The University of Maryland Graduate School Baltimore (UMGSB) offers 27 master's and doctoral programs in health, physical, biomedical, medical, and social sciences.

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  • 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.
  • Alveolar Bone Height Changes in Patients Treated with Conventional and Damon Brackets

    Akinwande, Akinwale Banji; Schneider, Monica, D.D.S., M.S. (2019)
    Objective: To compare the distance between the cementoenamel junction (CEJ) and marginal alveolar bone (MAB) on the buccal root surfaces of the maxilla and mandible after orthodontic treatment using Conventional bracket system (CS) and Damon bracket system (DS). Methods: The sample included 30 patients, 14- 46 years of age (13 treated with CB and 17 with DB) with moderate to severe crowding treated nonextraction. We compared pre and post-alignment cone beam computed tomography (CBCT) images to measure the bone height (BH) changes of 24 buccal surfaces for each subject. Results: Even though we found great variability in BH levels, there was a statistically significant difference (Paired t-test: p value ≤ 0.05) between pre and post- alignment CBCT images of teeth in both Conventional and Damon groups. There was no statistically significant difference (independent t-test: p value ≤ 0.05) in BH changes when comparing the 2 bracket systems. Conclusion: The bone height levels changed after orthodontic treatment with both Conventional and Damon bracket systems, but there appears to be no significant difference in alveolar bone height changes between the two brackets in this study.
  • The Effect of Malocclusion Severity and Treatment Duration on Patient Satisfaction with Clear Aligner Therapy

    Wiese, Lauren Elizabeth; Bosio, Jose A.; Williams, Robert E., D.M.D., M.A.; 0000-0003-0335-7606 (2019)
    Objective: Determine if a patient’s initial malocclusion and treatment duration influenced satisfaction with clear aligner (Invisalign®) therapy. Methods: Thirty-three orthodontic patients treated with clear aligners responded to a satisfaction questionnaire containing twenty-one questions relating to their satisfaction with treatment, approximately two years after completing treatment. Overjet, overbite, and maxillary and mandibular anterior crowding/spacing were measured to determine the initial severity of their malocclusion. Logistic regression analyses with satisfaction factors as the dependent variable were used to quantify associations between patient satisfaction regarding both the initial severity of malocclusion and treatment duration. Results: Overall, patients were satisfied with aligner treatment. However, no significant associations were observed between patient satisfaction and either the initial severity of the malocclusion or treatment duration. Conclusion: The current study does not find that initial malocclusion or treatment duration significantly affects patient satisfaction. Patient satisfaction with clear aligner therapy is generally very high, and multi-factorial.
  • Development of the Drude Polarizable Force Field for Molecular Dynamics Simulation

    Lin, Fang-Yu; MacKerell, Alexander D., Jr.; 0000-0003-0103-0167 (2019)
    Molecular dynamics (MD) simulations have been widely applied to study biomolecular systems. While MD simulations have been used in a variety of applications, the accuracy of the results depends strongly on the force field (FF) used. The commonly used FFs are additive or non-polarizable FFs. However, one limitation of the additive FFs is the lack of electronic polarizability to treat molecules. To overcome this, an attractive approach is to introduce the explicit treatment of electronic polarizability into the potential energy function known as polarizable FFs. In Chapter 1, an overview of the CHARMM empirical FF as well as the development and application of the polarizable CHARMM FF based on the classical Drude oscillator is presented. As noncovalent halogen bonding interactions between halogenated ligands and proteins are important in drug design, a well-parametrized polarizable FF for halogen containing compounds is required to perform more accurate modeling of halogenated molecules. During the course of this development, a significant contribution of halogen-hydrogen bond donor (X-HBD) interactions to ligand-protein complexes was uncovered in addition to halogen bond (XB) interactions (Chapter 2). In Chapter 3, the development of halogen polarizable FF to both aliphatic and aromatic systems using halogenated ethane and benzene model compounds is illustrated, with emphasis on optimizing both X-HBD and XB interactions as discussed in Chapter 2. To assure good reproduction of X-HBD interactions with proteins, further optimization of atom pair-specific Lennard-Jones (LJ) parameters is required for both the polarizable and non-polarizable halogen FFs (Chapter 4). Finally, Chapter 5 presents the current optimization of polarizable protein FF to yield a more accurate description of the polarization response in simulations. The resulting protein FF, referred to as Drude-2019, will be more applicable in the study of biomolecular systems. Overall, the advances made in this dissertation will facilitate important discoveries of a range of chemical and biological phenomena.
  • Examining Dietary Patterns and Relationship to Caloric Intake in a Sample of Youth with Antipsychotic Induced Weight Gain

    Bussell, Kristin Lynn; Scrandis, Debra; Friedmann, Erika; 0000-0002-3128-0178 (2019)
    ABSTRACT BACKGROUND: Antipsychotic medication (APM) treatment has risen dramatically over the past 15 years in children/adolescents with serious mental illness, increasing risk for serious cardiometabolic sides effects such as rapid weight gain, increased lipids/triglycerides, reduced insulin sensitivity and hyperglycemia. Given the long-term nature of childhood onset mental illness these youths are at considerable high risk for early onset diabetes, cardiovascular disease, chronic morbidity and shortened lifespan. Although clinical practice guidelines for assessment/monitoring have been established none have been developed for dietary assessment/interventions in weight management. Studies examining APM effects eating behaviors and nutritional composition/adequacy are lacking. METHODS: This dissertation includes a literature review related to weight loss strategies in youth treated with APM. Next a secondary analysis of dietary data collected from 117 overweight/obese youth treated with APM was conducted to examine baseline dietary status and change over 6 months after 8 session of healthy lifestyle education. Lastly, qualitative data collected from the parent/child regarding facilitators/barriers to healthy eating was analyzed. RESULTS: Literature review found primarily intervention studies with metformin, which was moderately effective, but without examining diet/exercise. Several dietary/exercise studies reported decreased weight/BMI but did not report dietary intake. Analysis in this study found excess consumption of carbohydrates, protein, fat and total/added sugar while deficient in fruits, vegetables, whole grains, fiber and water when compared to the USDA recommended daily allowance. Change in dietary intake over 6 months found significant decreases in calories, carbohydrates, protein, total/saturated/solid fat, total/added sugar, refined grains, total dairy and cheese with small increases in whole fruit, total vegetables and dark green/orange vegetables, although not statistically significant. Qualitative responses from youths/parents on barriers/facilitators to healthy eating identified barriers as excessive appetite/cravings for sweets, junk food in the home, fast food, disliking fruits/vegetables, and lack of meal planning and food shopping/preparation. Facilitators included encouragement from family, removing junk foods, eating home more, making healthy foods tasteful and including the child in meal planning. CONCLUSIONS: This study provides new information which contributes to understanding dietary intake/eating behaviors in youth treated with APM. Findings suggest a possible influence of a simple dietary intervention on changes in food intake.
  • The impact of organizational culture and climate in child welfare agencies on outcomes for children involved in the child welfare system: A multi-level analysis of a nationally representative sample

    Goering, Emily Smith; Hopkins, Karen M., 1954- (2019)
    Child welfare organizations in the U.S. are tasked with the overarching goal of protecting children from abuse and neglect. The achievement of this goal has been found to be difficult and some child welfare organizations seem to be more effective at reaching this goal than others. A dearth of empirical literature exists in understanding how child welfare organizational functioning impacts its ability to achieve positive outcomes for the children who come into contact with their local child welfare system. An extensive review of the literature revealed that culture and climate of organizations may play an important role, but the existing research is unclear about the extent and direction of that role. Additionally, methodological issues with the existing studies threaten the validity of the results. The present dissertation builds on existing research and conducts secondary analysis using a nationally representative sample. The study applied theories of organizational social context and ecological model to answer the research question: When controlling for risk factors related to child characteristics and organizational contextual characteristics, to what extent do the culture and climate of the child welfare agency impact child-level outcomes? Using the National Survey of Child and Adolescent Wellbeing (NSCAW II), bivariate and multivariate analyses were conducted to answer the research question. Results indicate that individual, agency, and local context characteristics impact recurrence of abuse during the study period. At the individual level, living in a poor household and having prior substantiated maltreatment increased the odds of recurrence. At the agency-level, of the six culture and climate variables, only the climate score of functionality had an impact on risk of recurrence. The agency-level local context variable of county child poverty had the largest effect on recurrence and added explained variance to the model. However, both significant agency-level variables did not impact recurrence in the expected direction. Future research should continue to focus on research methods, better conceptualization and measurement of organizational constructs, and utilize an ecological perspective approach.
  • A Comparison of the Levels of Salivary Biomarkers between Conventional Smokers and Electronic Cigarette Users (A Pilot Study)

    Faridoun, Afnan; Meiller, Timothy F. (2019)
    Cigarette smoking is known to alter host response and the release of inflammatory mediators and cytokines in the body. Electronic cigarettes were marketed to provide the same sensation of conventional smoking without detrimental health consequences; however, the safety of their use is an area where more research is needed. Utilizing saliva as a diagnostic medium has been rising, however, there is a lack of studies investigating the effect of e-cigarettes on the salivary biomarker profile. The purpose of this pilot project was to evaluate the salivary cytokines, associated with inflammation, and CRP profile in e-cigarette users, and compare that to cigarette smokers and controls in an attempt to assess the influence of the use of e-cigarettes on salivary biomarkers. Statistically significant elevated levels of IL- 1β accompanied by lower mean levels of IL-1RA in e-cigarette users were detected. TNF- α showed elevated values in e-cigarette users similar to conventional smokers.
  • Molecular Mechanisms Underlying the Metastasis Suppressor Activity of NME1 in Malignant Melanoma

    Pamidimukkala, Nidhi Vela; Kaetzel, David M. (2019)
    Metastatic melanoma is exceedingly lethal and is responsible for the majority of skin cancer related deaths. Impactful research on melanoma metastasis is crucial to improving prognosis. The discovery of metastasis suppressor genes, genes that inhibit metastasis but do not affect tumor growth, have advanced the field of metastasis research. NME1 (also referred to as NDPK-A or NM23-H1) was the first identified metastasis suppressor gene. NME1 is a multifaceted molecule, executing numerous cellular functions which are attributable to the suppressor phenotype. Our lab previously identified a signature of NME1-regualted genes which have prognostic value in human melanoma. As such, we hypothesize that NME1 possess an understudied transcriptional activity to regulate one or more of these signature genes. In the present study, we identified Aldolase C (ALDOC) from the gene signature as an ideal candidate to assess NME1 transcriptional activity. NME1 induced mRNA and protein expression of ALDOC in multiple melanoma cell lines. Transcriptional regulation was evidenced by elevated expression of ALDOC pre-mRNA and activation of the ALDOC promoter in NME1-expressing melanoma cells. Employing chromatin immunoprecipitation, we demonstrated NME1 localizes to the ALDOC gene and enriches the presence of active transcription indicators at the ALDOC promoter. Together, we provide novel systematic evidence for NME1 transcriptional activity, which may further be explored as a mechanism for the metastasis suppressor function. Furthermore, we establish unequivocal metastasis suppressor capability of NME1 using a transgenic mouse model susceptible to developing melanoma upon exposure to ultraviolet (UV) radiation. Mice deficient in NME1 expression experienced significantly increased melanoma metastases to the lung and lymph nodes compared to wild-type mice. Taken together, our studies demonstrate NME1 as a robust metastasis suppressor of UV-induced melanoma, and provide evidence for novel transcription factor activity of NME1. We aim to enhance our understanding of melanoma metastasis through our continued research of the metastasis suppressor gene, NME1.
  • Regulation of CD8+ T cell differentiation by cytokines and T-box transcription factors

    Reiser, John Wyatt; Singh, Nevil (2019)
    A protective immune response requires naïve CD8+ T cells to differentiate into effector cells, some of which persist as a memory population to protect against future threats. This differentiation program is controlled by a transcriptional network involving the T-box transcription factors T-bet and Eomesodermin (Eomes). These factors are in turn differentially regulated by cytokines in the local milieu. The precise architecture of this regulatory network in terms of the individual, synergistic and redundant roles of T-bet, Eomes and critical cytokines is still poorly understood. Here we sought to clarify these regulatory pathways, using mice deficient in T-bet, Eomes or both. Our studies identify a key role for Eomes, but not T-bet, in regulating IL-10 expression by CD8+ T cells early after activation. We further reveal a feedback loop where Eomes and IL-10 cooperatively enhance the expression of the lymph-node homing selectin CD62L. Consistent with the importance of CD62L in promoting memory T cell homing to secondary lymphoid organs (SLOs), we identify a role for CD8+ T cell-intrinsic IL-10 in promoting the long-term persistence of memory cells in vivo. In contrast, T cells that home away from the SLOs become terminal effector T cells. Using tumor and infectious disease models, we report that these cells experience a second wave of T-bet induction in the tissue, which may contribute to a heightened effector response. We also define distinct roles for T-bet and Eomes in regulating effector and memory genes, highlighting their redundant role in repressing Tc2 and Tc17 differentiation in CD8+ T cells. Finally, we demonstrate that in the context of a tumor-specific immune response, the absence of both T-box transcription factors severely limits tumor infiltration without significant alteration in early proliferation or effector functions. Taken together, these findings offer new insights into how T-bet, Eomes and IL-10 regulate effector and memory responses in distinct tissues during the immune response. Future developments targeting individual aspects of T-box and cytokine signaling in T cells may help engineer precise outcomes in the context of tumor immunotherapies, vaccination and transplantation.
  • Pseudomonas aeruginosa adaptation, pathogenicity, and persistence in the environment and cystic fibrosis airway.

    Chandler, Courtney; Ernst, Robert K.; 0000-0003-2076-3665 (2019)
    Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium associated with airway infections of patients with cystic fibrosis (CF). Lipid A is the membrane anchor of lipopolysaccharide, the dominant component of the outer leaflet of the outer membrane of Gram-negative bacteria. Lipid A structure can be modified via a number of biosynthetic enzymes. Here, we describe two -hydroxylase enzymes, LpxO1 and LpxO2, capable of catalyzing 2-hydroxylation of lipid A in P. aeruginosa. We additionally characterize their role in persistence and infectivity. Phylogenetic analysis suggests at least one lpxO originated from lateral gene transfer and the gene duplication is a recurring feature in Pseudomonas evolution. To determine the roles of LpxO1/2 in vivo, we used a rapid extraction from lavage fluid to describe the structure of P. aeruginosa lipid A isolated from the lungs of mice after intranasal infection. Lipid A is also known to be altered during adaptation to the lungs of CF patients, where P. aeruginosa can colonize and cause infection throughout a patient’s lifetime. This is a leading contributor of morbidity and mortality in this patient population, and thus the genetic and phenotypic adaptations that P. aeruginosa undergoes during CF airway infection are of interest. We used whole-genome sequencing of 130 P. aeruginosa isolates, including 81 from young children with CF, to define early-stage genetic adaptation events, including lipid A modification. We additionally investigate the influence of patient region of residence on such adaptation. With these data, we provide the first longitudinal analysis of P. aeruginosa genetics in young patients with CF in the United States. We additionally analyzed ten sublines of laboratory-adapted strain PAO1 to determine the level of microevolution experienced by this set of isolates. In total, our analyses contribute to our understanding of lipid A structure, synthesis, and modification in P. aeruginosa, and our sequencing data will serve as a resource for the entire CF and Pseudomonas community.
  • The Role of Testisin and PAR-2 Signaling in Ovarian Cancer Metastasis

    Conway, Gregory David; Antalis, Toni M. (2019)
    Ovarian cancer is the leading cause of death among gynecological cancers in the United States. Ovarian cancer employs a unique mode of metastasis, as tumor cells disseminate within the peritoneal cavity, colonizing in several sites and driving the accumulation of ascites. Tumor recurrence and metastasis are significant factors contributing to poor prognosis. The membrane-anchored serine protease testisin is aberrantly expressed in ovarian tumors and it’s only known substrate, protease activated receptor-2 (PAR-2), is also overexpressed in ovarian tumors. In this study I have examined 1) the role of testisin and PAR-2 signaling in ovarian tumor metastasis and 2) determined whether testisin and other membrane-anchored serine proteases may be anti-cancer therapeutic targets. We generated human ovarian ES-2 tumor lines that express testisin or the catalytically inactive testisin mutant S238A and explored the role of constitutive testisin expression in late stage ovarian cancer. We show that testisin stimulates the activation and internalization of PAR-2 resulting in decreased expression of ANG2 and ANGPTL4. Using a preclinical xenograft model of late stage ovarian cancer, we find that testisin activity in ovarian cancer cells reduces intra-peritoneal tumor dissemination, tumor burden and ascites formation. Analyses of the tumors showed that testisin activity downregulates the expression of ANG2 and ANGPTL4 in vivo as well as in vitro. To examine membrane-anchored serine proteases as therapeutic targets in cancer, we utilized an engineered anthrax toxin pro-drug strategy requiring proteolytic cleavage of a protective antigen (PrAg) for activation. We explored the efficacy of these engineered PrAg toxins in several intraperitoneal models of ovarian cancer metastasis and show PrAg treatment reduced tumor burden in both an intraperitoneal NCI/ADR-Res xenograft and an ES-2 minimal residual disease model, which replicates debulking surgery in ovarian cancer patients, but had no effect in a syngeneic ID-8-Luc xenograft model of ovarian cancer. Additionally, we found that engineered PrAg toxins are capable of inducing lung and pancreatic cancer cellular death in vitro. Data presented herein provide new insight into the potential role of testisin and PAR-2 signaling in ovarian cancer metastasis and suggests membrane-anchored serine proteases as a potential therapeutic target for metastatic ovarian cancer.
  • Characterization of PfCRT F145I in piperaquine-resistant Plasmodium falciparum isolates from Cambodia through zinc-finger nuclease-mediated gene editing

    Shrestha, Biraj; Takala-Harrison, Shannon (2019)
    ACTs are the first-line treatment for clinical malaria in the malaria-endemic world and have reduced malaria-associated mortality and morbidity. However, the recent emergence of Plasmodium falciparum that is resistant to both the artemisinins and key partner drugs, piperaquine, in Cambodia and nearby countries in GMS poses a threat to the control and elimination of malaria. Identification and validation of molecular markers of antimalarial drug resistance provide surveillance tools to monitor resistance and inform drug policy decisions and insights into the molecular mechanisms underlying resistance. Previous studies have found that F145I mutation within the PfCRT and plasmepsin2/3 gene copy number are associated with resistance to piperaquine. When PfCRT F145I is introduced into Dd2 of P. falciparum, it confers piperaquine resistance. In this study, we will use gene-editing approaches to remove F145I from field isolates that contain both this mutation and amplified plasmepsin2/3, to quantify the effect on malaria parasite susceptibility to piperaquine.
  • Smoking Cessation Among People With Severe Mental Illness

    Alghzawi, Hamzah Mohammad; Storr, Carla L. (2019)
    Introduction: People living with mental illnesses have a high rate of smoking and make up over half of those dependent on nicotine. A considerable body of research has shown that social support, stressful life events (SLE), receiving help for tobacco/nicotine use, intention to quit, and smoking use-related factors are associated with smoking cessation in the general population. Yet, little is known about these factors among people with severe mental illness (SMI). Purpose: This study aims to: 1) examine gender differences in the interrelations among social support, SLEs, and smoking cessation, 2) estimate the probability of remission from NUD by type of help/services received for tobacco/nicotine use (pharmacological, non-pharmacological, and both), and 3) estimate gender and racial/ethnic differences in the probability of smoking cessation among those with a history of intention to quit. Methods: A sample of 4610 people with SMI and a history of tobacco/nicotine use were identified in a public limited dataset of the 2012-2013 National Epidemiologic Survey on Alcohol and Related Conditions (NESARC-III). Four mediation and moderated mediation models were used in the first manuscript, whereas survival analyses were used in the second and third manuscripts. All analyses took into account the complex sampling design and controlled for possible confounders (i.e. sociodemographic characteristics) and covariates (i.e. comorbidity with another mental illness). Results: Total, appraisal, and tangible support in females exerted indirect effects on improving smoking cessation via decreased SLEs (total=.0094, appraisal=.0229, tangible=.0298; p<.05). The probability of remission from NUD was higher among those who received non-pharmacological services (28.5%, HR=1.95, p<.05) or those who received both services (19.6%, HR=1.52, p<.05) compared to those who only had pharmacological services (17.6%). Among those with a history of intention to quit, 31.7% had stopped. The probability of smoking cessation was highest for Hispanic females (HR=2.07, p<.05), non-Hispanic other females (HR=1.59, p<.05), non-Hispanic other males (HR=1.45, p<.05), Hispanic males (HR=1.40, p<.05), and non-Hispanic Black females (HR=1.35, p<.05) compared to non-Hispanic Black males. Conclusion: A greater understanding of subgroup differences and the correlates of smoking cessation among tobacco/nicotine users with SMI can enhance efforts to design and implement smoking cessation programs for people with SMI.

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