perturbations by abnormal stimuli (i.e. inflammation). Though neuro-immune cooperation between resident microglia and neurons is essential during critical periods of brain development, the contribution of infiltrating immune cells to neurodevelopmental processes is largely elusive. We discovered a critical and sensitive period created by a population of peri-hippocampal mast cells (phMCs) in the lateral ventricles abundant from birth through 2-weeks-old in the neonatal rat but absent thereafter. This epoch of phMCs is maintained by self-replication inside the brain, creating a mix of mature and immature mast cells which constitutively piecemeal degranulate. phMCs harbor a unique transcriptomic identity to skin, bone marrow and other brain mast cells including transcripts encoding colony-stimulating factors (Csf1, Csf2) essential for microglial development. Pharmacological inhibition of phMC degranulation and secretion stunts microglial maturation, illuminating the role of phMC-derived factors in driving hippocampal microglial development. In contrast, activation of phMCs releases proinflammatory chemokines, compromises the blood-brain-barrier (BBB), and recruits peripheral immune cells across the CNS for at least two days. Together, these findings indicate that a transient population of peri-hippocampal mast cells creates a critical period for microglial maturation via piecemeal degranulation in the healthy brain and a sensitive period to inflammatory stimuli. The dual role of phMCs in homeostasis and inflammation highlight an essential need to understand how the immune system can adapt its function to serve neuronal populations and prevent inflammatory disturbances that lead to neurologic disease.
  • Unraveling Cochlear Otic Mesenchyme Cells: The Role of POU3F4 During Cochlear Development

    Rose, Kevin; Hertzano, Ronna P. (2023)
    The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. One such cell type are otic mesenchyme cells (OMCs), which are a specialized type of neural crest and cranial paraxial mesoderm that express multiple unique transcription factors (e.g., POU3F4), all of which are known deafness genes, highlighting the importance of OMCs in auditory function. OMCs are known to terminally differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Interestingly, consequences of Pou3f4 mutations are diverse and include a complete loss of endocochlear potential, shortening of the cochlear duct, and defective pathfinding and survival of spiral ganglion neurons (SGNs), indicating diverse roles of POU3F4 in each OMC-derived cell type. Here, we aim to illuminate the molecular distinctness and functionality of OMCs and show how loss of Pou3f4 impacts cochlear development. By utilizing scRNA sequencing, we elucidated that OMCs divide into four transcriptionally distinct subpopulations well before the onset of hearing, each of which corresponding to one of the OMC-derived cochlear structures. Furthermore, we show OMC subpopulations display distinct functional roles corresponding to their spatial localization. We also unravel the cochlear cellular communication pathways showcasing OMCs are the main contributors of outgoing signaling during cochlear development, including both global and subpopulation specifying signaling pathways. Finally, we indicate how Pou3f4 expression regulates gene expression in each OMC subpopulation and which signaling pathways are lost in Pou3f4 mutants which may be the cause of the defects in surrounding cell types. Our data suggest that OMC diversification occurs not long after the formation of the otocyst with further refinement until the onset of hearing, well before terminal differentiation. Our data also suggests OMCs are the main contributors of paracrine signaling during cochlear development, showcasing their importance in influencing surrounding cochlear cell types. Finally, we show how loss of Pou3f4 affects each OMC subpopulation differently, leading to diverse phenotypes in Pou3f4 mutants. Without cochlear OMCs and their later terminally differentiated cell types, normal auditory function would not be feasible highlighting the importance of tissue specific mesenchymal cells in cochlear development.
  • Bacterial enzymatic combinatorial chemistry (BECC) enabled targeted lipid A modification of Shigella vaccine strains to reduce endotoxicity without compromising immunogenicity or invasiveness

    Sherman, Matthew; Ernst, Robert K. (2023)
    Shigella spp. are Gram-negative bacteria that cause severe diarrheal disease, contributing significantly to morbidity and mortality worldwide. High transmissibility and increased antibiotic resistance have propelled the development of Shigella vaccine candidates; however, no FDA-approved vaccine exists to date. We have collaborated with Walter Reed Army Institute of Research (WRAIR) to improve their live-attenuated vaccine candidates, whose only drawback during clinical trials was febrile symptoms experienced in a few individuals. In this study, we sought to resolve those unwanted side effects by detoxifying the lipid A moiety (also known as endotoxin) that is thought to contribute to the febrile symptoms observed. To do so, we employed bacterial enzymatic combinatorial chemistry (BECC), whereby lipid A modification enzymes were ectopically expressed to induce structural alterations known to dampen lipid A signaling capacity. The enzymes LpxE (phosphatase) and PagL (deacylase) were expressed in both virulent and attenuated vaccine strains of Shigella to modify the bis-phosphorylated hexa-acylated lipid A structure ordinarily present in the Shigella outer membrane. The expected modifications were confirmed using mass spectrometric and gas chromatographic analyses when the enzymes were expressed individually or in combination (both LpxE and PagL) using a construct we refer to as “Dual”. These enzyme/enzyme combinations were subsequently integrated into the chromosome using Tn7 transposition to avoid the possibility of plasmid loss during production. The impact of the induced lipid A structural alterations on innate immune signaling was assessed by stimulation of NF-?B reporter cell lines and human PBMCs with the extracted lipopolysaccharide (LPS). Additionally, in vivo reactogenicity of the LPS in a murine acute endotoxemia model was assessed. We found that dephosphorylation, but not deacylation, of lipid A, was a powerful tool to reduce LPS-mediated signaling in live-attenuated Shigella vaccines, resulting in reduced toxicity of Shigella LPS in vivo. Additionally, we found that dephosphorylation of the lipid A moiety did not impair invasion of colonic epithelia or immunogenicity in a mouse pulmonary model. Overall, this study generated Shigella vaccine candidates with reduced endotoxicity, which upon oral ingestion in humans, will ultimately have reduced reactogenicity.
  • The predictive accuracy of Shock Index in trauma outcomes in older injured patients

    Bulatao, Ilynn; Smith, Gordon S., M.B., Ch.B., M.P.H. (2023)
    The elderly is an increasing proportion of all cases treated at trauma centers. Shock index (SI) calculated as heart rate (HR) divided by systolic blood pressure (SBP), has been shown to be a good predictor of mortality and transfusion in injured patients. One limitation of SI is that its accuracy in different age groups, especially the elderly has not been fully evaluated. We studied the accuracy of admission SI in predicting early, 48-hour and in-hospital mortality, and major interventions (massive transfusion, ICU admission and surgery in 24 hours) in trauma patients admitted to a major trauma center. We examined whether age, injury severity, injury type, blood alcohol and comorbidities affected the predictive accuracy of SI. Of particular interest is the accuracy of SI in the elderly. We also compared the predictive accuracy of SI, HR and SBP. Optimal cut-points for SI were determined. SI had acceptable accuracy in predicting mortality outcomes, and ICU admission overall. Accuracy was good in the prediction of massive transfusion, and poor in the prediction of surgery in 24 hours. SI was better than HR or SBP in predicting mortality outcomes (all ages, elderly, and younger patients). However, in older patients, accuracy of SI in predicting major interventions was not different from that of SBP. Accuracy of SI in predicting 48-hour and all in-hospital mortality, and ICU admission was better in younger patients. Accuracy was also better among those with lower injury severity than in those who were more severely injured. Accuracy of SI in predicting massive transfusion was similar in older and younger trauma patients. Optimal cut-offs for predicting outcomes were lower for older patients (0.5-0.7 for mortality and major interventions) than in younger patients (0.6-0.9 for mortality and 0.6-0.8 for major interventions). Accuracy of SI in predicting all in-hospital death and massive transfusion was less among patients with elevated blood alcohol while comorbidities did not affect accuracy. In conclusion, SI is less accurate in in predicting mortality among older patients and is less accurate in predicting mortality and massive transfusion among blood alcohol-positive patients, potentially affecting its utility in triage and clinical management.

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