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

  • 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.
  • Characterizing the Signaling, Crosstalk, and Trafficking of Adhesion G Protein-Coupled Receptor G2 and the Calcium-Sensing Receptor in Parathyroid Adenoma Models

    Gad, Abanoub; Balenga, Nariman; Olson, John A. Jr.; 0000-0002-8225-5298 (2022)
    The parathyroid glands sense serum calcium levels and release parathyroid hormone (PTH) in response to hypocalcemia. PTH acts at bone, intestine, and kidney to increase serum calcium. Primary hyperparathyroidism (PHPT) is an endocrine disorder characterized by neoplasia of one or more glands, dysregulated PTH release, and hypercalcemia. The mechanisms of dysregulated calcium-sensing in parathyroid adenomas are not well understood. The literature suggests that the expression of the calcium-sensing receptor (CaSR) is diminished in parathyroid adenomas. We have found that a subset of PHPT patients do not show a significant change in CaSR expression. Therefore, we hypothesized that other molecules that modify the action of CaSR might inhibit its ability to sense calcium properly. We identified ADGRG2, an adhesion G protein-coupled receptor (aGPCR), in a transcriptome screen of parathyroid tissue and hypothesized that it might serve as a modifier of CaSR function. A transgenic mouse with parathyroid-restricted ADGRG2 overexpression develops parathyroid neoplasia, hypercalcemia, and hyperparathyroidism. To understand crosstalk mechanisms between ADGRG2 and CaSR, we developed a double-expressing ADGRG2-CaSR stable cell line (FL-CaSR). Activation of ADGRG2 in this novel cell line completely ablated the Gαi and Gαq signaling pathways downstream of CaSR. We also developed a SNAP and CLIP tag system to ascertain changes in receptor trafficking. Live cell imaging studies showed that in cells expressing both ADGRG2 and CaSR, co-stimulation caused the receptors to remain on the cell surface and not traffic into the early endosome. Given the important role that the ADGRG2 plays in inhibiting CaSR activity we sought to identify targetable
  • ALS/FTD-Linked UBQLN2 Mutations Result in Aberrant Accumulation and Aggregation of Serine Protease Inhibitor Proteins

    Higgins, Nicole; Monteiro, Mervyn J.; 0000-0001-8523-054X (2022)
    Mutations in the protein UBQLN2, which functions in protein quality control, cause amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). However, the mechanism(s) by which the mutations cause disease remains unclear. In this study, we used proteomic profiles generated from 8-week-old transgenic mice expressing either P497S mutant (P497S line) or wildtype UBQLN2 (WT356 line), and non-transgenic (Non-Tg) mice to gain insight into the pathophysiologic mechanisms driving disease. The P497S line recapitulates key features of human ALS/FTD, including motor neuron disease, cognitive impairments, and pathologic accumulation of UBQLN2 inclusions; symptoms and pathology which are largely absent in the WT356 line. Comparison of proteomic changes in the hippocampus and spinal cord of these animals revealed several members of the serine protease inhibitor (serpin) protein family are more highly expressed in P497S animals compared to either WT356 or Non-Tg controls. Serpins function in the regulation of proteolytic cascades by entrapping and destroying proteases through a conformational switch of the protein. However, this metastability makes serpins highly prone to misfolding and polymerization, placing a high demand on protein degradation systems to remove the misfolded proteins. Thus, we tested the hypothesis that ALS/FTD mutations in UBQLN2 perturb proteostasis, causing aberrant accumulation of misfolded serpin proteins. Double immunofluorescent staining revealed colocalization of Serpin A1, C1 and I1 with UBQLN2 inclusions that form in the brain and spinal cord of P497S mice. To further investigate the aberrant accumulation and mislocalization of serpins, we performed aggregation assays on cortical tissue from 8- and 32-week-old P497S, WT356, and Non-Tg animals, which revealed that serpin proteins have an increased tendency to aggregate in P497S animals compared to control mice. We also studied the effects of altered UBQLN2 expression on serpin protein levels and aggregation and found that UBQLN2 knockout induces serpin aggregation while overexpression of WT or mutant UBQLN2 oppositely affected serpin protein levels. These results are consistent with ALS/FTD mutations causing aberrant serpin accumulation through a loss-of-function mechanism. Taken together, this study identifies a novel role for UBQLN2 in the proper regulation of serpin expression and suggests that misaggregation of serpin proteins may be a pathologic consequence of UBQLN2 mutations.
  • Inhibition of Autophagy in Microglia and Infiltrating Monocytes/Macrophages Exacerbates Neuroinflammation and Functional Defects Following Traumatic Brain Injury

    Hegdekar, Nivedita Uday; Lipinski, Marta M.; 0000-0002-3633-899X (2022)
    The autophagy-lysosomal pathway serves an important role in cellular homeostasis and protection against neurodegeneration. Recently autophagy has been also implicated in regulation of immune and inflammatory responses. Specifically, high levels of autophagy flux – the progress of substrates through autophagic compartments leading to their delivery and degradation in the lysosomes – are generally associated with anti-inflammatory, and inhibition of flux with pro-inflammatory phenotypes. To determine if autophagy may be involved in modulation of brain inflammation after traumatic brain injury (TBI), we assessed the levels of autophagy in resident microglia and infiltrating macrophages following moderate controlled cortical impact (CCI) in C57Bl/6 mice. Consistent with a potential function in neuroinflammation, our data demonstrated accumulation of autophagosomes and inhibition of autophagy flux specifically in the activated microglia and infiltrating macrophages starting by day 1 and continuing through day 28 post-CCI. Our immunofluorescence studies in transgenic Cx3Cr1-GFP microglial and Ccr2-RFP monocyte reporter mice confirmed inhibition of autophagy flux and demonstrated that while both activated resident microglia and infiltrating macrophages are affected, inhibition of autophagy is much more prominent in macrophages. We used flow cytometry analysis to demonstrate that immune cells with inhibited autophagy flux after CCI expressed increased levels of pro-inflammatory markers, including IL-1 and TNF-, as compared to corresponding immune cells with normal levels of autophagy. Furthermore, inhibition of autophagy flux correlated with impaired phagocytic function, indicating microglial/macrophage dysfunction. These trends persisted through day 28- post CCI, suggesting that autophagy flux impairment following TBI has long-term consequences. Consistent with ability of autophagy to affect inflammation, our in vitro experiments in IMG microglial and RAW 246.7 macrophage cell lines demonstrated that inhibition of autophagy can potentiate pro-inflammatory activation induced by lipopolysaccharide (LPS) treatment. In vivo, mice with microglia/macrophage-specific knockout of the autophagy gene Becn1 (Beclin1flox/flox,LysMCre/Cre) subjected to CCI showed increased expression of pro-inflammatory genes and proteins as compared to LysMCre/Cre controls. This included a marked exacerbation of the innate immune responses, including activation of the NLRP3 inflammasome and the type-I IFN pathways, indicating the importance of autophagy in suppression of innate-immunity mediated inflammation. Finally, Beclin1flox/flox, LysMCre/Cre mice performed worse than LysMCre/Cre controls on motor and cognitive behavioral tasks after CCI. Overall, these findings indicate that inhibition of autophagy in microglia/macrophages exacerbates neuroinflammation after TBI and contributes to functional deficits.
  • Synaptic Mechanisms Underlying the Rapid Antidepressant Properties of the (2R,6R)-Hydroxynorketamine Ketamine Metabolite

    Riggs, Lace Marie; Gould, Todd D.; 0000-0003-4368-4089 (2022)
    Ketamine rapidly alleviates symptoms of treatment-resistant major depression within hours of a single administration. While this may offer a new and promising trajectory for novel therapeutic development, rapid antidepressant mechanisms of action have proven especially enigmatic. Here, I applied an in vitro-à-in vivo approach to identify the cellular actions of (2R,6R)-hydroxynorketamine (HNK) – a secondary metabolite of ketamine that exerts rapid antidepressant-like effects sine dissociative properties and abuse potential. I found that (2R,6R)-HNK promotes a rapid potentiation of glutamate release at hippocampal Schaffer collateral synapses, an important site of antidepressant action that serves as a conduit in the regulation of mood and cognition. This potentiation proved to be a synapse-selective calcium-mediated phenomenon that involves the rapid induction of cyclic adenosine monophosphate dependent protein kinase, known to actuate vesicle-associated phosphoproteins to enhance glutamate release. These properties confer (2R,6R)-HNK with an ability to restore Schaffer collateral-dependent plasticity and cognitive function in a preclinical model of treatment-resistant major depression. My findings are the first to establish a cellular mechanism by which (2R,6R)-HNK gives rise to sustained adaptations in synaptic transmission and behavior and provides a novel framework for the development of improved therapeutic options for depression.
  • The Effect of ZSCAN4 on the Long Non-Coding RNA TERRA

    Benyamien Roufaeil, Daniel; Zalzman, Michal; 0000-0001-6176-4109 (2022)
    Telomeres are DNA repeats located at the ends of chromosomes that shorten with every cell division. As such, they function as biological clocks to limit the ability of cells to replicate indefinitely. Telomeres were regarded as transcriptionally silent; however, evidence suggests that as telomeres shorten, their chromatin converts to accessible euchromatin. Telomeric repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from the sub-telomeres to the ends of chromosomes and has been suggested to be involved in telomere length regulation. Similarly, murine Zscan4 is involved in the regulation of telomere length, genomic stability, and the maintenance of the replicative lifespan of pluripotent stem cells. Our novel findings indicate that ZSCAN4 regulates TERRA expression and affects histone modifications in TERRA regions. Studying the effects of ZSCAN4 on TERRA will potentially uncover a novel mechanism in telomere maintenance and may hold implications for regenerative medicine and cancer therapeutics.
  • Fasting-induced decrease in mouse cardiac pyruvate dehydrogenase enzyme activity associated with increased tolerance to lethal hypoxia

    Carter, Zakiya; Fiskum, Gary; 0000-0003-4067-7166 (2022)
    The global mortality of patients with cardiovascular diseases annually exceeds tens of millions. This study tested the hypothesis that fasting-induced down-regulation of pyruvate dehydrogenase (PDH) enzyme activity preconditions mice to improve survival during acute hypoxia. PDH is a major supplier of substrates to the TCA cycle but also generates toxic reactive oxygen species (ROS). PDH inhibition could inhibit ROS production, thereby contributing to fasting-induced protection against hypoxic death. FVB mice fasted for 48 hr displayed 100% survival during 1 hr 5% oxygen exposure compared to their fed counterparts that perished within 20 min, however, fasted C57BL/6J (B6) survival was minimal. Fasting induced an upregulation of PDH kinase 4 (PDK4) gene expression, increased PDH E1? site 3 phosphorylation, and decreased PDH activity in cardiac tissue of both mouse strains. Therefore, although PDH likely plays a role in hypoxia tolerance, it is not the primary mechanism of survival of the FVB mice.
  • Mast Cells & Microglia “organize” preoptic area neuronal circuitry during perinatal brain development for adult sexual behavior

    Pickett, Lindsay A.; McCarthy, Margaret M., 1958- (2022)
    Organization of neuronal circuitry required for adult sexual behavior occurs during perinatal development and is largely carried out by resident innate immune cells, mast cells and microglia, just prior to birth and during the first week of life in the rodent, when the male brain makes estradiol from precursor androgens secreted by the testes. Mast cells degranulate in response to estradiol in the preoptic area (POA), releasing histamine, which in turn stimulates prostaglandin E2 (PGE2) production by microglia. The mechanism of PGE2-mediated differentiation of excitatory synaptic density in the preoptic area (POA) around birth is known and results in male sexual behavior in adulthood. Aberrant mast cell activation masculinizes POA spine density and sexual behavior of females and demasculinizes these measures in males. Despite its dependence on gonadal hormones for differentiation, the sexually dimorphic nucleus (SDN) of the POA, does not begin differentiating until brain hormone levels are no longer different between the sexes, on postnatal day 5 (PN5). In the SDN, males and females are born with the same number of neurons, but they selectively die off in females, whereas the production of estradiol at birth in males protects them. The SDN is located within the central medial preoptic nucleus (cMPN), where we discovered females have more phagocytic microglia than males during the first postnatal week, a phenomenon that peaked on postnatal day 8 (PN8). Inhibition of microglial phagocytosis by intracerebral injections of an antibody to CD11b (complement receptor 3 CR3) or mast cell degranulator, c48/80, from PN5-7 reduced microglial phagocytosis and increased the volume of the SDN in both sexes on PN8, demonstrating that microglia are engaging in phagoptosis (engulfment of stressed, but viable cells) to shape the size of the SDN. Females treated neonatally with the CD11b (CR3) antibody blockade lost their typical sexual preference for male odor in adulthood. This discovery challenges the dogma that estradiol prevents neuronal apoptosis in the male SDN and reveals novel hormone and neuroimmune mechanisms that regulate phagocytic and neuroprotective cascades during normal brain development.
  • The Genomics and Epidemiology of Typhoid Fever in Samoa

    Sikorski, Michael Joseph; Levine, Myron M. (Myron Max), 1944-; Rasko, David A.; 0000-0002-3811-7285 (2022)
    Typhoid fever is a human host-restricted systemic infection caused by ingestion of fecally-contaminated food or water bearing Salmonella enterica serovar Typhi (S. Typhi). For decades, the Pacific Island nation of Samoa (population ~200,000) has faced unexplained, persistently endemic typhoid fever, despite improvements in water quality, sanitation, and economic development. Herein, epidemiologic analysis of surveillance data from 2008-2019 revealed that 53-193 blood culture-confirmed typhoid fever cases occurred annually in Samoa without seasonality. The greatest annual burden (number of cases/year) and incidence (cases/100,000 persons/year) occurred in primarily urban and peri-urban regions, where piped treated water reaches most households. Phylogenetic analyses of whole genome sequences (WGS) of 306 S. Typhi isolates from Samoa collected between 1983-2020 identified a dominant population of rare, Samoa-exclusive genotypes 3.5.3 and 3.5.4, which could be further divided into local sub-lineages. These Samoan genotypes are calculated to have most likely emerged in the 1970s with a shared common ancestry with other parent clade 3.5 isolates from South America, Southeast Asia, and Oceania. Furthermore, Samoan S. Typhi were reported susceptible to all clinically-relevant antibiotics, including those that are no longer effective in many other typhoid-endemic regions; indeed, resistance-conferring polymorphisms or genes were detected in <5% of isolates spanning decades of endemicity and despite suboptimal antibiotic prescribing practices. Finally, to elucidate patterns of transmission, point pattern spatial statistics and the high-resolution discerning power of WGS were integrated with detailed epidemiological data generated through household investigations of typhoid cases. Patterns identified were consistent with exposure within households in rural regions, dispersion of infections among a wider geographic area in urban regions, and transmission of genetically similar isolates among cases and asymptomatic shedders (carriers) identified through the household investigations. Notably, cases peaked during a period of increased population movement and dramatically decreased during two national lockdowns, indicating possible roles for population mobility and interaction in propagating typhoid fever. Together, these studies characterize the epidemiology and genomics of S. Typhi in Samoa, an endemic island setting in an understudied global region, and can directly assist the Samoa Ministry of Health and other similarly resourced Pacific Island public health programs in their typhoid control efforts.
  • Can you hear me now? Towards a model of age-related difficulty in noisy environments

    Shilling-Scrivo, Kelson; Kanold, Patrick; 0000-0003-2622-3459 (2022)
    The National Institute on Deafness and Other Communication Disorders estimates that there are 40-million Americans who report having hearing difficulty. One of the chief complaints is the inability to hear in noisy environments. We currently do not understand how the brain separates background noise from attended sounds. The goal of this thesis is to observe how the brain detects sounds in noise and how these mechanisms are disrupted during normal aging. In the first chapter, we review the relevant literature surrounding what is known about how sound suppression occurs at the cortical level. We then review the literature surrounding what is known about cortical decline during healthy aging. In the second chapter, examine the differences in sound-encoding by recording from auditory cortex while animals passively listen to tones in noise. From this work, we see that old animals have deficits in temporal and spectral encoding. At the population level, we find that there are increased noise correlations, which we find limit the ability of cortex to encode the tone identity. In the third chapter, we examine the effect of behavior by recording from old and young animals while performing a tone detection task in noise. We confirm the temporal deficits and increased noise correlation seen in passive listening are still present. We additionally find that aging animals are unable to fully suppress their responses to the noisy background. This increased activity to the background leads the old animals to have more false alarms. In the fourth chapter, we examine how life-long auditory training can mitigate the problems found in chapters two and three. We train animals on a tone detection task for a period of 6 months and then see how their passive responses to noise differ from an animal that has not been trained. In the final chapter, we take the results from the three chapters in order to form a working model of how healthy aging affects the auditory cortex and leads to disruptions in encoding tones in noise. We then suggest future work that could be done to further test the predictions of this model.
  • Addressing Bias in Genomics: Genome-Wide Association Studies and Polygenic Risk Scores in Latinx Cohorts

    Loesch, Douglas Paul; O'Connor, Timothy D. (2022)
    Genome-wide association analyses (GWAS) are an important tool for uncovering gene-trait associations and, increasingly, for genetic risk prediction. Differences in allele frequencies and linkage disequilibrium patterns, though, can lead to divergent GWAS results and predictive performances. Despite this, GWAS cohorts have been overwhelmingly of European ancestry. This means that the genetic architecture of many traits is poorly understood in many other populations and genetic risk models underperform for non-Europeans, potentially perpetuating existing health disparities. My current research seeks to shrink these biased knowledge gaps by improving representation of Latinx populations in GWAS data and identifying best practices for polygenic risk prediction in Latinx cohorts. In chapter 2, I performed the first GWAS in a Latinx Parkinson’s disease (PD) cohort (the Latin American Research Consortium on the Genetics of Parkinson’s Disease, or LARGE-PD), reaffirming the important of the SNCA locus for PD etiology and finding a potentially novel locus near the NRROS gene. Then, in chapter 3, I demonstrated the challenges of performing polygenic risk prediction for PD using European ancestry GWAS data. I found that the distribution of the European-ancestry polygenic risk score (PRS) exhibited shifts according to ancestry, potentially limiting its clinical utility. I then further explored the rs356182 variant in the SNCA locus as it is a major component of the PD PRS. By performing a haplotype analysis, I found that population-specific haplotypes with little shared variation other than rs356182 were associated with PD, providing orthogonal evidence that the variant rs256182 is functional. Finally, in chapter 4, I extended my focus to six additional complex traits such as height and type 2 diabetes and found that including non-European GWAS data improves predictive performance in Latinx cohorts even if that GWAS data did not contain Latinx subjects. In general, increasing PRS model complexity results in improved predictive performance, though the genetic architecture of a trait can alter this pattern, as is the case for prostate cancer. Overall, this work sheds light onto the genetic architecture of PD in Latinx cohorts, provides guidance for performing PRS-based prediction in non-Europeans, and helps move the field towards a more equitable future
  • Investigating the Regulation and Metastasis Suppressor Function of Obscurin in Breast Cancer

    Guardia, Talia; Kontrogianni-Konstantopoulos, Aikaterini; 0000-0003-0077-3460 (2022)
    Mounting evidence has implicated the giant cytoskeletal protein obscurin (720-870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work from our group has shown that downregulation of OBSCN in immortalized normal breast epithelial cells promotes survival and chemoresistance, induces cytoskeletal alterations, and enhances their migratory, invasive, and metastatic potentials. Consistent with these observations, analysis of Kaplan-Meier-Plotter data sets reveals that low OBSCN levels correlate with significantly reduced survival and relapse-free survival in breast cancer patients. Despite the compelling evidence linking OBSCN loss in breast tumorigenesis and progression, its regulation has remained elusive, limiting any efforts to target and potentially restore its expression a major challenge given its molecular complexity and gigantic size (~170 kb). Our studies revealed that OBSCN-AS1, a novel nuclear long noncoding RNA (lncRNA) gene, and OBSCN display positively correlated expression and are downregulated in breast cancer. OBSCN-AS1 regulates OBSCN expression in a cis-fashion through chromatin remodeling involving H3 lysine 4-trimethylation enrichment, associated with an open chromatin conformation, and RNA polymerase-II recruitment. CRISPR-activation of OBSCN-AS1 in triple negative breast cancer cells effectively and specifically restores OBSCN expression, and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Furthermore, given the associations of OBSCN dysregulation and silencing in cancer formation and progression, we aimed to decipher the downstream molecular consequences of obscurin loss in breast epithelial cells. Our findings revealed that OBSCN knockout in MCF10A breast epithelial cells leads to upregulation of the PI3K/AKT/NFkB signaling cascade and alters the expression of genes involved in important biological processes including extracellular matrix organization, angiogenesis, and cell adhesion. Importantly, loss of obscurin expression promotes malignant transformation of immortalized normal breast epithelial cells. Taken together, our studies reveal the previously unknown regulation of OBSCN by an antisense lncRNA, the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, and the downstream activation of the PI3K/AKT/NFkB signaling pathway with OBSCN deletion. These discoveries expose fundamental insights into the regulation and function of obscurin in breast cancer and pave the way for future research with significant implications in cancer.
  • Association Between Maternal Experience of Intimate Partner Violence and Under-5 Child Health in Egypt, Jordan, Pakistan, and Afghanistan

    Motawi, Basant; Hirshon, Jon Mark (2022)
    Background : Intimate partner violence (IPV) is the most common type of violence experienced by women globally. The Middle East has some of the highest IPV prevalence worldwide, in particular, Afghanistan, Egypt, Jordan and Pakistan with prevalence ranging from 39% to 87%. The relationship between IPV and childhood morbidity is not well understood. Nonetheless, poor child health outcomes have been linked to homes with IPV. There also is a general lack of research that considers childcare practices when examining the relationship between IPV and child health. The study aims to investigate the association between maternal exposure to IPV and under-5 childhood morbidity taking into account childcare practices. Study Design: Secondary data analysis utilizing data from Demographic and Health Surveys collected in the 2015, 2014, 2017-2018 and 2017-2018 for Afghanistan, Egypt, Jordan, and Pakistan respectively. The study examined the association between IPV and under-5 years childhood morbidity in Afghanistan, Egypt, Jordan, and Pakistan, taking into account the mediating effect of childcare practices. The IPV exposure was assessed through the DHS domestic violence module while the outcome was defined as diarrhea, fever, or ARI experience by the child in the two weeks preceding the survey. Results: The study identified several contextual and individual level factors associated with IPV. Younger, less educated women, with less educated husbands, who are less wealthy and have more children were more likely to experience IPV. The study identified a strong association between IPV and under-5 child illness signs in Afghanistan (OR 2.32, 95 % CI 2.05-2.63) , Egypt (OR 2.02, 95 % CI 1.65-2.48) and Jordan (OR 2.08, 95 % CI 1.54- 2.83).This relationship was not observed in Pakistan (OR 1.17, 95 % CI 0.93-1.47).Childcare practices were found to have no mediation effect on the association between IPV and child illness. Conclusion: The study provides evidence that IPV is a major public health problem affecting not only women, but also their children. Our finding supports designing IPV interventions that integrate IPV screening into routine neonatal care and childcare visits. This approach will not only contribute to monitoring and prevention of IPV but could also reduce child morbidity.

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