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

  • The Clinical Translation of Cardiac Xenotransplantation

    Goerlich, Corbin; Mohiuddin, Muhammad M.; Singh, Nevil (2022)
    Patients with end-stage heart failure requiring heart transplantation die simply because allografts are in short supply. Cardiac xenotransplantation from genetically-modified pig source animals has been proposed to bridge the gap between supply and demand. However, there are two predominant barriers to clinical translation and are poorly understood. The first, is a type of primary graft dysfunction, termed perioperative cardiac xenograft dysfunction (PCXD), which causes failure of the xenograft within 48 hours after transplantation. The second, post-transplantation xenograft growth, which causes a life-limiting diastolic heart failure within the first month after transplantation. We demonstrate that PCXD can be overcome with cardiac preservation techniques that minimize ischemia, either with blood-based cardioplegia induction or non-ischemic continuous preservation (NICP). In a heterotopic PCXD model, we further demonstrate that PCXD is likely a phenomenon rooted in xenograft dysfunction resulting from activation of innate immunity within the xenograft. While further studies need to be done, we demonstrate evidence that PCXD is augmented by the synergistic inflammatory effects of both cardiopulmonary bypass and cross-species transplantation. We also provide evidence that polymorphisms from TLR4 of S. scrofa (swine) compared to H. sapiens, may explain a possible target for the unique inflammatory signaling, that results in xenograft dysfunction after cardiac xenotransplantation. We also demonstrate that post-transplantation xenograft growth is multifactorial, but can be limited by growth hormone receptor knockout donors, along with genetic modifications that reduce immunogenicity of the xenograft. This growth, within 6 months after transplantation, is not a result of physiologic mismatch or cardiomyocyte hypertrophy. Therefore, recipients do not need to be treated for tachycardia and hypertension as previously thought. Lastly, we demonstrate the clinical translation of cardiac xenotransplantation by applying knowledge and expertise obtained from these studies. Expanded access (“compassionate use”) FDA authorization was based on demonstration of principle from our preclinical model. An ECMO-dependent patient without other therapeutic options was transplanted a genetically-modified cardiac xenograft, with 10-gene edits, combined with non-ischemic cardiac preservation and anti-CD40 monoclonal antibody-based immunosuppression. The patient was able to successfully wean from ECMO, participate in active rehabilitation and survived 60 days after transplantation without evidence of rejection.
  • Investigating the Role of Microglia and Extracellular Vesicles in Spinal Cord Injury-Induced Brain Dysfunction

    Khan, Niaz; Faden, A. I. (2022)
    Spinal cord injury (SCI) causes brain neurodegeneration leading to cognitive and affective changes, including memory loss and mood alterations. Using SCI models, we and others have demonstrated that progressive neurodegeneration is accompanied by neuroinflammation, including sustained microglial activation. The primary goal of this dissertation was to test the hypothesis that SCI triggers brain microglia-mediated neuroinflammation and secondary neurological dysfunction and to study the underpinning mechanisms including changes in systemic and central extracellular vesicles (EVs). First, we probed the mechanisms responsible for microglia activation and examined the effect of pharmacological depletion of microglia on posttraumatic neuropathology and cognitive/depressive-like behavior in a mouse SCI model. Microglial depletion significantly improved neuronal cell loss in key brain regions and associated cognitive/depressive-like behavioral outcomes after SCI. The transcriptomes of the spinal cord and brain were also substantially altered, supporting our hypothesis that microglia significantly contribute to changes related to inflammation, neurotransmission, and apoptosis after SCI. Second, we studied changes in circulating EVs after SCI. EVs are biological nanoparticles released from cells that contribute to intercellular communication and can become altered with disease. We found a significant increase in plasma tetraspanin CD81+ EVs after SCI at 1d post-injury. Surface CD81 was decreased on astrocytes at the injury site, suggesting that these cells may release CD81+ EVs into circulation. Total plasma EV microRNA content was also significantly modified, similar to the profile previously described in inflammatory astrocyte EVs. Notably, when injected into the cerebroventricular system, plasma EVs from SCI mice increased brain expression of several inflammatory genes, including markers of astrocyte reactivity. Finally, we examined the brain transcriptional profile and EV changes 19 months post-SCI in male and female mice. While we observed strong sex-dependent differences in the overall brain transcriptome after SCI, the homeostatic microglial phenotype was reduced in both sexes. Chronic SCI increased EV count in the brain and modified their microRNA content, which may explain the observed transcriptional changes. Plasma EV markers were also elevated late after injury, especially in males. Collectively, these experiments are the first to characterize EV dynamics after SCI and suggest that EVs may be involved in posttraumatic brain inflammation.
  • Characterization of Temperature Dependent Activity in a Model Polyextremophilic Beta-galactosidase Enzyme Through Kinetic and Structural Analysis

    Laye, Victoria; DasSarma, Shiladitya (2022)
    The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for polyextremophilic enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. Double-mutated enzymes were constructed and tested for temperature effects from 0-50 ºC, including two new tandem residue pairs, two potential loop insertions, and pairwise combination of the single residue mutations. The observed temperature and salinity dependent kinetic effects were compared to root-mean-square fluctuations to determine structural mechanisms of polyextremophilic activity. All the mutated enzymes were found to be more catalytically active at higher and/or less active at colder temperatures compared to the wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface-exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface-exposed residue, I476V, was paired with N251D or V482L, primarily a kcat effect was observed. The temperature dependent kinetic effects were continued to 25-50 ºC with a dominant Km effect observed in all mutated enzymes. By deleting the identified insertions/loops, we are able to determine the kinetic effects of these insertions. The domain B deletion resulted in a less active enzyme overall converging with the wild-type at higher temperatures while the domain C deletion resulted in reduced cold-activity and improved activity at higher temperatures. Domain B may confer cold-activity without changing the thermal stability while domain C confers cold activity, at least in part, at the expense of activity at higher temperatures. With molecular dynamics simulations, increased flexibility was observed with higher temperature and salinity and in the mutations, indicating that the enzyme’s function is related to its flexibility and there is a balance required for optimal polyextremophilic activity.
  • Visual Deficits in a Model of Gestational Hypothyroidism

    Tarasiewicz, Agnieszka; Medina, Alexandre E. (2022)
    Hypothyroidism prevalence among pregnant women is between 0.5 to 4% (Carney et al., 2014). Most studies look at the prolonged or severe reduction in thyroid hormone (TH) levels. We looked at how the reduced levels of TH during the third trimester of human gestation and the first weeks after birth in rodents impact the visual system. Additionally, we try to answer whether PTU treatment would affect neuronal plasticity in the visual cortex. We used the Visual Evoked Potential recordings to assess contrast sensitivity, spatial frequency acuity, and ocular dominance plasticity. In addition, we look into the expression of the photoreceptors in the retina. PTU exposure impacts the contrast sensitivity but not the spatial frequency acuity or ocular dominance plasticity. The expression level of the photoreceptor Opsin-M was also impacted. The reduced levels of the thyroid hormones during this crucial time have long-lasting consequences for the proper visual system processing.
  • The Role of Colonization Factors CFA/I and CS21 in Enterotoxigenic E.coli (ETEC) Pathogenesis in the Human Enteroid Model

    Smith, Emily; Barry, Eileen M. (2022)
    Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and in young children in low-to-middle income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF and are prime targets as vaccine antigens. Many clinical isolates express CFA/I and CS21; however, a role for CS21 alone or with CFA/I has not been defined. We hypothesize that expression of both CFs confers increased adherence and toxin delivery to the human enteroid. Clinical strains expressing CFA/I and/or CS21 were evaluated, and CF-deficient mutants were engineered. After confirming CF expression using western blot and electron microscopy, assays demonstrated CFA/I was important for CFA/I-CS21 ETEC adherence, as CFA/I-deficient mutants and strains pre-incubated with anti-CFA/I antibody had significantly reduced adherence to enteroid monolayers compared to wildtype. In contrast, CS21 was not required as CS21-deficient mutants and strains pre-incubated with anti-CS21 antibody adhered at similar levels as wildtype. These data demonstrate that targeting CFA/I in CFA/I-CS21 ETEC is sufficient for significant adherence reduction. Delivery of ST by CFA/I-CS21 ETEC was evaluated. Strain-specific levels of toxin delivery were detected but CF-dependent ST delivery was not observed, which may reflect the lack of flow and stretch in the current enteroid model. Upon investigation of host responses to ETEC, the enteroid monolayer integrity was not disrupted, as shown by the increase in transepithelial electrical resistance and the lack of inflammatory cytokines produced. Infection with ETEC strains resulted in decreased mucus (MUC2) production, but this was not CF-dependent. Further studies of strain-specific CFA/I expression revealed that it may be transcriptionally or post-transcriptionally regulated, following observation of nearly identical CFA/I operon sequences and many shared CF-specific regulators at the genomic level. Overall, these data support the role of CFA/I in CFA/I-CS21 ETEC adherence and reinforces CFA/I as a main target for vaccines. These data also highlight the human enteroid model to study ETEC pathogenesis and for evaluation of preclinical therapeutics.
  • Novel Modules in the T Cell Signaling Circuit Which Enable Synergy Between Responses to Self and Foreign Peptides

    Wolf, Gideon; Singh, Nevil (2022)
    T cell activation occurs when a T cell receptor (TCR) engages with cognate agonistic peptides in the context of Major Histocompatibility Complexes (pMHCs) on antigen presenting cells (APCs). This initiates a series of intracellular signaling events proximal to the TCR and associated CD3 complexes, mediated by unique kinases together with scaffolding and lattice molecules. The downstream cascades ultimately lead to transcriptional changes that promote the cellular program of conventional T cell activation—for example, cytoskeletal rearrangement, cytokine production, cellular proliferation, and differentiation. Understanding signaling mechanisms not only allows us to decipher the regulation of T cell activation but also to manipulate immune responses pharmacologically. TCR signaling by agonistic pMHCs is well studied, but much less is known about signaling by a parallel universe of self-peptides that also engage TCRs in vivo. The focus of this thesis is to understand how T cells perceive these signals without fully acquiring effector responses as a result. The central hypothesis of my thesis is that self-peptide ligands signal uniquely through the TCR to alter the fate and function of a T cell both with and without presence of their cognate agonist. We evaluated this hypothesis using approaches that globally increased self-peptide presentation in vivo (using FLT3L to generate more DCs), deprived T cells of self-peptide (by culturing away from APCs) or stimulated a TCR with a known self-peptide in the presence or absence of the strong agonist. The significant findings from these studies are that (i) boosting self-peptide presentation transiently increases a narrow T cell effector subset; (ii) depriving T cells of self-engagement lowers basal phosphorylation in the key signaling adapter LAT; (iii) self-peptides do not trigger cellular activation on their own, but synergize to enhance activation as measured by CD69, pERK, and several other parameters (iv) self-peptides initiate TCR signaling up to the level of pMEK and (v) self-peptides elicit a unique transcriptional profile. Together, these results not only define the unique contributions of self-peptides to T cell activation but also demonstrate a distinct wiring profile in the TCR-signaling network that limits self-peptide sensing at the ERK step.
  • The Role of Granzyme B in Tumor Migration and Metastasis

    Ellis, Tibbs; Cao, Xuefang (2022)
    The granule exocytosis pathway has classically been described as our most potent response to pathogens, cancers, as well as autoreactive disorders. This is executed by the action of perforin and a family of granule-associated serine proteases known as granzymes (Gzm). Many studies have shown that these proteases exert their effects by killing affected cells, creating an obvious correlation with cytotoxic T cells (CTLs) and natural killer cells (NK) greatly expressing GzmB. Studies have found that the tumor cells ability to create a suppressive environment is dependent on Gzm expression by regulatory T lymphocytes (Treg). Research has shown GzmB can target and cleave extracellular molecules such as fibronectin, vitronectin and laminin. Bird et al published data showing that GzmB aids in the transmigration of CTLs, however the ability of GzmB to aid in the migration of non-T cells has not been investigated. We hypothesize that GzmB secretion may cause extracellular matrix (ECM) remodeling in the tumor microenvironment, aiding in the outgrowth of the tumor cells via promoting higher rates of invasion and/or metastasis. First, we designed experiments to test in-vivo and in-vitro conditions that generate GzmB production in multiple cell types. We were able to induce GzmB production by Tregs both in-vitro and in-vivo. We also identified multiple mechanisms that allow for CD8 T cells to produce GzmB. We next investigated mechanisms that would allow for GzmB to be found in the extracellular environment. We found that CD8 T cells have the ability to secrete GzmB without the need of an immunological synapse. In fact, GzmB secretion is not dependent on exogenous IL-2. GzmB improves the migration and invasive potential of tumor cells. In Treg-specific GzmBko (Foxp3creGzmBfl/fl) mice, GzmB+ Tregs increase the rate of tumor metastasis in the lungs of the mice. In addition, GzmB+ CD8 T cells can potentiate tumor metastasis, in contrast to GzmB-deficient CD8 T cells. These findings shed light on the protumorigenic potential of secretory GzmB, which could serve as a unique biomarker for predicting metastasis.
  • Using PacBio SMRT platform to assess the genetic variation of Plasmodium falciparum Circumsporozoite Protein (PfCSP) in Kalifabougou, Mali

    Ouedraogo, Nadiatou; Laurens, Matthew B. (2022)
    Objectives: In this study, we assessed the genetic variation in CSP sequences from Kalifabougou, Mali, by evaluating the diversity of haplotypes in the N- and C- Terminal region, the repeat regions, and the T- and B-cell epitopes. Methods: Sequences used for the study were generated from 59 sequences collected during a malaria transmission season conducted in Kalifabougou, Mali. Generated using the PacBio SMRT platform. Results: Results showed that the central repeat and C-terminal Th2R/Th3R epitope regions were highly polymorphic in this study, whereas the N-terminal non-repeat region was less variable. Furthermore findings highlighted CSP polymorphism and suggested that the repeat and B-cell epitope of our samples is similar to those of 7G8 PfCSP, whereas the C-terminal is similar to that of the 3D7 PfCSP strain. Finally, findings the CSP sequences from the Kalifabougou site are more similar to those from Navrongo than to those from Cape Coast.
  • Membrane-Anchored Serine Proteases in Ovarian Cancer Dissemination and Metastasis

    Pawar, Nisha; Antalis, Toni M. (2022)
    Ovarian cancer (OvCa) is the most fatal gynecological malignancy due to delayed clinical presentation, passive metastasis, frequent tumor recurrence, and lack of effective targeted therapies. OvCa tumors predominantly shed as single cells or spheroids and disseminate throughout the peritoneal cavity. Spheroids are considered critical metastatic units that contribute to disease recurrence and chemoresistance. Despite recent advances in treatment strategies, there has been little improvement in patient survival. There is an urgent need to develop a better understanding of molecular mechanisms that are fundamental for OvCa dissemination, in order to provide alternative therapeutic options for long-term remission. Matriptase, a membrane-anchored serine protease (MASP), and its substrate protease-activated receptor-2 (PAR-2) both exhibit elevated expression in OvCa compared to normal ovary tissues which worsens patient survival, suggesting a critical role for activation of this pathway in OvCa progression. Overactive matriptase, induced by an imbalanced ratio with its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) has been implicated in disruption of barrier integrity and epithelial carcinogenesis, although the mechanism is not known. This study, demonstrates a functional role for an imbalanced matriptase:HAI-1 ratio in OvCa progression, and identifies a matriptase/PAR-2/PI3K/Akt/MMP-9/E-cadherin signaling axis that disrupts cell-cell interactions and promotes formation of pro-metastatic loose spheroids in vitro, in in vivo orthotopic xenograft models, and in patient-derived tumor cells. This matriptase/PAR-2 pro-metastatic signaling is in direct contrast to another MASP family member, testisin, suggesting a novel mechanism of biased agonism that orchestrates OvCa dissemination. Since over-activity of MASPs is associated with advanced OvCa, we have developed a MASP-activated pro-drug based on a re-engineered anthrax toxin (PAS:LF) that is selectively activated by zymogen-activating proteases on the tumor cell surface to inhibit tumor cell-survival pathways. This therapeutic strategy is radically different from anti-proliferative mechanisms of standard chemotherapeutic agents. We have demonstrated anti-tumor efficacy in a range of OvCa cells and spheroids in vitro, in various in vivo orthotopic xenograft models of OvCa dissemination, in patient ascites-derived tumor cells and in patient-derived xenograft models in vivo, with no off-target adverse effects. Clinical translation of these preclinical findings could establish PAS:LF as a promising treatment strategy to improve patient outcomes.
  • The Effects of HIV Exposure and Maternal Antibodies to Hepatitis B Virus on the Immune Response of Nigerian Infants to Hepatitis B Vaccine.

    Bada, Florence; Stafford, Kristen Alyce; Campbell, James Daniel (2022)
    Introduction: Chronic hepatitis B virus (HBV) infection leads to considerable morbidity and early mortality. Development of chronic HBV infection can be prevented by a three to four dose schedule of Hepatitis B vaccines in immunocompetent infants. However, HIV exposed uninfected infants (HEU) are thought to exhibit an attenuated immune response to some vaccines. In addition, a significant proportion of pregnant women in Nigeria have antibodies to HBV, specifically Hepatitis B surface antibodies (HBsAb). Maternal antibodies inhibit infant immune responses in some instances. Objective: To estimate the effect of antenatal and perinatal exposure to maternal HIV, and the effects of maternal HBsAb on the immune response of Nigerian infants to hepatitis B vaccine. Methods: Using a retrospective cohort design, we determined the relationship between infant HIV-exposure status, and infant exposure to detectable concentrations of maternal HBsAb, and infant immune response to Hepatitis B vaccine separately using general linear models adjusted for potential confounders. Subsequently, we used Fisher’s Exact tests to compare the proportion of infants with HBsAb concentrations above 10mIU/mL: for HEU as compared to HIV unexposed uninfected infants (HUU), and for infants exposed to detectable concentrations of maternal HBsAb as compared to infants unexposed to detectable concentrations of maternal HBsAb at 24 and 52 weeks of age separately. Results: We found HIV exposure to be associated with infant immune response to hepatitis B vaccine at birth, Weeks 4 and 52 (P<0.0001, P=0.05 and P<0.0001) and also to be associated with the proportion of infants with HBsAb concentrations ≥ 10mIU/mL at Week 52 (P=0.04). Exposure to detectable concentrations of maternal HBsAb was also associated with infant immune response at Weeks 24 and 52 respectively. Conclusion: Though antenatal and perinatal exposure to HIV, and to detectable concentrations of maternal HBsAb were found to be associated with infant immune response to hepatitis B vaccine, these exposures did not appear to attenuate immune response. In addition, differences observed in the proportion of infants with HBsAb concentrations ≥ 10mIU/mL were small and do not appear to be clinically relevant.
  • Hippocampal Contributions to Stress: What the Hippocampus Tells the HPA Axis and What Could Go Wrong?

    Cole, Anthony; Thompson, Scott M. (2022)
    The links between stress and psychiatric illnesses are numerous and bidirectional. Stress is highly correlated to psychiatric illness, increasing the risk of illness and often precipitating its onset. The primary class of neuroendocrine stress hormones, glucocorticoids, is often dysregulated in patients with major depression. Understanding the neurobiological changes underlying these findings is crucial for the development of improved therapeutic strategies and better patient outcomes. Prior work suggests that the hippocampus is stress-sensitive, substantially altered in major depression, and plays an important regulatory role in HPA axis function. Our understanding of the processes regulating the stress response is incomplete, particularly in regards to the various brain regions responsible for its central regulation. In this document I strive to clarify our understanding of a specific component of stress regulation, namely the hippocampal contribution, which could prove important to understanding the various links between stress and psychiatric illness. To do this, I targeted the ventral hippocampus with injections containing virus that expressed the light-sensitive ion channel channelrhodopsin (ChR-EYFP) in glutamatergic neurons. I determined the extent of glutamatergic hippocampal terminal expression in brain regions thought to regulate the paraventricular nucleus of the hypothalamus (PVN), and then stimulated those terminals while recording from corticotropin-releasing factor-positive (CRF+) neurons in the PVN in a whole-cell voltage clamp recording configuration. Using parasaggital brain sections, I was able to induce optically-evoked inhibitory post-synaptic currents in CRF+ neurons of the PVN, demonstrating the first direct functional evidence of hippocampal inhibition of the PVN. I used a similar injection scheme to record GAD+ neurons in the BNST. Finally, I utilized in vivo optical stimulation of hippocampal terminals within the BNST during acute restraint stress and observed a significant decrease in circulating levels of corticosterone. My data indicates that the hippocampus sends dense projections to the BNST and optical excitation of these hippocampal terminals produce inhibitory currents in CRF+ neurons of the PVN. Our data suggests a disynaptic hippocampus-BNST-PVN circuit of regulation by which the hippocampus inhibits the CRF+ PVN cells. The in vivo results indicate that the hippocampal inhibition of these neurons is capable of functionally inhibiting the HPA axis.
  • Pharmacometabolomics of clopidogrel: Determining the genetic and metabolic contributors to clopidogrel response

    Bromberek, Sarah Katherine; Beitelshees, Amber L. (2021)
    Clopidogrel is a commonly prescribed antiplatelet drug and there is considerable variability in response. The PAPI study was conducted in 687 individuals to determine the genetic predictors of clopidogrel response. Targeted metabolomic profiling of 42 amines was performed in a subset of 198 PAPI subjects with genetics data available. We identified the metabolic signature of clopidogrel and determined metabolites associated with clopidogrel-induced changes in platelet reactivity. We found tyrosine was most significantly changed after exposure to clopidogrel, and BCAAs baseline levels were associated with clopidogrel-induced changes in platelet aggregation. Gaining insights into factors that influence variability in antiplatelet response is important for identifying novel antiplatelet mechanisms or biomarkers for predicting response. These findings can have long-term implications toward advancing precision medicine in antiplatelet therapy.
  • Investigating the Role of Acetylated Tubulin on Microtubule Dependent Mechanotransduction in Striated Muscle

    Coleman, Andrew; Ward, Christopher, Ph.D.; Lederer, W. Jonathan (2022)
    Mechanotransduction is critical to the maintenance and development of striated muscle in response to a change in workload. Mechanical sensors within muscle respond to muscle movement to regulate EC coupling, gene expression, and signal propagation. Our lab has investigated the role of the cytoskeleton as a mechanosensor in striated muscle. Biophysical properties of microtubules (MTs) allow for mechanical energy created during sarcomere shortening to be transferred to the cytoskeleton network and associated proteins. Upon contraction or stretch, mechanical energy is transferred through the MT network to the membrane bound NADPH oxidase 2 (Nox2) to trigger a localized increase in reactive oxygen species (ROS) production that regulates calcium channels at the triad junction. This mechanotransduction pathway is regulated not only by the abundance of MT and Nox2, but also the biophysical properties of MTs. Post translational modifications to tubulin have various consequences to polymerized tubulin and create distinct subsets of MT populations within muscle. While MT acetylation of lysine 40 within the lumen of polymerized MTs is abundant in muscle, little is known about its function in muscle. Recent investigations that explored the biophysical properties of MT acetylation in vitro have shown that acetylation increases the resistance of MTs to damage by repeated mechanical insults. Here we sought to investigate the role of MT acetylation in muscle mechanotransduction in health and disease. Using Pharmacologic and genetic strategies, we show that microtubules enriched in acetylated α-tubulin increase cytoskeletal stiffness and viscoelastic resistance. These changes slow rates of contraction and relaxation during unloaded contraction and increased activation oof Nox2 by mechanotransduction. Importantly, MT acetylation had no effect on tension produced during contraction of intact muscle enabling enhanced mechanotransduction without altering force production. Furthermore, we show that microtubule PTMs are elevated in heart failure, muscular dystrophy, and aging. These changes translated to excess mechanotransduction and are potential therapeutic targets to diminish oxidative stress associated with muscle disease. Together, these findings add to growing evidence that microtubules contribute to the mechanobiology of striated muscle and are detrimental muscle disease modifiers.
  • The Evolution of B Cell Selection and Affinity Maturation in Cartilaginous Fishes

    Matz, Hanover Christian; Dooley, Helen, Ph.D.; 0000-0002-0669-1696 (2022)
    Affinity maturation of the B cell immunoglobulin (Ig) repertoire occurs through coordinated somatic hypermutation (SHM) and Darwinian selection of clones in specialized microanatomical structures known as germinal centers (GCs). GCs have only been identified in the endothermic vertebrates, and so it was long presumed that the antigen (Ag)-specific Ig responses of ectothermic vertebrate lineages were “primitive”. However, affinity maturation and immunological memory have subsequently been demonstrated for the oldest extant vertebrate class with Ig-based adaptive immunity, the cartilaginous fishes (Chondrichthyes). In this dissertation, I investigated the cellular model of B cell selection in the nurse shark (Ginglymostoma cirratum) spleen and determined how it influences the dynamics of the Ig repertoire. I found that shark splenic B cell follicles possess many functional analogs of mammalian GCs: (1) segregation of SHM and selection regions by CXCR4/CXCR5 expression in B cells, (2) functional T follicular helper-like cells, (3) presentation of nondegraded Ag, and (4) Ag-driven selection of mutated Ig clones. I also demonstrated that the transcription factor BCL6 likely regulates the shark B cell response. Through a long-term immunization study, I demonstrated that this selection model can generate IgNAR repertoires that are both diverse and of high affinity. In multiple animals immunized by the same methods, I observed uncoupling of the T-dependent isotypes, IgNAR and monomeric IgM. Sharks that produced robust IgNAR titers matured their polyclonal repertoires to subnanomolar binding affinities and generated a diverse pool of memory clones. Together, this suggests that B cell selection in cartilaginous fishes evolved to support both affinity maturation and Ig repertoire diversification, possibly utilizing SHM to anticipate future pathogen variants. Finally, I developed a method of magnetic nanoparticle enrichment to isolate Ag-specific B cell clones directly from the peripheral blood of sharks. Overall, the data presented in this dissertation indicate that all the fundamental components of B cell selection were present at the advent of adaptive immunity in jawed vertebrates. Furthermore, these components were capable of affinity maturation of the B cell repertoire without sacrificing receptor diversity. My findings have many implications for our understanding of the evolution of the B cell response in vertebrate lineages.
  • The Role of CCAAT/Enhancer Binding Protein (C/EBP) Homologous Protein (CHOP) in the Antileukemic Activity of Artemisinins

    Tabassum, Sumiya; Civin, Curt I.; 0000-0002-2086-9737 (2022)
    Current chemotherapy options for acute myeloid leukemia (AML) are still limited, despite recent efforts to develop novel drugs for AML with greater efficacy and acceptable toxicity. Several antimalarial analogs of the compound artemisinin (ARTs) possess antineoplastic activity across many cancer cell types, with highest potency against leukemia cells, but their detailed molecular mechanisms of action (MOA) are inconclusively established. This study leveraged our previous findings that ARTs downregulated the antiapoptotic protein, myeloid cell leukemia-1 (MCL1), and upregulated the transcription factor, CCATT/enhancer-binding protein homologous protein (CHOP), in human AML cells. We assessed the roles of these molecules in the antileukemic MOA of the highly potent ART analog, ART838, in the human MOLM14 AML cell line. We found that enforced MCL1 overexpression rescues from ART838-mediated cell death. However, neither CHOP overexpression nor CRISPR-Cas9-mediated CHOP knockout affected growth/survival or cellular levels of MCL1 protein, in the absence or presence of ART838.
  • Disynaptic Prefrontal Cortical Input to the Dorsolateral Striatum

    Harris, Morgan; Mathur, Brian N. (Brian Neil); 0000-0002-8424-7822 (2022)
    The dorsomedial striatum (DMS) is responsible for actions that are reversible, goal-directed, and require attentional oversight. In contrast, the dorsolateral striatum (DLS) is responsible for relatively irreversible, but precise habitual actions that need little attentional oversight. In addiction, the balance between these two action strategies is shifted toward habits, leading to compulsive behaviors. The DMS is activated by input from executive, prefrontal cortical areas of the brain, while the DLS is activated by sensorimotor cortices. A major gap in knowledge is how executive cortical centers may mediate shifts away from habitually performed actions. While no executive cortical areas monosynaptically activate the DLS, we hypothesize the existence of disynaptic circuits allowing executive cortical control of the DLS. To test this, we utilized trans-synaptic target specific tracing (TranSTart) to reveal that executive cortices disynaptically project to the DLS through the basolateral amygdala (BLA) and the rostral intralaminar nuclei of the thalamus (rILN).
  • Associations of Rare Variants Underlying Depressive Symptoms in the Old Order Amish Founder Population

    Choe, Jayme Hyowon; Ament, Seth A.; 0000-0003-3453-8944 (2022)
    Depressive disorders are among the leading causes of disability worldwide. Genome-wide association studies of common variants of depressive disorders have identified 178 risk loci, yet mechanisms remain elusive due to the very small effects of common variants. Certain rare variants may have larger effects, but exome and genome sequencing studies to date have been underpowered to detect effects of specific rare variants. One approach to address these limitations is to utilize population isolates, like the Old Order Amish (OOA), in which certain rare variants become enriched due to the population bottleneck effect. This study aimed to identify rare variants associated with depressive symptoms, utilizing whole exome sequencing (WES) and phenotypic data from two OOA cohorts (N = 5,052), the Amish Wellness Study and the Amish Connectome Project. We identified five significant SNP-depressive symptoms associations. Case-series phenotyping revealed high depressive symptoms screening scores across carriers of each variant compared to non-carriers.
  • Identifying targets of sulforaphane in mesothelioma

    Ezeka, Geraldine; Eckert, Richard (Richard L.); 0000-0002-9222-0566 (2022)
    Mesothelioma is a fatal cancer of the mesothelial lining that is caused by asbestos exposure. The most common forms of mesothelioma arise in the pleural and peritoneal cavities of the lung and abdomen. Current treatment involves surgical resection and chemotherapy, but this approach is marginally successful and leads to drug resistant disease. We study sulforaphane (SFN), a nature derived anti-cancer agent that has high bioavailability and low toxicity. Our goal is to identify sulforaphane responsive targets in mesothelioma. Protein arginine methyltransferase 5 (PRMT5) is an epigenetic modifier that acts with methylosome protein 50 (MEP50) to symmetrically dimethylate arginine residues on histones H3 and H4 to silence target gene expression. PRMT5/MEP50 histone methylation has been implicated in cancer and is associated with silencing of tumor suppressors leading to enhanced cancer development. Our studies show that PRMT5/MEP50 knockdown reduces H4R3me2s and attenuates the cancer phenotype. Moreover, SFN reduces PRMT5/MEP50 function and cancer cell proliferation, spheroid formation, invasion and migration. Further, forced expression of PRMT5/MEP50 antagonizes SFN suppression of the cancer phenotype, suggesting that loss of PRMT5/MEP50 is required for SFN action. SFN suppression of mesothelioma tumor formation is associated with reduced PRMT5/MEP50 levels and activity. These findings suggest that SFN treatment suppresses PRMT5/MEP50 activity to attenuate the cancer phenotype. We also examined SFN impact on AKT/mTOR and MEK/ERK1/2 signaling which act together to activate translation of selected mRNA species via regulation of the eIF4F complex. AKT/mTOR activity leads to 4E-BP1 phosphorylation, leading to the release of eIF4E, which binds to eIF4G to assemble the eIF4F complex. MEK/ERK1/2 signaling activates MNK1/2 which activates eIF4E in the eIF4F complex. These events result in a selective increase in the translation of a subset of mRNAs. We show that SFN treatment suppresses AKT/mTOR activity leading to reduced phosphorylation of 4E-BP1 which would be expected to reduce assembly of the eIF4F complex. However, we also observe an unexpected increase in MEK/ERK1/2 activity and MNK1/2 phosphorylation which we propose is a compensatory response to the inhibition of activity of the eIF4F complex. These findings suggest that SFN may suppress eIF4E-dependent translation to attenuate the mesothelioma cancer phenotype.
  • Impact of mobile elements on human traits and diseases

    Chuang, Nelson Ta-Ching; Devine, Scott E.; 0000-0002-2015-8935 (2022)
    Several large-scale Illumina whole genome sequencing (WGS) and whole exome sequencing (WES) projects have emerged recently that have provided exceptional opportunities to discover mobile element insertions (MEIs) and study the impact of these MEIs on human genomes. However, these projects also have presented major challenges with respect to the scalability and computational costs associated with performing MEI discovery on tens or even hundreds of thousands of samples. To meet these challenges, we have developed a more efficient and scalable version of our Mobile Element Locator Tool (MELT) called CloudMELT. We then used MELT and CloudMELT to perform MEI discovery in 57,919 human genomes and exomes, leading to the discovery of 104,350 non-redundant MEIs. We leveraged this collection: 1) to examine the population distributions and subfamilies of these MEIs, 2) to examine the mutagenesis of GENCODE genes, ENCODE-annotated features, and disease genes by these MEIs, and 3) to examine the potentially active L1 source elements that drive mobilization of new Alu, L1, and SVA MEIs in humans. Our study provides new insights on the L1 source elements that drive MEI mutagenesis and brings forth a better understanding of how this mutagenesis impacts human genomes.
  • Characterization of filarial parasite evolution through genome assembly and transcriptomic analysis of Brugia malayi and Brugia pahangi

    Mattick, John Stocker Antalis; Dunning Hotopp, Julie C.; 0000-0002-8743-1158 (2022)
    To study the chromosomal structure of filarial parasites, the genomes of Brugia malayi and Brugia pahangi were assembled using a combination of Illumina, PacBio and Oxford Nanopore sequencing. The assembly was able to reconstruct all of the major chromosomes, including the X chromosome, which comprised over 20% of the genome in both species. Male specific sequencing was used to identify contigs associated with the Y chromosome of B. malayi, and showed that these contigs contained the majority of the repetitive sequences in the genome. A chromosomal fusion of the sex chromosomes of these species that created a pseudoautosomal region of the X chromosome and a haploid region in males was also identified. Analysis of other filarial parasites revealed that while not all species had the same fusion, this haploid region was consistently conserved. In order to compare the chromosomes of these filarial species to other species whose genomes had not been assembled into chromosome form, a classification system called Nigon elements was created to assign large conserved syntenic blocks to specific Nigon elements that were comparable across species. A large reduction in nucleotide diversity across this region of the sex chromosome was also identified, indicating that the fusions were recent. Finally, short read transcriptomics identified novel microRNAs in B. malayi that originate from both the parasite and its Wolbachia endosymbiont. Target prediction across the mammalian portion of the parasite life cycle revealed seven major clusters of co-regulated genes, including a number of developmental and adult-specific gene pathways. Furthermore, in situ hybridization imaging confirmed that the microRNAs predicted to originate from the Wolbachia were not present in the parasite nuclei, suggesting that these sequences are bacterial in origin. These findings reveal a new co-evolving pathway for endosymbiont and parasite communication.

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