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

Recent Submissions

  • Biases and caveats to implementing genomic medicine in diverse populations

    Kessler, Michael D.; O'Connor, Timothy D.; 0000-0003-1258-5221 (2019)
    Genetic science has traditionally focused on the study of European populations, which has resulted in the under-representation of other ancestral backgrounds across the vast majority of genetic resources. Since data derived from a limited number of ancestral backgrounds are unlikely to represent the biological variation inherent to diverse populations, genetic and genomic methods and models predicated on this Eurocentric data will have questionable accuracy and limited value when applied to diverse patient populations. This can drive scientific and/or clinical disparities, which can then seem opaque and be difficult to resolve. The work outlined in this thesis aims to increase the prevalence of ancestrally informed genetic science by characterizing bias deriving from a lack of ancestry awareness, evaluating ancestral representation in genomic resources, and identifying novel ancestry-informed biological signal. I first characterize bias by evaluating a typical clinical variant prioritization pipeline, and I demonstrate a significant positive correlation between African ancestry proportion and the identified number of clinically evaluable variants. I then more directly explore ancestral representation across translational resources by estimating the genetic ancestry for 1,018 common cancer cell lines. This analysis highlights the marked ancestral underrepresentation that exists among preclinical cancer cell line models, and identifies novel signals of ancestry-specific gene expression and somatic mutation. Finally, I evaluate how de novo mutation rates vary across diverse human populations from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. I find associations with heterozygosity, a reduced mutation rate in the Amish founder population, and near zero estimates of narrow-sense heritability. On the whole, these findings help to quantify the effects of ancestral diversity and under-representation on the application of genomic medicine.
  • The Design and Development of Dual MCL-1/BCL-2 and HDM2/Bcl-2 Protein Family Inhibitors Using a Polypharmacology Approach

    Drennen, Brandon; Fletcher, Steven (2019)
    Apoptosis, a cellular process that leads to cell death, is a vital signaling pathway for maintaining homeostasis. Intracellular-activated apoptosis is regulated by the B-cell lymphoma 2 (BCL-2) family of proteins, which encompasses two classes of proteins: the pro-apoptotic and anti-apoptotic members. Apoptosis is controlled by a protein-protein interaction (PPI) between the two members. Specifically, the anti-apoptotic proteins’ surface hydrophobic binding groove binds to the α-helical Bcl-2 homology 3 (BH3) domain of the pro-apoptotic proteins, thus inhibiting apoptosis. During apoptotic conditions, BH3 activator proteins are expressed and disrupt the PPI, initiating apoptosis. During tumorigenesis, the anti-apoptotic proteins are overexpressed and capture the activator proteins before they can act, progressing tumor development. A strategy developed to overcome this oncogenic transformation is BH3 mimicry, the design of small molecules that behave like BH3 activators to free the pro-apoptotic proteins. Though potent BH3 mimetics have been synthesized, cytotoxic and resistance issues have arisen. Specifically, BCL-XL inhibition causes thrombocytopenia within patients and BCL-2 inhibition causes resistance mechanisms to emerge that involve the upregulation of MCL-1. Presently, there are no potent dual inhibitors of BCL-2 and MCL-1 to overcome these issues. Additionally, p53 has been shown to regulate apoptosis through the Bcl-2 family by either direct interactions or increasing their expression. P53 is rapidly degraded due to the overexpression of HDM2, a ubiquitin ligase, within cancer cells. The PPI between p53 and HDM2 resembles the PPI between the members of the Bcl-2 family. Also, Venetoclax (BCL-2 inhibitor) and idasanutlin (HDM2 inhibitor) act synergistically in combination therapies. Thus, we followed a polypharmacology approach to synthesize dual BCL-2/MCL-1 and dual HDM2/Bcl-2 family inhibitors. We were able to create potent dual MCL-1/BCL-2 indazole inhibitors (Ki MCL-1 < 1.50 µM, BCL-2 < 0.050 µM, BCL-XL > 10.00 µM), dual HDM2/Bcl-2 family pyrazole and imidazole inhibitors (Ki MCL-1 < 0.050 µM, HDM2 < 25.00 µM), HSQC-confirmed nicotinate-based MCL-1 inhibitors (Ki MCL-1 < 3.00 µM) and a new alpha-helix mimetic scaffold for disrupting PPIs. Further optimization of these inhibitors is planned, along with cell viability studies. Overall, these inhibitors can serve as starting points for future experiments and polypharmacology designs.
  • Bioinformatic Analysis of Single Nucleus Transcriptome Data of Huntington's Disease

    Malaiya, Sonia; Ament, Seth A.; 0000-0002-0010-3259 (2019)
    Huntington’s Disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. The earliest changes in HD are observed in the striatum, prior to the onset of symptoms. Here we use a knock in HttQ175/+ mouse model to perform single nucleus RNA sequencing (snRNAseq) of the striatums of four HttQ175/+ and three wild type 14 and 15 month old mice and obtain their expression profiles. Using available snRNAseq quality control and analysis methods, we identify eleven cell types within our samples, including the newly discovered “Eccentric MSNs”. We compute the differentially expressed genes between the two genotypes and find significant lowering of cell type specific markers in most cells with the HttQ175/+ mutation. Trajectory analyses reveal stages of HttQ175/+ MSNs that range from identical to extremely distinguished form the wild type MSNs, supporting the length dependent somatic expansion hypothesis.
  • Sex differences in soman-induced toxicity and response to medical countermeasures in serum carboxylesterase knockout mice.

    Kundrick, Erica; Pereira, Edna; Lumley, Lucille A.; 0000-0002-4222-1528 (2019)
    In rodents, exposure to chemical warfare nerve agent soman leads to status epilepticus and extensive neuronal loss. Mice and rats are less sensitive to nerve agent toxicity compared to primates since high levels of plasma carboxylesterase, which acts as a bioscavenger against soman, increase resistance of these rodents to organophosphorus poisoning. One objective of this research project was to determine the LD50s of soman in female plasma carboxylesterase knockout (ES1-/-) mice at the different stages of their estrous cycle and to compare toxicity across estrous and with male mice. Female mice in estrus were less susceptible to the soman lethality compared to female mice in proestrus and to male mice. The second objective was to evaluate dose-response effects of delayed midazolam treatment in soman-exposed ES1-/- mice. Delayed midazolam dose-dependently increased survival and reduced seizure severity but did not prevent epileptogenesis or brain pathology in seizure-sensitive brain regions, independent of sex.
  • The Roles of Autophagic SNARE proteins SNAP29 and SNAP47 in Autophagy and Enterovirus D68 Replication

    Corona, Abigail; Jackson, William T.; 0000-0003-2271-4541 (2019)
    Enterovirus-D68 (EV-D68) is a positive-sense, single-stranded RNA virus of the Picornaviridae family that causes respiratory disease in children and has been implicated in recent outbreaks of acute flaccid myelitis, a severe paralysis syndrome. We have demonstrated that EV-D68 induces autophagy upon infection and modifies the autophagic process to benefit its own replication. Autophagy is a regulated process of cytosolic degradation in eukaryotic cells which maintains cellular homeostasis by degrading damaged organelles, protein aggregates, microbes and other xenobiotics in the cytoplasm. The autophagic process is characterized by the formation of double-membraned autophagosomes around cytosolic cargo, which then undergo a series of fusion steps with endosomes and lysosomes to degrade the vesicle’s contents. The autophagy pathway is targeted by many pathogens, either to protect themselves from degradation or to utilize components to benefit replication. EV-D68 uses virally-encoded proteases to cleave an autophagosome fusion SNARE protein, SNAP29, blocking delivery of autophagosome contents, including nascent viruses, to the lysosome. Our data show that relocalization occurs for SNAP47 during autophagy induction, and is required for normal virus replication. SNAP47 plays a major role in acidification of autophagosomes into amphisomes, with binding partner VAMP7, which we hypothesize promotes maturation of virions into infectious particles. Using both viral- and non-viral forms of autophagy induction, these data suggest that the cellular network of SNARE proteins is being redirected during infection to promote EV-D68 replication and egress from the cell.
  • Designing Next Generation Genomics and Serological Tools for Surveillance of Plasmodium vivax Malaria to Guide Elimination Efforts in Southeast Asia

    Agrawal, Sonia; Plowe, Christopher V.; 0000-0003-4484-7433 (2019)
    Malaria is a major global health problem caused by mosquito-borne, protozoan parasites belonging to the genus Plasmodium. Plasmodium vivax, the human malaria parasite with the widest global distribution, accounts for majority of the total malaria cases outside sub-Saharan Africa. The inability to establish long term in vitro culture system and low parasite densities, combined with high levels of human genomic DNA isolated from patient samples with P. vivax infections, makes it difficult to obtain sufficient amounts of parasite DNA for whole genome sequencing (WGS). New, reliable, highly sensitive and specific methods are needed to produce high quality P. vivax WGS data. Genome-wide analyses of the parasite, using WGS, have the potential to improve our understanding of parasite population dynamics and help identifying locations that serve as possible transmission sources and sinks. Additionally, protein microarrays that can simultaneously measure human antibody responses to a large number of Plasmodium antigens have the potential to identify P. vivax specific biomarkers to detect not only current but also past malaria infections, providing a more sensitive surveillance tool for identifying human populations at risk. To address these needs, using Roche/NimbleGen SeqCap EZ whole genome capture technology, high quality WGS data was generated from P. vivax clinical samples collected from the China-Myanmar border. This new genome-wide data along with publicly available WGS from circulating isolates in Southeast Asia were utilized to characterize parasite genetic diversity and relatedness, population structure, complexity of infection, and distinguish locally transmitted infections from imported P. vivax infections revealing clonal parasite population on the China-Myanmar border. Using protein microarray analyses, several P. vivax specific serologic markers during active infection were identified that may serve as useful biomarkers of current or recent P. vivax infection supporting the possibility of serology as a tool for estimating species-specific malaria exposure to P. vivax in heterogeneous malaria transmission settings. The combination of next generation tools attempted to be designed as part of this dissertation will help improve the understanding of the genomic epidemiology and estimates of transmission patterns of this human malaria parasite, thus, guiding rational P. vivax malaria control and elimination policies in Southeast Asia.
  • Depletion of endoplasmic reticulum calcium triggers the loss of ER resident proteins

    Trychta, Kathleen Anne; Harvey, Brandon K. (2019)
    The endoplasmic reticulum (ER) contains proteins that carry out the diverse functions of the ER including calcium storage, protein folding, modification, and trafficking, lipid metabolism, and drug detoxification. When soluble ER resident proteins with an ER retention sequence (ERS) depart from the ER they interact with KDEL receptors in the Golgi membrane and are retrogradely transported to the ER lumen via the KDEL receptor retrieval pathway. ER calcium depletion disrupts this process resulting in the mass departure of ERS-containing proteins into the extracellular space. Such a loss of ER resident proteins has two potential consequences to an affected cell. First, there is a loss of proteins involved in the aforementioned critical ER functions. Second, the relocation of such proteins and their associated functions outside of the cell may cause changes in the extracellular environment. This dissertation describes the identification and characterization of a phenomenon whereby ER resident protein secretion is triggered by pathophysiological ER calcium depletion. By exploiting the enzymatic activity of one of the identified ERS-containing proteins we developed an assay to monitor changes in the ER proteome. We also developed a high-throughput screen that identified drugs that could prevent the release of ER resident proteins following ER calcium depletion and showed that several of these compounds have therapeutic potential in models of ER stress and ischemia. Taken together, the work described in my dissertation identifies a novel molecular mechanism of cellular dysfunction for which I have identified both endogenous biomarkers and possible therapeutics.
  • Community-level and Individual-level Predictors of Variation in Rates of Homelessness among Youth Transitioning Out of Foster Care

    Sneddon, Dori; Bright, Charlotte Lyn (2019)
    Youth who age out of foster care are a known high-risk subgroup for homelessness. Studies estimate between 19% and 36% of youth experience homelessness shortly after emancipation. This study examined homelessness among youth transitioning out of foster care by incorporating individual-level and county-level influences to better understand the risk of homelessness among this population. Multilevel models and generalized estimating equation models were constructed to include both individual- and county-level variables. Data were obtained from multiple national datasets: the 2011-2015 National Youth in Transition Database (NYTD), 2011 Adoption and Foster Care Analysis and Reporting System (AFCARS), University of Wisconsin’s County Health Rankings & Roadmaps Data and the 2013 U.S. Department of Agriculture (USDA) Rural-Urban Continuum Code data file. The analytic sample included 3,968 youth who responded to the NYTD Wave 1 (age 17), Wave 2 (age 19) and Wave 3 (age 21) survey. Of the sample, 35.3% experienced homelessness between 17 – 21 years old. Findings indicated statistically significant variation between counties in the proportion of youth who become homeless. Multiple individual-level factors were found to predict homelessness between ages 17 – 21. Prior homelessness, substance use history, and incarceration had a positive relationship with risk of homelessness. Connection with a caring adult, enrollment in school, and employment were inversely related to risk of homelessness. Specific to foster care experience, number of placements and age of entry had a positive relationship with risk of homelessness. Being in foster care at age 19 and at age 21 were related to a reduced risk of homelessness. Not as hypothesized, receipt of independent living services had a positive relationship with homelessness. None of the county-level indicators had a statistically significant relationship to the homelessness outcome. Policy and practice implications for child welfare include extending foster care, capturing housing histories and prioritizing housing plans for youth, and targeting intensive services to youth at the highest risk of homelessness. Future research to further examine socioeconomic community- and state-level predictors of homelessness among this population inform homelessness prevention and housing strategies for youth aging out of foster care. Suggested areas for improvement in NYTD data are also discussed.
  • Identification of the Exocrine Pancreas Proteases as Novel Regulators of β-cell Production

    Hostelley, Timothy Lain; Zaghloul, Norann A.
    The rising rate of type 2 diabetes (T2D) has made it a major health burden. This can largely be attributed to high rates of obesity, one of the greatest contributing factors towards the development of T2D. Despite this, the majority of obese individuals are not diabetic. This raises the question of what mechanisms mediate differential susceptibility to T2D in the presence of obesity. This project seeks to address this question by studying the mechanisms of differential susceptibility to T2D in two rare obesity ciliopathies, Alström Syndrome (AS) and Bardet-Biedl Syndrome (BBS). Both of these ciliopathies are characterized by near-complete penetrance of early onset obesity, but they exhibit markedly different rates of T2D. AS patients are far more susceptible, with the majority developing T2D by age 20, compared to a much smaller subset of BBS patients that are afflicted. In light of the likely contribution of β-cells to T2D susceptibility, we set to identify mechanisms of β-cell production in them. Here we have shown β-cell loss in AS and increase in BBS as a likely contributor towards the discrepant rate of T2D. Next, RNA-Sequencing of these models identified several exocrine pancreas proteases that were differentially expressed between AS and BBS. Thus, we hypothesized that these proteases affect endocrine pancreas function. To test this, we overexpressed each protease in transgenic zebrafish embryos in which β-cells can be visualized. Overexpression significantly increased the β-cell number and rescued the loss of β-cells observed in animals depleted of alms1. We found ctrb1 overexpression led to increased β-cell proliferation in transgenic larvae and rescued the AS model reduction in proliferation. In addition, we found that the proteases are taken up by β-cells in vitro and induce proliferation in cultured β-cells and freshly isolated ex vivo islet cultures, suggesting this effect is conserved in mammalian systems. Finally, endogenous uptake of CTRB1 was found in both zebrafish and mouse models, suggesting a direct interaction between the exocrine and endocrine pancreas. These data support an important role for exocrine pancreatic enzymes in the modulation of β-cells in diabetes.
  • Development of Novel Nanostructured Therapeutic Root Canal Dental Sealers with Strong Antibacterial and Remineralization Capabilities

    Baras, Bashayer Hussain; Xu, Huakun H. (2019)
    Root canal therapy aims to remove microorganism or at least reduce them to subcritical levels that permit the host’s immunity to eliminate infection and regenerate damaged tissues. However, due to the complex and variable root canal anatomy and the resistant nature of root canal biofilm, complete elimination of root canal microorganisms is rarely accomplished. In addition, it has been frequently reported that some of the most commonly used irrigating solutions, such as, sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) can adversely alter the chemical and mechanical properties of dentin, resulting in a brittle dentin structure that is more susceptible to root fracture. This dissertation aims to develop a therapeutic root canal sealing material with potent antibacterial properties and remineralizaition capabilities through the incorporation of dimethylaminohexadecyl methacrylate (DMAHDM) to provide bacterial contact killing in case of micro leakage, nanoparticles of silver ions (NAg) to eliminate bacteria in the more complex root canal anatomy through release of silver ions, and nanoparticles of amorphous calcium and phosphate (NACP) to reverse the action of NaOCl and EDTA on root dentin and strengthen the root structure through the release of Ca and P ions. In this dissertation projects, the effects of incorporating DMAHDM, NAg, and NACP on the physical and sealing properties were evaluated. The antibiofilm properties were assessed by polysaccharide production, live/dead, and colony-forming units (CFU) assays. The antibiofilm properties of the developed sealer were assessed on cured sealer disks and utilizing a human dentin model. In addition, the effects of NACP on the Ca and P ion release, pH-alkalizing properties, and influence on dentin hardness were all measured. The triple incorporation of DMAHDM, NAg, and NACP did not compromise the physical properties of the root canal sealer and demonstrated sealing properties that were similar to that of a commercial control material. The incorporation of DMAHDM and NAg alone into the root canal sealer demonstrated great reductions in bacterial viability and quantity. However, when both agents were combined the antibiofilm effects were maximized, resulting in CFU reductions of 6 orders of magnitude. The DMAHDM NAg containing root canal sealer was able to kill bacteria not only on the surface of resin disks but also bacteria impregnated inside human dentin. The incorporation of NACP into the respective sealer allowed for the release of high levels of Ca and P ions, neutralized the acid and increased the solution pH, and increased the dentin hardness to match that of sound dentin. This bioactive antibacterial and remineralizing root canal sealer is promising to prevent endodontic treatment failure and secondary endodontic infections while releasing high levels of Ca and P ions that could remineralize and strengthen the tooth structures and potentially prevent future root fractures and teeth extractions.
  • The Characterization of the PARK10 Gene USP24 in Autophagy and Mitophagy

    Thayer, Julia; Lipinski, Marta; 0000-0002-8607-5075 (2019)
    Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world and there is currently no cure, just treatments to mask symptoms. PD results in loss or damage to dopaminergic (DA) neurons within the substantia nigra pars compacta. Autophagy, a lysosome-dependent degradation pathway, is essential for neuronal survival. Dysregulation of this pathway has been linked to several neurodegenerative diseases. Defects in autophagy and mitochondrial specific autophagy (mitophagy) have been implicated in PD. The goal of this project was to characterize the function of the deubiquitinating enzyme (DUB) ubiquitin-specific peptidase 24 (USP24) in autophagy as well as in mitophagy. USP24 is located on chromosome 1p of the PARK10 locus that has been associated with late-onset PD. As a DUB USP24 is responsible for removing ubiquitin chains from target substrates, thus altering their stability and function. The precise role of USP24 in PD is not yet known. My data demonstrate that USP24 is a negative regulator of autophagy that is downstream of MTROC1 and acts via the class III PtdIns3K and ULK1 complexes. Specifically, USP24 negatively regulates ubiquitination levels and stability of the ULK1 protein, thus decreasing autophagy flux. I have also shown that USP24 is able to regulate autophagy in induced pluripotent stem cells (iPSC)-derived DA neurons with no adverse effects. In fact, knockdown of USP24 led to increased neurite length in aged iPSC-DA neurons, suggesting a potential neuroprotective effect. Furthermore, in addition to ULK1, USP24 may regulate levels of the PD-associated Parkin protein. Recruitment of Parkin to the outer mitochondrial membrane is necessary for initiation of mitophagy. My data demonstrate that similarly to ULK1, stability of Parkin is increased following USP24 knockdown, suggesting that USP24 may negatively regulate Parkin-dependent mitophagy. Together, my data provide a better understanding of USP24 function in regulation of autophagy, mitophagy and PD. In addition, the data suggest that USP24 could be a possible new therapeutic target for PD.
  • Natural Killer T Cell Development and Activation: Implications in Cancer Immunotherapy

    Shissler, Susannah; Webb, Tonya J.; 0000-0002-0660-9955 (2019)
    Natural killer T (NKT) cells are a population of innate-like lymphocytes that express both NK and T cell markers. Following activation by the recognition of glycolipid antigen presented in the context of CD1d, NKT cells have been shown to play important roles in infection, autoimmunity, and cancer. The research undertaken herein sought to determine factors important for NKT cell development and activation to further understand the potential of NKT cells in cancer immunotherapy. We characterized thymic NKT subsets in different strains of mice and demonstrated that both the size and the subset distribution of the NKT cell population is dependent on genetic factors. By developing sensitive methods to track cellular proliferation, we identified baseline differences in NKT cell subset activation parameters. Specifically, NK1.1+ NKT cells were less likely to enter cell cycle after stimulation, divided less when activated, and were more adversely affected by CD28 costimulatory blockade compared to NK1.1- NKT cells. In our lymphoma and breast cancer studies, we show that NKT cell recognition of cancer cells can be enhanced by the presence of an activating ligand, but not by PI3K inhibitors or CARP-1 functional mimetics. Additionally, NKT cell-mediated cytotoxicity is enhanced by, but not dependent on, CD1d engagement. Whereas NK1.1+ NKT cells are the optimal subset of NKT cells for use in cancer immunotherapy because of their potent cytolytic abilities, activation of NK1.1- NKT cells could pose a threat to anti-tumor immunity, attributable to their secretion of immunosuppressive or regulatory cytokines. Collectively, our data demonstrate a critical role for CD1d engagement and costimulation for optimal activation of NK1.1+ NKT cells. Due to the fact that cancers often downregulate CD1d and do not express costimulatory molecules, our data highlight the need for the development of strategies focused on inducing the activation of specific subsets of NKT cells for cancer immunotherapy.
  • Neighborhood Disorder and Mental Health Outcomes Among a Sample of Baltimore City Residents: The Influence of Urban Parks, Social Cohesion, and Social Control

    Mattocks, Nicole; Meyer, Megan; 0000-0002-4935-0859 (2019)
    The physical and social environments have increasingly received attention as key factors that explain health outcomes and health disparities for individuals. Recent studies have shown that being exposed to high levels of physical and social disorder (i.e., crime, vandalism, vacant buildings, drug activity) in the neighborhood environment can compromise mental health by generating fear, stress, anxiety and depression. Residents of urban disadvantaged neighborhoods are most at risk of exposure to disorder, and also experience higher rates of anxiety and depression, compared to those living in other settings. Some studies suggest green spaces (e.g., parks, gardens, tree canopy) and collective efficacy (i.e., social cohesion and informal social control) provide mental health benefits in the urban neighborhood environment. However, the relationships among these factors, and how they impact mental health in urban environments is complex, and research in this area is limited. The purpose of this study was to elucidate the relationships between these factors by first examining how one indicator of green space, proximity to parks, moderates the relationship between neighborhood disorder and mental health, and second testing whether social cohesion and informal social control mediate this relationship, for a sample of adults living in Baltimore City. Results from multilevel models demonstrated that neighborhood disorder was associated with anxiety and depression symptoms, consistent with theory and prior research. Proximity to parks did not moderate this relationship; however, social cohesion partially mediated the associations between disorder and both depression and anxiety, while social control partially mediated the association between disorder and anxiety, but not depression. Contrary to expectations, higher levels of perceived social control were linked to higher levels of anxiety symptoms. This study demonstrates that neighborhood conditions matter to individual mental health, and perceptions of the social environment act as an important pathway through which the environment influences mental health for Baltimore residents. More study is needed to understand the relationship between exposure to urban parks and mental health. Findings from this study contribute to our understanding of the social determinants of health, and provide further evidence that the neighborhood context is critical to the mental health of urban residents.
  • Defining the Mechanisms That Mediate Sexual Differentiation of the Developing Hippocampus

    Kight, Katherine; McCarthy, Margaret M., 1958-; 0000-0001-9382-4700 (2019)
    Studying the processes by which male and female brains develop differently is not only a rich source for understanding the contrasting mechanisms of brain development that enable an organism to respond appropriately as an adult to intrinsic and extrinsic factors, it is also important for understanding the etiology of the numerous neurodevelopmental disorders that exhibit a sex bias in prevalence or presentation. The hippocampus is an area of the brain responsible for context-dependent memory and regulation of the stress axis, and as such is implicated in many sex-biased neurodevelopmental disorders. There are two striking sex differences in the hippocampus of neonatal rats which may fundamentally shape the circuitry of this region of the brain differently between males and females. First, roughly twice as many proliferating cells are present in the hippocampus during the first week of life in males, compared to females, and second, the timing of the developmental shift in which GABA signaling switches from depolarizing to hyperpolarizing occurs later in males. This thesis sought to determine the mechanisms that promote the sex difference in depolarizing GABA in the neonatal hippocampus of rats, and whether there is a causal relationship between depolarizing GABA and cell genesis in this context. One set of experiments tested the role of the neurotrophin BDNF. Analyses of Bdnf gene expression patterns revealed a baseline sex difference that mirrored the sex difference in cell proliferation. However, Bdnf content in response to steroid hormone signaling in the neonatal hippocampus showed subregion-specific expression patterns that did not correlate with cell proliferation, indicating cell-type specificity of BDNF function in the developing hippocampus. A second set of experiments found female-biased expression in the neonatal hippocampus of several microRNAs known to regulate cell proliferation and neurogenesis. One of these microRNAs, mir124, was tested for its potential role in regulating cell proliferation and the depolarizing response to GABA, using a combination of in vitro and in vivo approaches. Functional studies also tested the role of miR124 in regulating the expression of NKCC1, a key chloride channel involved in regulating depolarizing GABA and proliferation.
  • In Vitro and in Vivo Characterization of Candida albicans and Streptococcus mutans Interactions

    Khoury, Zaid; Jabra-Rizk, Mary Ann; 0000-0001-9596-3560 (2019)
    The oral cavity is a complex environment harboring diverse microbial species that often co-exist within biofilms formed on oral surfaces. Within a biofilm, inter species interactions can be synergistic in that the presence of one organism generates a niche for another enhancing colonization. Among these species are the opportunistic fungal pathogen Candida albicans and the bacterial species Streptococcus mutans, the causative microorganisms strongly linked to the development of oral candidiasis and dental caries, respectively. Recent studies have reported the enhanced prevalence of C. albicans in children with early childhood caries indicating that this fungal-bacterial interaction may have clinical implications. In this study, we aimed to elucidate and characterize this interaction between these diverse species. Specifically, we designed in vitro and in vivo studies to validate the hypothesis that the presence of C. albicans in the oral cavity augments S. mutans colonization, potentially mediating dental caries development. Using various C. albicans mutant strains and a GFP-tagged S. mutans, metabolic viability and fluorescent biofilm assays were performed to assess S. mutans recovery from mixed biofilms and to elucidate the mechanisms of interactions. Additionally, to visualize the architecture of formed biofilms, confocal scanning laser fluorescent and electron scanning microscopy were used. Importantly, a clinically relevant mouse model of oral co-infection was developed to demonstrate C. albicans mediated enhanced S. mutans colonization in a host. The findings demonstrated significantly higher recovery of S. mutans from biofilms with C. albicans in vitro. Images revealed a high bacterial affinity to C. albicans, and secreted fungal cell wall polysaccharides were identified as the key factor mediating biofilm formation, particularly mannans. Importantly, analyses of harvested tissue demonstrated significantly higher S. mutans recovery from teeth of co-infected mice compared to mice infected only with S. mutans. Collectively, the findings strongly indicate that the presence of C. albicans in the oral environment may impact the development of dental caries and should be considered as a factor in evaluating the risk of caries. Results obtained in this thesis will support future studies using animal models of dental caries to further characterize this relationship in a closely related model in our laboratory.
  • Potential for School-based Malaria Treatment to Reduce P. falciparum Transmission

    Cohee, Lauren; Laufer, Miriam K.; 0000-0002-7575-630X (2019)
    School-age children bear an under-appreciated burden of malaria and are a key reservoir for the spread of P. falciparum. We conducted school-based cohort studies to measure the impact of treating students with positive malaria rapid diagnostic tests on subsequent gametocyte, the parasite stage required for human-to-mosquito transmission, prevalence and density. We concomitantly quantified the proportion of gametocyte burden in school-age children and compared it to that of other age groups in household-based surveys in the school catchment area. Treatment reduced the prevalence and density of gametocytes by 79% and 89%, respectively. Half of all gametocyte-containing infections were detected in school-age children. We estimated that school-based malaria treatment could reduce overall gametocyte prevalence in the community by 26% and 34% in the rainy and dry seasons, respectively. These results suggest that school-based malaria treatment could further decrease the burden of malaria in areas where malaria has remained entrenched despite current control measures.
  • Novel Cholinergics for Treatment of Central Nervous System Disorders

    Johnson, Chad; Coop, Andrew; 0000-0001-7584-3000 (2019)
    Approximately 16% of Americans are diagnosed with major depressive disorder, a mental disorder thought be caused by a combination of characterized by genetic, biological, environmental, and psychological factors. It can be accompanied by low self-esteem, loss of interest in normally enjoyable activities, low energy, and diminished quality of life. Between 2-7% of adults with this disorder die by suicide. In addition, almost half of patients who are treated initially with an SSRI do not achieve complete remission, and nearly a third after four different treatment regimens (nimh.nih.gov). While counseling and antidepressant medication can be effective treatments, current selective serotonin re-uptake inhibitors (SSRI's) take weeks before therapeutic effects are observed. This "delay" period of action is not well understood and presents a significant challenge for medical professionals in the management of major depression. Mechanisms of anti-depressants have been a major focus of both current/past research in hopes of developing more effective and faster acting drugs. Directly related to this, clinical data (nimh.nih.gov) that oral and intravenous treatment with the muscarinic cholinergic antagonist scopolamine had rapid anti-depressant effects in humans--likely mediated through an antimuscarinic effect. Unfortunately, scopolamine can produce cognitive impairment including memory disturbances due to its anticholinergic properties. Since major depressive disorder is associated with deficits in cognition, this would produce an undesired additive effect that would only exacerbate the problem. It is our goal to identify a muscarinic antagonist that may be able to relieve depression and have little to no effect on memory or cognition. The 3-exo-1-azabicyclo[2.2.1]heptane, 1-azabicyclo[2.2.2]octane, 1-azabicyclo[3.2.1]octane, and N-methyltetrahydropyidine 3 (and 4)-substituted-1,2,4-oxadiazoles appear to be excellent chemical scaffolds for the generation of potent muscarinic agonists/antagonists. In order to probe the orthosteric site of the mAChRs we designed a large library of compounds and evaluated them via a battery of pharmacological assays to confirm both their antidepressant and cognitive effects. This resulted in the identification of lead compound (CJ2100) that showed potent antidepressant activity without cognitive impairment. (Supported by NIMH Grant 107499)
  • Examining the Assisted Living Environment and Residents’ Satisfaction with Assisted Living

    Holmes, Sarah; Resnick, Barbara; Galik, Elizabeth; 0000-0002-5949-2463 (2019)
    Background: Understanding satisfaction with assisted living (AL) is essential for creating supportive environments that are specifically targeted toward the needs and desires of residents. Moreover, the AL environment may play an important role in residents’ satisfaction with AL. Unfortunately, few measures have been developed and tested to evaluate residents’ satisfaction with living in these settings. Additionally, limited research has examined the impact of the AL environment on residents’ satisfaction with AL. Purpose: The purpose of this dissertation was to: (1) develop and test a comprehensive AL environment measurement model; (2) evaluate the psychometric properties of the Resident Satisfaction Index (RSI) in a sample of AL residents; and (3) test the impact of the AL environment on residents’ satisfaction with AL. Guided by the ecological theory of aging, it was hypothesized that controlling for residents’ age, gender, functional level, cognition, and comorbidities, the AL environment would be significantly associated with residents’ satisfaction with AL. Methods: Secondary data analysis using baseline data from a study testing the Dissemination and Implementation of Function Focused Care in AL. A total of 501 residents in 54 AL facilities across three states were included in the sample. Structural equation modeling was used to test the AL environment model and examine associations between the AL environment and residents’ satisfaction with AL. Rasch analysis and differential item functioning (DIF) analysis was used to evaluate the RSI. Results: Findings showed that the AL environment model fit the data (2/df=1.861, p<.05; CFI=.858, RMSEA=.126). In addition, the RSI is a reliable and valid measure. Significant and substantive differences were noted on 6 items by cognition and gender. Finally, gender and function were significantly associated with residents’ satisfaction with AL and accounted for 2.6% of the variance. The AL environment, based on staffing, health care services, amenities, and physical environment, was not related to residents’ satisfaction with AL. Conclusions: Understanding the interplay between individual and environmental factors that influence residents’ satisfaction with AL will inform strategies to modify the environment to target the needs and preferences of residents and thereby improve residents’ satisfaction with AL.
  • Viral and Cellular Determinants of Picornavirus-mediated Autophagy Induction

    Corona Velazquez, Angel; Jackson, William T; 0000-0002-1157-6908 (2019)
    Macro-autophagy is a basal cellular process that involves the degradation and turnover of cytosolic components, including elimination of damaged organelles and cytosolic cargo. In response to cell stressors, such as but not limited to, starvation and infection from xenobiotics, autophagy is upregulated. The process is controlled by the upstream autophagy signaling ULK complex, composed of the kinases ULK1 or ULK2, and the scaffold proteins ATG13, RB1CC1, and ATG101. This complex serves as a nexus for signaling pathways from nutrient sensitive kinase complexes such as MTORC1 or AMPK. Poliovirus (PV) has been shown to induce autophagy in infected cells, but the mechanism of initiation has not been completely elucidated. Furthermore, the host cellular factors that are involved in this virus-induced autophagy are unknown. We recently have shown that PV does not require the ULK1/2 complex for replication or autophagic signal induction during infection, demonstrating a novel ULK1/2-independent autophagic signaling pathway. We show that knockdown of RB1CC1, a vital scaffold protein for the ULK1/2 complexes, has no effect on PV replication and does not impede the ability of the virus to induce autophagic signals. Furthermore, PV mediates the elimination of this complex during infection in a mechanism that is not dependent on the proteasome. PV causes the cleavage of an autophagic cargo receptor SQSTM1, which was previously described in CV-B3, and therefore impairs our ability to measure bona fide autophagy during infection. We have also found that several members of the Enterovirus genus: Enterovirus D68 (EV-D68), Coxsackievirus B3 (CV-B3), and Rhinovirus A1 (RV-A1) also do not require the ULK complex for replication or with their respective effects on autophagy. We have evidence that suggests that the BECN1 complex, downstream of the ULK complex, is dispensable for PV. Exogenous expression of viral proteins 2BC and 3A from PV and CV-B3 increase the presence of LC3+ puncta but show no acidification of autophagosomes, suggesting the presence of a secondary acidification signal. We discuss the implications of these findings in regards to the ability of picornaviruses to reformat the induction process for their own benefit.
  • Single Cell Tracking of Breast Cancer Cells Removes Aggregation and Allows Prediction of Sphere Formation Using Information From Early Cell Divisions

    Bailey, Patrick Christian; Martin, Stuart S. (2019)
    The mammosphere assay has become widely employed to quantify stem-like cells in a population. Problematically, there is no standard protocol employed by the field. Cell seeding densities of 1000 to 100,000 cells/mL have been reported. These high densities lead to cellular aggregation. To address this, we have individually tracked 1,127 single MCF-7 and 696 single T47D human breast tumor cells over the course of 14 days. This tracking has given us detailed information for the commonly used endpoints of 5, 7 and 14 days that is unclouded by cellular aggregation. This includes mean sphere sizes, sphere forming efficiencies and a well-defined minimum size for both lines. Importantly, we have correlated early cell division with eventual sphere formation. At 24 hours post seeding, we can predict total spheres on day 14 with 98% accuracy in both lines. This approach removes cell aggregation and potentially shortens a 5-14 day assay to 24 hours. To increase the throughput of the assay while still addressing the effects of aggregation, we incorporated a technology that uses lipid tethering on a low attach surface. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. We observed that Day 7 spheres in lipid-coated wells contained significantly more clonal spheres than traditional low attachment plates. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

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