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

  • The Muscle Xenograft Model of Facioscapulohumeral Muscular Dystrophy: Development, Optimization, and Novel Applications

    Mueller, Amber; Bloch, Robert J.; 0000-0002-2842-1072 (2019)
    Aberrant expression of DUX4 in human muscle causes Facioscapulohumeral Muscular dystrophy (FSHD) which affects about 1 in 8,333 individuals worldwide, yet the mechanism by which DUX4 causes muscle wasting is unknown. The DUX4 gene is unique to humans and transgenic animal models have largely failed to exhibit the dystrophic phenotype and endogenous molecular changes characteristic of FSHD. Therefore, studies of DUX4 signaling in human muscle have been limited to patient biopsies and cultures of myogenic cells in vitro. There are no therapies that target the mechanism of disease, thus there is a pressing need for studies of DUX4 in mature human muscle. We have developed a method to xenograft human-derived muscle precursor cells, isolated from patients with FSHD and controls, into the tibialis anterior of immune-deficient mice to form xenografts of pure human muscle tissue. The FSHD xenografts are robust, mature, and well organized. Human myofibers are innervated and associate with human satellite cells, making the xenografts structurally comparable to intact human skeletal muscle. The FSHD but not control xenografts express DUX4 and DUX4 gene targets and have 4q35 methylation profiles typical of FSHD. The FSHD grafts also display a novel biomarker of FSHD, SLC34A2, measurable by immunofluorescence, which will provide a quantifiable metric for future therapeutic studies. We have described several modifications to the engraftment strategy that can be used to answer complex mechanistic and functional questions regarding the FSHD pathophysiology. Finally, we report promising data from a collaborative study with Fulcrum Therapeutics in which we were able to repress the DUX4-signaling pathway by administering a targeted small molecule therapy to FSHD grafts. Ours is the first scalable and reproducible in vivo model of FSHD muscle. Future studies will include those aimed at continuing to delineate the molecular pathogenesis of FSHD, performing functional tests to define the pathophysiology of FSHD, and testing new and exciting therapeutic strategies aimed at reducing the DUX4 program in human FSHD xenografts.
  • Multiple Challenges in Kinship Families: How Are They Associated with Children’s Behavioral Health in Kinship Care?

    Xu, Yanfeng; Bright, Charlotte Lyn (2019)
    The use of kinship care has increased in the United States. This dissertation, comprised of three papers, aims to understand multiple challenges in kinship care and their associations with children’s behavioral health using data from the second National Survey of Child and Adolescent Well-being (NSCAW II). Paper 1 developed a new kinship typology based on financial assistance and examined factors associated with receiving Temporary Assistance for Needy Families (TANF) and foster care payments. Results from logistic regression models showed that child maltreatment, children’s externalizing problems, and receiving social services were significantly associated with receiving foster care payments. Living in poverty and a single-adult household were associated with receiving TANF. The results of paper 1 imply that child welfare workers need to increase kinship caregivers’ awareness of financial resources and to make the right resources accessible for them. Paper 2 examined longitudinal relations among economic hardship, economic pressure, TANF, foster care payments, and children’s behavioral problems in kinship care and non-relative foster care. Results of multi-level mixed-effects generalized linear models indicated that economic pressure was associated with children’s internalizing and externalizing problems, as was receiving TANF. Receiving foster care payments was associated with lower externalizing problems. Significant interaction terms showed that foster care payments had positive effects on children’s behavioral health among families without economic hardship and families with economic pressure. The results of paper 2 imply that assessing caregivers’ subjective economic experiences is important to promoting child wellbeing. Findings point to the hardships of families that receive TANF and suggest providing financial and non-financial services to these families. Paper 3 examined the association between neighborhood disorder and children’s behavioral problems and tested the mediating role of social support and the moderating role of race/ethnicity. Results of moderated mediation regression models showed that neighborhood disorder was associated with lower social support, while more social support predicted lower children’s internalizing and externalizing problems. Social support mediated the relation between neighborhood disorder and children’s behavioral problems, but race/ethnicity did not significantly moderate the pathways. The findings of paper 3 imply that interventions are needed to enhance kinship caregivers’ social support and neighborhood quality.
  • Molecular Mechanisms of Enzymes in Infection and Immunity

    Klontz, Erik; Sundberg, Eric J. (2019)
    Enzymes are biological catalysts that enable life by accelerating specific reactions. As new infectious diseases are discovered, the enzymes produced by these organisms require characterization. In some cases, they can be inhibited to prevent disease; in other cases, they can be coopted or altered to serve as diagnostic or therapeutic tools. In this work, we explore the mechanisms of several enzymes involved in infection and immunity. In the first major theme, we characterize the mechanisms of fosfomycin resistance proteins from Escherichia coli (FosA3) and Klebsiella pneumoniae (FosAKP), which degrade the antibiotic fosfomycin. Although the active sites of FosA enzymes are well conserved, differences in activity between enzymes may be due to the presence of an allosteric site at the dimer interface of these enzymes. We solved crystal structures with a novel small molecule FosA inhibitor, termed ANY1, which binds with high affinity to the active site and acts as a competitive inhibitor of fosfomycin binding. By inhibiting FosA, ANY1 potentiates the effects of fosfomycin in several priority Gram-negative pathogens, and may serve as a suitable lead candidate for structure-guided drug design and pre-clinical development. In the second major theme, we describe the mechanisms of enzymes with activity on the carbohydrates of IgG antibodies. EndoS and EndoS2 are enzymes produced by Streptococcus pyogenes, which remove the conserved glycan on Asn297 of IgG to evade the host immune system. We discovered that these enzymes share a conserved mechanism of substrate recognition, binding primarily to the core and α(1,3) antenna of the IgG glycan. EndoS2 recognizes a more diverse set of glycans through differences in the both the active site and a coevolved carbohydrate binding module. We then describe the structure and function of AlfC, an α(1,6)-specific fucosidase from Lactobacillus casei with activity on the core fucose of antibodies. We identified D200 and D242 as the most likely catalytic residues, and provide a structural basis for the remarkable specificity of this enzyme and transfucosidase mutants. Finally, we describe a novel application for these carbohydrate-active enzymes in the directed evolution of antibodies based on yeast display.
  • Health Savings Account Effects on Health and Debt

    Hageman, Sally; Shaw, Terry V. (2019)
    Title of Dissertation: Health Savings Account Effects on Health and Debt Sally Anne Hageman, Doctor of Philosophy, 2019 Dissertation Directed by: Terry Shaw, Ph.D. More than a decade ago Health Savings Accounts (HSAs) were deemed contrary to social work values and practice (Gorin, 2006). More recent research, however, demonstrated HSAs may help individuals’ access financial resources when encountering financial barriers (Hageman & St. George, 2019). To further examine the potential of HSAs, this study examines HSA effects on health and debt outcomes. Applying the framework of the social determinants of health (Dahlgren & Whitehead, 1991) and the health lifestyles theory (Cockerham, 2005), a subset of 12,686 respondents from three years (2010, 2012, and 2014) of secondary quantitative data from the National Longitudinal Surveys of Youth (NLSY) was drawn. The sample included respondents who answered survey questions about owning an HSA, chronic disease status, health behavior, and health-related debt. Descriptive, bivariate, weighted logistic regression, and generalized estimating equation (GEE) analyses were conducted. Descriptive analyses indicated about 47% of HSA owners were male, 64% were Non-Black/Non-Hispanic race/ethnicity, with an average age of 53.34 (SD=2.26) years old, 99% owned their home, and had an average income of $126,853 (SD=$122,994). About 75% of HSA owners reported they did not have a chronic disease and 70% reported they did not have health-related debt. Weighted logistic regression was conducted to determine if Chronic Disease status was associated with HSA ownership status. Results indicated Chronic Disease status (p=.88) was not significantly associated with owning an HSA. GEE was conducted to determine whether HSA ownership status was associated with respondent debt. Results of the GEE analysis indicated HSA ownership status (p=.76) was not significantly associated with reporting Debt.
  • Functional characterization of Myosin Binding Protein-C slow in health and disease

    Geist Hauserman, Janelle; Kontrogianni-Konstantopoulos, Aikaterini (2019)
    Myosin Binding Protein-C (MyBP-C) comprises a family of proteins with structural and regulatory roles in muscle. There are three MyBP-C isoforms in the family, encoded by different genes. Although the isoforms share significant structural and sequence homology, slow skeletal MyBP-C (sMyBP-C), encoded by MYBPC1, is unique as it is heavily spliced in both the NH2 and COOH-termini. To study the role of sMyBP-C in healthy, adult skeletal muscles, in vivo gene transfer and CRISPR plasmids were used to knock down sMyBP-C. Decreased sMyBP-C levels resulted in significantly decreased levels of thick, but not thin, filament proteins. The reduced levels of thick filament proteins were accompanied by disorganized A- and M-bands. Moreover, examination of the contractile activity of treated muscles demonstrated that downregulation of sMyBP-C resulted in significantly decreased force production and velocities of contraction and relaxation. In addition to the extensive exon shuffling that takes place in the NH2-terminus of sMyBP-C, it also undergoes PKA and PKC mediated phosphorylation within two motifs, which flank the first Ig domain of the protein. Recombinant NH2-terminal sMyBP-C phosphomimetic peptides were tested in co-sedimentation and in vitro motility assays, indicating that phosphorylation of sMyBP-C variants regulates actomyosin binding and sliding velocity. Mutations in MYBPC1 have been implicated in the development of distal arthrogryposis, while four recently discovered mutations (Y247H, E248K, L259P, and L263R) co-segregate with the development of a new myopathy characterized by muscle weakness, hypotonia, skeletal deformities, and tremor. In vitro studies and computational modeling suggest altered myosin binding and/or protein instability for the four mutations. Further in vivo evaluation of the E248K mutation in a heterozygous knock-in mouse model revealed significant biochemical, morphological, and behavioral deficits compared to wild type littermates. Additionally, functional assessment of heterozygous E248K muscles demonstrated decreased force and power production, as well as decreased cross bridge cycling kinetics, indicating the tremor may begin at the level of the sarcomere. My studies therefore reveal that sMyBP-C has important structural and regulatory roles within the sarcomere, is modulated through phosphorylation, and that novel MYBPC1 mutations lead to the development of myopathy and tremor that is of myogenic origin.
  • Traumatic Brain Injury (TBI) Causes Alterations in Myeloid Cell Function: Role of Sex Differences and Lung Infection on Overall Outcomes following TBI

    Doran, Sarah Jean; Loane, David J.; Faden, A. I.
    Traumatic brain injury (TBI) is a major cause of morbidity and mortality. Males are nearly 3-times more likely to die from brain injury than females. However, the impact of sex differences in relation to clinical outcomes following TBI is conflicting. Recent studies report that men have an increased risk for lung infection after TBI. Importantly, hospitalized severely injured TBI patients have nosocomial infection rates of 50%. Acute-onset pneumonia represents 30-50% of infections after more severe TBI, leading to increased deaths and disability. The goal of this work was to investigate the role of sex differences and lung infection on overall outcomes following TBI. In a well-characterized murine TBI model, there are increased numbers of infiltrating myeloid cells in male brains as compared to females following injury, correlating with poorer neurological outcomes. However, myeloid cells and microglia in male and female animals showed a similar pattern of activation in response to brain injury- including increased pro-inflammatory cytokines (TNF-α, IL-1β), reactive oxygen species (ROS), and greater phagocytic activity. These these findings suggest that sex differences with regard to immune responses after TBI that may impact overall outcomes. We also examined potential bi-directional brain-lung interactions by examining posttraumatic lung infection at 3- or 60-days after injury. Animals subjected to TBI showed increased mortality after infection with Streptococcus pneumoniae Type 3 (Sp) at both 3 and 60 days post-injury. However, mortality was greater with infection at 60 days following TBI and exacerbated brain injury, with increased markers of neuroinflammation including TNF-α, IL-1β, and NADPH oxidase 2 (NOX2) in the injured cortex. In the lung, TBI followed by infection resulted increased bacterial burden and pathology. Analysis of Ly6C+ infiltrating monocytes in the lungs of mice infected with Sp 3 days post-injury demonstrated that these cells were less able to produce TNF-α, ROS, and IL-1β in response to infection. In contrast, lung-infiltrating Ly6C+ monocytes in mice subjected to infection 60 days post-TBI produced twice as much IL-1β and 50% more ROS as Ly6C+ monocytes from Sham-infected mice. These data show that TBI causes immune alterations, both acutely and chronically, that may contribute to susceptibility to infection.
  • Molecular level studies of the impact of poly (oxonorbornenes) on D. rerio. embryos

    Klutts, Jennifer N.; Rosenzweig, Zeev (2019)
    Poly (oxonorbornenes) (PONs) are amphiphilic cationic polymers that possess antimicrobial properties. Cationic polymers are proposed as an alternative to antimicrobial peptides and it is important to assess their impact on organisms. The two side chains of PONs that are responsible for these properties are a hydrophobic alkyl and a charged amine. In this study, we investigated how changing the amine/alkyl ratio and polymer length affects the activity of PONs on the model vertebrate organism, D. rerio. (zebrafish). Zebrafish embryo toxicity test were used to elucidate the LC50 of PONs. Whole-mount immunofluorescence with caspase-3 was used to analyze apoptotic cells. We hypothesize that PONs would interact with the cell membranes of embryos to induce toxicity and the level of toxicity would depend on the molecular structure of PONs. Our results indicate that increasing the hydrophobicity and polymer length decreases the viability of the embryos and number apoptotic cells in the embryos.
  • Advancing Drug and Biomaterial Design with Constant pH Molecular Dynamics Simulations

    Tsai, Cheng-chieh; Shen, Jana (2019)
    Molecular dynamics (MD) simulation is a valuable tool for investigating motions of macromolecular systems; however, solution pH, a critical factor for biological and chemical processes, is neglected in conventional MD simulations. To address this weakness, our group developed various continuous constant pH molecular dynamic (CpHMD) tools. In this dissertation, applications and new protocols that utilize CpHMD for drug and biomaterial design are presented. β-secretase 1 (BACE1, implicated in Alzheimers disease) and Src kinase (implicated in cancer) were utilized as model systems for drug design applications. In chapter 2, we applied CpHMD to understand the binding of BACE1 with two small-molecular inhibitors. We discovered that, despite the structural similarity of the two inhibitors, the titration behavior of the protein active site differs and this difference dramatically impacts the protein-ligand interactions and consequently the inhibitor affinity. Further, we tested a new protocol that combines CpHMD titration with free energy simulations to construct the pH-dependent binding free energy profiles. The resulting data showed excellent agreement with experiment and identified one potential allosteric site in BACE1. Next, in chapter 3, we simulated Src kinase to test the capability of CpHMD tostudy kinases. Starting from the crystal structure of the inactive Src, CpHMD can capture the conformational activation along the major inactive states without introducing any biasing potential or mutation. Starting from chapter 4, we studied the chitosan-based hydrogel systems to explore the detailed molecular mechanisms that give rise to macroscopic materials properties. We examined the flexibility of chitosan chains under different environmental conditions such as pH and salt concentration. Simulation data revealed that in addition to electrostatic screening, salt ions enhance the chain flexibility by interrupting the intra-molecular hydrogen bonds and thereby shifting the conformational populations between extended and bent states. In chapter 5, the atomic-level mechanism of pH-responsive chitosan-based hydrogels with switchable mechanical properties was investigated. Our data suggested that the electrostatic crosslinks are formed through the pH-dependent salt-bridge interactions between the chitosan glucosamines and surfactant headgroups. The pKa difference between the chitosan crystallite and the surfactant-bound chitosan is a key for the persistent but erasable gradient in the structural and mechanical properties between the two crosslinked regions. In summary, my work provids insights that will contribute to drug and biomaterial design and highlights the usefulness of CpHMD in these fields.
  • Abutment Material Affects the Attachment of Co-cultured Fibroblasts and Keratinocytes

    Tibbs, Maria; Saito, Hanae (2019)
    Creating a stronger transmucosal seal around the implant abutment may help prevent epithelial downgrowth and resultant crestal bone loss. Most studies focus on titanium and zirconium, but provisional restorations are gaining popularity and these materials require further study. Previous in vitro models utilize a monoculture technique to understand cell behavior, which makes direct intercellular comparisons difficult. Our first aim was to develop a co-culture of human gingival fibroblasts and human oral keratinocytes. Then, cell attachment, proliferation, and migration across six commercially available abutment materials was ascertained and comparisons drawn. Discs made of smooth titanium, (control), rough titanium, CAD/CAM poly (methylmethacrylate), poly ether etherketone, smooth zirconium, and rough zirconium were chosen. Preliminary results indicate that at various time points, significant differences in fibroblast and keratinocyte proliferation and attachment exist among abutment materials.
  • Temporary anchorage devices for ridge preservation after extraction

    Joseph, Dr. Surya Elizabeth; Oh, Se-Lim (2019)
    The objective of this study is to investigate effects of transcortical mini-screws placed in the buccal plate of the extraction socket following tooth extraction in human subjects compared to untreated sockets. Patients planned for extraction of two maxillary premolars were recruited. One mini-screw was placed in the buccal plate of the extraction side on the experimental side. The contralateral extraction side was left untreated as the control side. Intraoral digital scans and clinical photographs were obtained at baseline, 4 months, and 8 months. CBCTs were exposed after the extraction and before mini-screw placement and at 8 months. A considerable decrease in ridge width was observed in all three subjects at experimental and control sides. However, the experimental sides exhibited less bone loss compared to the control sides in two subjects. Transcortical mini-screw placement in the buccal plate might reduce dimensional changes at the extraction side.
  • Developing a patient-driven cost-effectiveness analysis of pharmacological treatments for patients with chronic hepatitis C

    Mattingly II, T. Joseph; Mullins, C. Daniel; 0000-0001-7786-5780 (2019)
    Background: Innovations in hepatitis C virus (HCV) drug therapy included in comparative clinical effectiveness evaluations focus on sustained virologic response (SVR) without consideration of socioeconomic or psychological outcomes. This study aimed to identify and prioritize variables important to patients and determine the impact of patient-centered parameters on the cost effectiveness of HCV treatments. Methods: An individual-based state-transition model was developed with the guidance of a patient-centered multi-stakeholder advisory board and patient-only Delphi panel. The model was used to perform a patient-driven cost-effectiveness analysis (CEA) of direct acting antivirals (DAAs) over 10 and 20 year time horizons from both health sector and societal perspectives. The patient-centered model and CEA results were then compared with recently published HCV CEAs. Results: Patients identified treatment effectiveness, longer life, fear of complications, financial issues, quality of life, and impact on society as important factors to include. Fear of harming others was considered more important than physical symptoms in terms of patient-reported problems caused by HCV. Total infected life-years (ILYs) and work days missed were reduced in the treatment group for both 10 and 20 year health sector models in addition to quality-adjusted life-year gains. Compared to no treatment, the incremental cost-effectiveness ratio for treatment would be $3,464/ILYs avoided, $715/work day missed, and $39,086/QALY gained. When costs of absenteeism, presenteeism, and patient/caregiver time were included, the DAA intervention was cost-saving at both 10 and 20 years. Very few traditional economic models for HCV treatments attempt to capture the indirect and non-medical costs or outcomes that may impact HCV patients. Conclusions: Treatment was cost-effective from a health sector perspective and cost-saving when including non-health costs such as patient/caregiver time and productivity. Compared to published HCV CEAs that focused mainly on SVR, our patient-centered CEA provides results that reflect the outcomes of interest informed by direct patient engagement.
  • Drug Repurposing for Lymphatic Filariasis Enabled with Multi-Species Genomics Approaches

    Chung, Matthew; Hotopp, Julie C. Dunning; 0000-0002-9545-523X (2019)
    Filarial diseases affect >200 million individuals worldwide, with another ~800 million individuals at risk across ~50 countries. The causative agents of filariasis are roundworms of the superfamily Filarioidea, also known as filarial nematodes. The most common human filarial disease is lymphatic filariasis, caused by one of three filarial nematodes: Wuchereria bancrofti, Brugia malayi, and Brugia timori. There are four different antihelminthic therapeutics for lymphatic filariasis—albendazole, diethylcarbamazine, ivermectin, and doxycycline. Three are primarily microfilaricidal and have limited efficacy on adult worms. The fourth therapeutic, doxycycline, adulticidal, but causes complications when administered to pregnant women or children <9 years of age. Combined with the emergence of drug resistance in filarial nematodes, new therapeutics are needed for the treatment for lymphatic filariasis. To identify potential drug targets, we conducted a multi-species transcriptomics analysis of B. malayi, its obligate mutualistic endosymbiont Wolbachia endosymbiont wBm, and its laboratory vector host A. aegypti across the entire B. malayi life cycle. We validated the use of the Agilent SureSelect RNA-Seq capture platform to enrich for B. malayi and wBm sequencing reads in low coverage samples, namely those taken during the B. malayi vector life stages in A. aegypti. For the analysis of wBm we developed FADU (Feature Aggregate Depth Utility), a quantification tool designed for prokaryotic RNA-Seq analyses, with an emphasis on accurately quantifying operonic genes. From our transcriptomics analysis, we identified an over-enrichment of the bromodomain and extra-terminal (BET) family of transcription factors, upregulated in the adult female, embryo, and microfilariae life stages. In previous studies, knockdowns of the BET proteins in Caenorhabiditis elegans lead to adult worm sterility and sometimes lethality. We treated adult female B. malayi in vitro with the BET inhibitor JQ1(+) and found that JQ1(+) induced sterility and worm death at lower concentrations than ivermectin, suggesting BET inhibitors may be promising antihelminthics. We also sequenced the genome of the Wolbachia endosymbiont of W. bancrofti, wWb, and used comparative genomics to identify conserved genes between lymphatic filariae Wolbachia. Additionally, we developed an approach for determining species using core genome alignments to reassess the supergroup designations in the genus Wolbachia.
  • Application of Machine Learning Algorithms for Predicting Missing Cost Data

    Rueda, Juan-David; Slejko, Julia; 0000-0002-0907-7106 (2019)
    Objective: To compare new alternatives to estimate health care costs in the presence of missing data using methods based on machine-learning (ML). Introduction: Costs must be correctly estimated for value assessment and budget calculations. Problems arise when they are not correctly estimated. Sometimes costs can be biased and lead to wrong decisions that affect population health. Cost estimation is a challenging task and it is more challenging in the presence of missing data. Methods: We used Surveillance, Epidemiology, and End Results program (SEER)-Medicare including patients with multiple myeloma newly diagnosed from 2007-2013. We explored the problem of missing data using different approaches creating artificial missing data. We hypothesized that the use of ML techniques improves the prediction of mean medical total costs in the presence of missingness. ML methods included support vector machines, boosting, random forest, and classification and regression trees. First, we analyzed the problem considering only one dimension, when one variable is missing in a cross-sectional scenario, using generalized linear models as a comparator against ML. Then, we added time as a factor for missingness, utilizing reweighted estimators against ML. Finally, we explored the different levels of censoring and determined how each censoring level affected our cost estimations. In this case, we created multiple linear spline models to establish the effect of censoring on the bias of the estimator. Results: We demonstrated that ML algorithms had better prediction when data were missing completely at random and missing at random. All the methods performed badly in the missing not at random scenario. In the second aim, we showed that ML-based methods predict just as well as reweighted estimators for the five-year total cost of a patient with multiple myeloma. Lastly, we found that ML methods are consistent and robust at low and moderate levels of censoring; however, we failed to prove that they are better than the reweighted estimators. Conclusions: ML-based methods are a good alternative for the prediction of missing cost data in the case of cross-sectional and longitudinal data.
  • An investigation into the behavioral effects of targeted memory reactivation during sleep on sensorimotor skill performance

    Johnson, Brian Philip; Westlake, Kelly P. (2019)
    Background. Memory consolidation occurs during sleep, providing an opportunity to enhance upper extremity (UE) function in people with residual impairments post-stroke. Targeted memory reactivation (TMR) has been used to enhance this process, which involves pairing auditory cues with task performance and subsequent cue replay during sleep. TMR application during sleep leads to increased task-related brain network connectivity and behavioral performance in healthy young adults. Yet it remains unknown whether TMR can enhance sensorimotor performance in individuals with stroke. Methods. Healthy younger and older adults and individuals with chronic stroke were trained on a non-dominant (or non-paretic) UE throwing task before a period of waking or sleeping consolidation, with some receiving TMR throughout the consolidation period. Study 1 involved the use of TMR throughout the first two slow wave sleep periods over a full night of sleep with young adults. Studies 2, 3, and 4 investigated whether TMR throughout a one-hour nap was sufficient to influence sensorimotor performance in young adults, older adults, and people with a history of stroke, respectively. Results. All studies found that TMR application during sleep enhanced sensorimotor performance. In addition, TMR during wake did not influence sensorimotor performance (Studies 1 and 2), and enhanced performance of a cognitive aspect of the trained task (Study 2). Additional generalization and transfer tests helped to support the hypothesis that TMR enhanced a task-specific motor program, as improvements were seen within the trained task but not un-trained, but similar tasks. Lastly, sleep alone appears to stabilize sensorimotor performance variability, but this process demonstrates an age-related decline. Conclusion. This dissertation has shown that the use of TMR during sleep is a useful method for enhancing sensorimotor performance in healthy young and old adults, as well as individuals with a history of stroke. Future research may lead to an adjunct to traditional physical rehabilitation protocols.
  • Spatiotemporal Regulation of Myosin II Dynamics during Cell Movement

    Snell, Nicole; Rizzo, Mark A. (2019)
    The goal of this research was to investigate how the phosphorylation of non-muscle myosin II (NMMII) by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) regulates cell motility. Cellular movement is important to both biological processes such as immune response, organism development, and axon guidance and to diseases such as cancer metastasis and hypertension. Movement requires a complex series of coordinated events involving the simultaneous buildup and tear down of the actomyosin cytoskeleton. The actomyosin cytoskeleton stabilizes cellular protrusions by joining with proteins in the extracellular matrix (EM), like fibronectin, and together they produce stress fibers that allow movement. NMMII regulatory light chain (RLC) phosphorylation at Serine 19 and Threonine 18 helps drive cell movement. Removing NMMII causes cells to lose structure and lose their migratory capabilities. Subsequently, phosphorylation allows NMMII to bind to actin filaments and create actomyosin crossbridges, the structural components, of the leading and lagging edges of moving cells. The dynamic activity of NMMII and MLCK at the leading edge remains undetermined in live cells, and it is also not well understood where MLCP influences cell movement on the motile edge. I investigated the moving edge of the cell using a multiparametric imaging approach with Förster resonance energy transfer (FRET) biosensors for NMMII, MLCK, and MLCP. Transfected NIH3T3 fibroblasts were imaged using fluorescence polarization microscopy. My results suggest that NMMII and MLCK activity are compartmentalized at the leading edge during cell motility and that there are differential phases of activity on a retracting membrane. We aim to understand the spatial relationship of NMMII phosphorylation with its regulators in different areas of the cell during movement. Taken together, this thesis work advances our understanding of non-muscle myosin II phosphorylation and regulation during random cell migration.
  • Computational Modeling of Behavior and Neural Mechanisms of Decision-Making Using Reinforcement Learning Theory

    Pietras, Bradley William; Schoenbaum, Geoffrey; Dayan, Peter, 1965- (2019)
    In the study of learning and decision-making in animals and humans, the field of Reinforcement Learning (RL) offers powerful ideas and tools for exploring the control mechanisms that underlie behavior. In this dissertation, we use RL to examine the questions of (i) how rats represent information about a complex, changing, task; (ii) what are the relevant variables underlying their decision-making processes; and (iii) whether those variables are encoded in the firing rates of neurons in the orbitofrontal cortex (OFC). We addressed these questions by making inquiries across three levels of understanding: computational theory, algorithmic representation, and physical implementation. Within this tri-level framework, we hypothesize that the subjects are engaged in a form of approximately optimal adaptive control. This involves their tracking critical, task-relevant, features of their environment as these features change, and then making appropriate choices accordingly. Two classes of RL algorithms were constructed, embodying different structural assumptions. One class of so-called return-based algorithms is based on elaborations of a standard Q-learning algorithm. The other, novel, class of income-based algorithms is based on a rather weaker notion of action-outcome contingency. Both classes of algorithm were parametrized and other factors were included such as perseveration. We t the algorithms to behavioral data from subjects using complexity-controlled empirical Bayesian inference. Internal variables from our algorithms were then used to predict neural ring rates of OFC neurons that were recorded as subjects performed the task. Linear regression, randomization testing, and false discovery rate analysis were used to determine statistically significant correlations between the predictors and neural activity. We found that income-class algorithms augmented with perseveration offered the best predictions of behavior. For the least restrictive statistical test (linear regression, p < 0.05), as many as 24% of the neurons were significantly correlated with variables associated with the best-fitting algorithm. By contrast, for our most restrictive test (randomized false discovery rate < 0.05), only 3% of the neurons passed as significant for one or more of our predictor variables. Other forms of neuronal dependence were apparent, including neurons that appeared to change their computational function dynamically depending on the state of the task.
  • Therapeutic Evaluation of a Novel Topical Antimicrobial Formulation against Candida-Associated Denture Stomatitis in an Experimental Rat Model

    Sultan, Ahmed; Jabra-Rizk, Mary Ann; 0000-0001-5286-4562 (2019)
    Candida-associated denture stomatitis (DS), caused by the fungal species Candida albicans, is the most common manifestation of oral candidiasis and is prevalent in up to 70% of denture wearers. DS tends to be a persistent and recurrent oral condition as a consequence of the ability of C. albicans to adhere to denture material and invade associated palatal tissue. There are currently no effective therapeutic strategies targeting DS, and despite antifungal therapy, infection is often re-established after treatment ceases. Therefore, it has become crucial to identify novel therapeutic approaches. Antimicrobial peptides have attracted significant attention as candidates for drug development due to their potent antimicrobial and anti-inflammatory properties, lack of toxicity and lack of development of drug resistance. Specifically, histatin-5 (Hst-5), naturally produced and secreted by host salivary glands, has demonstrated potent antifungal activity, including against strains resistant to traditional antifungals. However, our laboratory has previously demonstrated vulnerability for Hst-5 to proteolysis by C. albicans secreted proteolytic enzymes at specific amino acid residues. Therefore, to generate a resistant derivative of Hst-5, we engineered a variant (K11R-K17R) with substitutions in the amino acid residues at the cleavage sites. The new peptide proved to be more stable, and unlike the native Hst-5, resistant to proteolysis by C. albicans proteases. Importantly, for clinical application, we designed a polymer-based bioadhesive hydrogel as a delivery system for the peptide and developed a therapeutic formulation specifically designed for oral topical application. The potency of the new formulation in inhibiting C. albicans adherence and biofilm formation on denture acrylic material was demonstrated in vitro indicating a potential clinical applicability against DS. To that end, using 3D digital design and printing technology, we engineered and fabricated a universal intraoral device that was successfully used in the animals to develop clinical disease mimicking DS as in humans. Using the novel animal model, we established the clinical utility of the formulation for the prevention of biofilm formation on denture device and DS development. Importantly, in addition to DS, the formulation can also be used for treatment of other forms of candidiasis as well as serve in augmenting host natural immune defenses.
  • Neonatal Nurses' Work in a Single Family Room NICU

    Doede, Megan; Trinkoff, Alison M. (2019)
    Background: In the past twenty years, neonatal intensive care units (NICUs) have undergone changes in layout from open-bay (OPBY) to single family room (SFR). SFR layout may be advantageous to nurses’ work in that it improves the quality of the physical environment, patient care, and parent-nurse interactions. SFR layout may disadvantage nurses’ work in terms of decreased interaction among the NICU patient care team, increased nurse workload, and decreased visibility on the unit. It is unclear exactly how SFR layout is producing these changes. Purpose: This study asked: what is it like for neonatal nurses to work in a SFR NICU? Methods: Interpretive description, a qualitative methodology, guided this study. Interviews and observations were conducted in one SFR NICU over a six-month period. Data were coded broadly, then collapsed into themes as patterns within the data emerged. The Systems Engineering Initiative for Patient Safety model aided interpretation of nurses’ job demands. Emotional work was conceptualized as being preceded by emotional demands and anteceded by stress and burnout. Results: A total of 15 nurses participated. Overall, privacy, visibility, and proximity were integral in shaping nurses’ work. Regarding job demands, four themes emerged: challenges in infant surveillance and informal communication, alarm fatigue, and increased walking distances. Regarding emotional work, four themes emerged: families “living on the unit,” isolation of infants, ability to form trust and bonds, and sheltering. Emotional demands increased when families were living on the unit or when infants were left in isolation but were absent when nurses were able to form trusting relationships with parents and shelter them. Privacy gains on SFR NICUs may serve to balance losses in visibility and proximity for nurses. Conclusions: NICU layout impacts nurses’ job demands and emotional work. Future research should investigate unit layouts that maximize visibility and proximity for nurses while maintaining privacy. Neonatal clinicians transitioning to SFR layout should consider overall visibility and proximity of patients, equipment, and staff members from any point on the unit as a primary avenue for decreasing nurses’ work demands. Neonatal nurses will benefit from tactics that improve their communication skills with families.
  • Neuroprotective role of nicotinamide adenine dinucleotide precursor in modulation of mitochondrial fragmentation and brain energy metabolism

    Klimova, Nina; Kristian, Tibor (2019)
    Nicotinamide adenine dinucleotide (NAD+) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD+ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD+ results in impaired mitochondrial and cellular functions. Administration of NAD+ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age associated physiological decline, and inhibit post-ischemic NAD+ degradation and cellular death. In this work we identified a novel link between NAD+ metabolism and mitochondrial dynamics. A single dose (62.5mg/kg) of NMN, administered in naïve animals and after animals are subjected to transient forebrain ischemia, increases hippocampal mitochondria NAD+ pools and drives a sirtuin 3 (SIRT3) mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species (ROS) levels via SIRT3 driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 (S616)), with mitochondria. In conclusion, manipulation of mitochondrial NAD+ levels by NMN results in metabolic changes that protect mitochondria against ROS and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.
  • The Effects of Glossectomy on Airway to Tongue Ratio and Mandibular Morphology Using MRI

    Kim, Eric J.; Stone, Maureen L. (2019)
    Purpose: This study asked if glossectomy surgery causes anatomical changes of the surrounding structures and the airway by altering the balance of forces in the oral cavity. We predict that glossectomy patients will have proportionately larger pharyngeal air spaces than controls relative to the hard and soft structures around the mandible. Materials and Methods: Twenty subjects were studied, ten T1 or T2 SCC glossectomies and ten controls. The gathered MRI data sets were reconstructed into 3D volumes. Results: Mid-sagittal transpalatal airway lengths were significantly shorter for the glossectomy subjects. All other measurements were not statistically significant between the two groups. Discussion: A person may compensate for the reduction of tongue size following glossectomy, which may contribute to a shorter A-P airway distance at the transpalatal level. However, all other tests were not statistically significant, including the transpalatal area inidicating that objects in the oral cavity adapt to the reduction in tongue size and does not affect the established equilibrium. The overall transpalatal airway size may be maintained in post-glossectomy speakers by lateral expansion of the airway at the transpalatal level. Conclusion: This study concluded that the spatial relationships between airway and oral structures may change in dimension, but not in balance of forces following glossectomy. Second conclusion was that a 3-dimensional imaging is required for evaluation of the airway.

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