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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  • Atrial Fibrillation Risk-Stratification Schemes: Improving Patient-Centeredness and Precision

    Oehrlein, Elisabeth Maria; Perfetto, Eleanor M. (2018)
    Background: Despite treatment-guideline recommendations and availability medications to reduce stroke risk, widespread underutilization of oral anticoagulants (OACs) has been previously documented among individuals with atrial fibrillation (AF). Younger age and female gender are important in light of evidence that these groups, in particular, may not receive optimal AF care. The objective of this dissertation was to identify: 1) What are the barriers to patients initiating OACs? 2) Are providers aware of and using the RSSs and do disparities exist by age and gender? 3) Are RSSs predictive of stroke and OAC initiation among subpopulations (women and <65 years of age)? Methods: In Aim 1, we invited patients and health care providers (HCPs) to participate in in-depth interviews. In aims 2 and 3, we conducted retrospective cohort studies using Optum’s Clinformatic Data Mart (2008-2016). We used logistic regression to calculate odds ratios and 95% confidence intervals to identify whether RSSs were associated with OAC initiation and whether disparities exist by age or gender in aim 2. For Aim 3, we used a discrete time approach to estimate the risk of ischemic stroke associated with RSSs. Separately, we tested whether incorporating risk factors identified in the literature as predictive of ischemic stroke improved prediction among women and patients ≤65 years. Results: Themes from qualitative interviews include: specialists heightened perception of stroke risk compared to generalists and comorbidities/characteristics absent from RSSs also factor into risk consideration. The proportion of patients initiating OACs was only approximately 30%. CHADS2, but not CHA2DS2-VASc, scores corresponded with higher odds of OAC initiation. We found no statistically significant differences between odds of initiating OACs among OAC-recommended males/females or age categories. Among women and those ≤65 years, all CHA2DS2-VASc scores >1 and CHADS2 scores >0 were significant predictors of stroke. Prognostic models developed within subpopulations were no better at predicting stroke than existing RSSs. Conclusions: RSSs are associated with ischemic stroke among newly diagnosed females and <65 years of age patients. Initiation of OAC treatment was consistently low. More research is needed to more clearly understand why RSSs might not be followed and why OACs are not initiated.
  • Innovation of Vancomycin Treatment in Neonates Via A Bayesian Dose Optimization Toolkit For Adaptive Individualized Therapeutic Management

    Pastoor, Devin DeForest; Gobburu, Jogarao (2018)
    Personalized medicine continues to gain momentum as a topic for discussion, yet directly linking patient-level decision support to more advanced analytical techniques, such as nonlinear mixed effects modeling, is not being practiced in most hospitals. Current practice for Vancomycin therapy uses dosing nomograms to determine the dosing regimen for patients. For simplicity, these nomograms stratify patients into bins based on some combination of weight, serum creatinine, and/or age to adjust starting regimens. Yet, studies across the US and Europe have shown as few as 37% of neonates achieve recommended target concentrations using such nomograms. The purpose of this research was to develop a bayesian decision support toolkit to provide adaptive, individualized dose recommendations for neonates. First, a bayesian nonlinear mixed effect model was developed and qualified for predictive forecasting in individual patients. Second, this model was used to develop a novel algorithm for dose individualization. Finally, a web application was developed to allow clinicians to provide decision support for clinicians involved in vancomycin dosing decisions. The proposed strategy can decrease the number of patients improperly dosed up to 90%, drastically reducing the chance for treatment failure, toxicity-related adverse events, and resistance development.
  • Lower Gastrointestinal Tract Delivery of a Tetra-specific Antibody ABAB-IgG1 for the Treatment of Clostridium Difficile Infection (CDI)

    Jiang, Bowen; Stephen W. Hoag
    More than 246 protein or peptide therapeutics are on the market, including 47 monoclonal antibodies approved since early 1980s. Although most protein therapeutics are administered parentally, treatments for gastrointestinal diseases such as Clostridium difficile infection (CDI), ulcerative colitis and Crohn’s Disease would benefit from an oral delivery system that can target biologics to a site in the GI tract, with less systematic exposure and therefore less systematic toxicity, given the targets of disease are in the GI tract. This research focused on local delivery of a novel antibody therapeutic, ABAB-IgG1, for the treatment of CDI. The antibody was fabricated into a multi-particulate delivery system comprised of nonpareil beads and functional polymers using spray coating techniques. A proof-of-concept study was conducted using BSA as a model protein. BSA was first spray layered onto beads, then coated with pH sensitive polymers. There was no significant change in BSA conformation and aggregation profiles after the spray layering process. BSA multi-particulates were stable for at least 1 month stored at 4 ⁰C. In vitro dissolution testing showed that the enteric coated BSA beads remained intact in acidic media, while releasing BSA in higher pH buffers. A Design of Experiments strategy was used to understand how the formulation and process parameters impacted antibody stability during spray coating process and during accelerated stability studies. The formulation of novel structured antibody ABAB-IgG1 was also optimized based on the conformational and colloidal stabilities using various high throughput biophysical characterization techniques. The multi-particulate delivery system of ABAB-IgG1 was evaluated both in vitro and in vivo, and showed the feasibility of delivering ABAB-IgG1 to the lower GI tract using the multi-particulate system. The multi-particulate delivery system, which is well-studied for small molecule drugs, can be adopted to biologics without modification of existing fluid bed processing equipment, which implies the possibility of efficient scale up of this technique in existing industrial-scale equipment.
  • Neuroimaging in Headache Patients: The Sensitivity of Computerized Tomography (CT) in Missed Stroke Diagnoses

    Heetderks, Elizabeth; Johantgen, Mary E. (2018)
    Background: Stroke is the leading cause of disability in the US, costing $34 billion a year and affecting 800,000 patients. Early detection and treatment is the best way to improve outcomes. Yet, 12.5% of strokes are discharged from the ED within the prior 30 days, with headache the most common diagnosis. Neuroimaging, ideally, would catch an impending stroke, but head CT has variable sensitivity based on onset of symptoms and there are both Federal and provider-led (including Choosing Wisely and the American College of Radiology Appropriateness Criteria (ACR-AC) initiatives to reduce overuse of imaging. Purpose: This study examined variation in ED treatment for patients presenting with a headache, particularly focusing on use of neuroimaging. Potential missed strokes were identified to determine if CT or MRI could have captured stroke. Methods: Using HCUP 2013 Maryland State Emergency Department Dataset, and State Inpatient Data, patients who were seen in the ED within 30 days of a stroke with a complaint of headache were identified. Generalized linear mixed modeling determined if neuroimaging predicted stroke bounce back while controlling for patient and hospital variables. Results: Of the 63,942 headache visits in Maryland EDs, 337 patients presented with a stroke within 30 days of ED discharge. Half (54%) were seen in the ED the day of their stroke and 72% were seen within 7 days. A large majority of the stroke patients (82%) underwent CT for their ED headache visit. Patients who underwent CT for their headache were 2.5 times more likely to return with ischemic stroke, and 7.7 times more likely to return with hemorrhagic. Patients who underwent MRI were 1.7 times more likely to return with any stroke, and 2.8 times more likely to return with ischemic stroke. Conclusions: Providers were concerned about pathology, given the large percentage of patients imaged; however, imaging did not catch active ischemia or bleeding. The negative predictive value of imaging for headache may need to be reconsidered. Patients with high suspicion of pathology should be placed in observation and have appropriate follow up testing. The ACR-AC should be incorporated into diagnostic pathways to optimize use.
  • Cortical enhancement of posture and movement planning, initiation, and execution during standing voluntary reach following stroke

    Yang, Chieh-ling; McCombe-Waller, Sandra (2018)
    Stroke is the leading cause of disability that frequently includes impairments of postural control and upper extremity function. The ability to maintain balance while performing reaching during everyday activities requires appropriate sequencing of anticipatory postural adjustments (APAs) and the goal-directed reaching movement. Although studies have shown that these abilities are frequently compromised following stroke, the interaction of posture and upper extremity movement coupling (APA-reach sequence) regarding movement planning, preparation, and execution is not well understood. We investigated posture and movement planning, preparation, and execution in individuals with chronic hemiparesis and healthy controls. Movement planning and preparation of APA-reach sequence were examined by StartReact (SR) responses elicited by a loud acoustic stimulus (LAS). After an instructed delayed period, subjects performed a standing reaching task in response to a "go" light cue. An LAS was randomly delivered at - 500, - 200, and 0ms relative to the "go" cue. Kinetic, kinematic, and electromyography data were recorded to characterize APA-reach movement response. Subjects with stroke demonstrated a marked reduction in the occurrence of the SR responses and impaired APA-reach performance during not only the paretic but also the nonparetic arm reaching compared to healthy controls. This indicates that the deficits in posture and movement planning, preparation, and execution are not specific to the lesioned side only but system-wide to both the lesioned and contralesional sides. Moreover, compensation strategy characterized by greater involvement of trunk and pelvic rotation was utilized during the paretic arm reaching compared to the nonparetic arm reaching and healthy controls. Lastly, we tested how transcranial direct current stimulation (tDCS) applied over the region of premotor areas and primary motor cortex (M1) affect movement planning and preparation of a standing reaching task in individuals with stroke and healthy subjects. The main finding was that stroke-related deficits in movement planning and preparation as shown by an abnormal absence of SR responses during the paretic arm reaching could be improved by application of anodal tDCS over the region of lesioned M1 and the enhancement effects are depending on the timing of the LAS.
  • The Claustrum: From Top to Bottom

    White, Michael; Mathur, Brian N. (Brian Neil); 0000-0002-7202-7515 (2018)
    Despite much speculation, the function of the claustrum, a thin telencephalic brain nucleus, remains largely unknown. The claustrum is reciprocally connected with seemingly the entire cortical mantle, which motivates hypotheses of claustrum function that include multi-sensory integration and top-down/bottom-up attention. In order to discriminate these hypotheses, we examined the claustrum at multiple levels of analysis in rodents. We find that the claustrum is well-connected with executive areas of cortex, such as anterior cingulate cortex (ACC), and highly responsive to stimulation of ACC inputs in acute brain slices. In contrast, claustrum exhibits less connectivity with and responsivity to inputs from hierarchically lower cortices. Using in vivo circuit monitoring and manipulation, we find that ACC inputs to claustrum and claustrum projection neurons mediate top-down cognitive control relative to other basic brain functions, such as motor control and stimulus-response action strategy. Consistent with a role in cognitive control, claustrum neurons projecting to visual cortices and parietal association cortex faithfully propagate ACC input, and claustrum afferents provide strong excitatory drive across cortical layers. To understand how claustrum processes top-down input, we identified claustrum neuron subtypes using membrane properties and examined claustrum microcircuit responsivity to ACC input. We find that one of two claustrum projection neuron subtypes preferentially burst fires in response to ACC input, that the two subtypes differentially target cortex, and that recruitment of inhibitory microcircuits constrains claustrum output. These findings support a model wherein claustrum mediates synchronization of cortices for cognitive control.
  • Identification and Characterization of Factors Associated with Biofilm Formation in Acinetobacter baumannii Surveillance Isolates

    Wallace, Lalena; Rasko, David A.; 0000-0002-7105-3607 (2018)
    Acinetobacter baumannii is a Gram-negative nosocomial pathogen. It is responsible for a variety of infections and is among the five most common pathogens in U.S. hospitals associated with ventilator-associated pneumonia. It has been estimated that A. baumannii is responsible for approximately 45,000 hospital infections in the U.S. per year. The two main characteristics that make this organism a formidable threat in health care settings is its ability to acquire multidrug resistance and to persist on surfaces. A better understanding of the genetic factors responsible for these virulence traits is needed. Genomic comparisons of 203 A. baumannii strains, collected as part of a surveillance study at the University of Maryland Medical Center, were performed using Large-Scale BLAST Score Ratio (LS-BSR) analysis. For these genomic comparisons, the genomes were grouped according to the date and source of the strain, as well as the carbapenem resistance status. This analysis resulted in the identification of genes unique to specific phylogenomic group, site of isolation, or resistance phenotypes. In addition, this work included identification of genes exclusive to other Acinetobacter species, which may be useful in the future to positively identify A. baumannii as this is currently a difficult clinical task. Phenotypic characterization of the A. baumannii strains resulted in the identification of a phylogenetic cluster of strains that exhibited robust biofilm formation. Genomic analysis of these strains revealed several genes unique to this group and potentially associated with biofilm formation. Mutagenesis of three of these genes was performed and the ability to form robust biofilms was determined. In particular, mutagenesis of a putative pilus assembly gene resulted in significantly decreased biofilm formation, suggesting that this gene plays a key role in the robust biofilm phenotype. In addition, a transcriptional regulator was identified that may play a role in the regulation of genes associated with biofilm formation. Overall, the work presented herein has broadened our understanding of the vast diversity among surveillance isolates of A. baumannii in a single healthcare setting, and has demonstrated the utility of genotypic/phenotypic correlations to identify novel virulence factors.
  • The Role of ZSCAN4 in Cancer Stemness

    Portney, Benjamin Andrew; Zalzman, Michal (2018)
    Cancer stem cells (CSCs) represent a subset of cells within tumors that maintain the ability to self-renew, drive tumor heterogeneity, and contribute to therapeutic resistance and cancer recurrence. Over the past decade, embryonic factors have emerged as key regulators of cancer stemness and as therapeutic targets. Zscan4 is an early embryonic gene expressed in mouse embryonic and induced pluripotent stem cells where it plays critical roles in genomic stability, telomere maintenance, and pluripotency. Like other embryonic factors, ZSCAN4 is reactivated in cancer. In these studies, we define for the first time the role of ZSCAN4 in human CSCs of head and neck squamous cell carcinoma (HNSCC). We find that ZSCAN4 is enriched for in and marks the HNSCC CSC population. Induction of ZSCAN4 promotes the CSC phenotype, increases CSC factors and alters the epigenetic profile. Importantly, extreme limiting dilution analyses both in vitro and in vivo indicate that ZSCAN4 induction significantly increases the frequency of CSC. Consistently, ZSCAN4 depletion leads to loss of the CSC phenotype including CSC marker expression, the ability to form spheroids in non-adherent culture conditions, and hypersensitivity to genotoxic drugs. Furthermore, loss of ZSCAN4 severely impairs tumor growth in vivo. As our findings indicated that ZSCAN4 promotes the CSC phenotype, we next chose to study its regulation and turnover in cancer cells. Expression of Zscan4 is transient, and characterized by infrequent high expression peaks that are quickly down-regulated, suggesting its expression is tightly controlled. However, little is known about the protein degradation pathway responsible for regulating the human ZSCAN4 protein levels. We first determined the protein half-life of ZSCAN4 and elucidated the role of the ubiquitin proteasome system in ZSCAN4 degradation. Importantly, our data indicate an interaction between ZSCAN4 and the E3 ubiquitin ligase RNF20. RNF20 depletion stabilizes the ZSCAN4 protein half-life, suggesting that RNF20 negatively regulates ZSCAN4 stability. Due to the crucial cellular functions of ZSCAN4, these results have important implications in telomere regulation, stem cell biology, and cancer. Overall, our study suggests that ZSCAN4 plays a critical role in maintaining the undifferentiated state and survival of CSCs, indicating that ZSCAN4 is a potential therapeutic target in HNSCC.
  • Novel WNT/beta-catenin Signaling Pathway Inhibitors for the Treatment of Metabolic Disorders

    Obianom, Obinna N.; Shu, Yan, Ph.D. (2018)
    The WNT/β-catenin signaling (β-cat) pathway is critical for embryonic development and tissue homeostasis. For this reason, alterations in the β-cat pathway are associated with many ailments including metabolic disorders, which may result from defects in the energy metabolism. The contribution of β-cat pathway to energy metabolism has become a subject of many investigations following the identification of polymorphisms in β-cat pathway components that predispose individuals to type-2-diabetes. Current evidence suggests that downregulation of the β-cat pathway activity may help treat metabolic disorders. Given these findings, the overarching goal of this thesis was to discover and develop novel β-cat pathway inhibitors and to examine their efficacy on glucose and lipid metabolism. We started with an FDA approved anthelmintic, pyrvinium, which is a potent inhibitor of the β-cat pathway. Our results showed that pyrvinium improved glucose tolerance by inhibiting glucose output, hepatic lipid accumulation and activating the AMPK pathway. Despite these beneficial effects, pyrvinium is unsuitable for repurposing to use orally in the treatment of metabolic disorders due to its almost zero bioavailability and other unspecific toxic effects in mice at higher doses. Based on the structure of pyrvinium, we decided to discover new potent β-cat pathway inhibitors with lower toxicity and improved bioavailability. Our screening of more than 150 newly synthesized pyrvinium derivatives led to the discovery of YW1128 as such a candidate having the aforementioned properties. Administration of YW1128 led to decreased lipid accumulation and improved glucose tolerance in the mice fed with high fat diet. Previous studies had suggested a critical role of hepatic β-cat pathway in determining the whole body metabolic homeostasis. So we next sought to achieve a selective delivery of the new derivatives to the liver without having significant disposition in other tissues. We performed a proof-of-concept study where we took advantage of high expression of organic cation transporter 1 (OCT1) in the liver to modify the compounds that were not specifically permeable to OCT1 expressing cells. We inserted a biguanide, which is a major backbone of several OCT1 substrates, into these compounds and showed that they became highly permeable to cells overexpressing OCT1. This suggests that insertion of the biguanide moiety into YW1128 may be an approach to improve its selective liver targeting. In conclusion, this thesis uncovered the efficacy by small molecule inhibition of β-cat pathway in the treatment of metabolic disorders and established that incorporating a biguanide moiety to the compounds may serve as a strategy to achieve selective liver targeting.
  • Neighborhood and Cumulative Ecological Risk: Predicting Physical Abuse and Neglect in an Urban Longitudinal Studies of Child Abuse and Neglect (LONGSCAN) Sample

    Moon, Catherine Anne; Harrington, Donna; Shaw, Terry V.; 0000-0001-9301-2606 (2018)
    The "role of cultural processes, social stratification influences, [and] ecological variations" (Institute of Medicine and National Research Council, 2014, p. 12) in the etiology of child abuse and neglect remains elusive despite a proliferation of studies over the past 40 years. This study seeks to further the knowledge base by longitudinally modeling cumulative ecological risk for child physical abuse and neglect, with a specific focus on the additive contribution of neighborhood structure and process. The Ecological-Transactional Framework (Cicchetti and Lynch, 1993) is used to conceptualize and organize the place of risk markers where cumulative risk theory (Rutter, 1979) is used to guide the analysis. This study combines two sources of secondary data (1) individual risk markers and parents' perceptions of their neighborhood process from the LONGSCAN Eastern site with (2) contemporaneous (1990) census indicators of neighborhood social disorganization. The risk markers and neighborhood structure and process at age six are used to predict child physical abuse and neglect from age 6 through 18. Participants (n=188) were experiencing elevated levels of ecological cumulative risk on the ontogenic and microsystem levels, per the sampling design of the Eastern study site. Further, the characteristics participants' exosystem, or neighborhood level, indicated elevated social distress as measured by The Social Distress Scale (Hyde, 2002). Neighborhood process at age 6 were associated with child report of neglect between ages 13 and 16, however, neighborhood structure was not associated with neglect and physical abuse. The findings suggest the necessity for further study with more diverse samples.
  • Rural Veterans: Pathways to Homelessness

    Jorden, Brenda J.; Belcher, John R.; DeForge, Bruce R. (2018)
    Veterans are overrepresented in homeless populations compared to non-veterans (Gamache, Rosenheck, & Tessler, 2001; Perl, 2015). Most homeless individuals live in urban centers and as a result most research studies have focused on urban homelessness leaving the rural homeless less studied and understood (Knopf-Amelung, 2013). The purpose of the current qualitative study was to explore the pathways to homelessness for a sample of rural homeless veterans. Fifteen veterans and 16 staff/administrators were interviewed to determine the causes of homelessness for veterans living in a rural area. A limited grounded theory approach was used to develop themes identified as causing homelessness. The primary pathway according to both groups was chronic substance abuse. Mental health and economic problems, and adverse childhood events were also major factors in veterans experiencing homelessness. Social support appeared to delay the onset of homelessness for some individuals. Not all veterans qualify for VA services, which leaves a segment of the population without access to VA services. Overcoming barriers to substance abuse and mental health services for rural homeless veterans is an area to focus research efforts. Creative ways to provide outreach to the hidden homeless are needed.
  • Differential FOXO1 Localization in SLE and Healthy Human Lymphocyte Subsets

    Hritzo, Molly K.; Golding, Amit; 0000-0001-9614-732X (2018)
    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by elevated levels of circulating autoantibodies and multi-organ damage. Although SLE is a highly heterogeneous disease, one factor unifies it: lymphocyte hyperactivity driving immunopathogenesis. This involves CD4 helper T cells potentiating autoreactive B cells to produce pathogenic autoantibodies. In healthy individuals, lymphocyte activation is a closely regulated kinetic process controlled by key transcription factors (TF) signaling downstream of the T cell (TCR) and B cell receptor (BCR). Forkhead box O1 (FOXO1) is one such TF that integrates activation and differentiation signals in human lymphocytes. When active, it remains in the nucleus, but upon Akt phosphorylation downstream of TCR or BCR signaling, FOXO1 is inactivated and shuttles to the cytoplasm, linking FOXO1 localization to function. In SLE, both T and B cells are hyperactive, and respond more quickly and strongly to antigen, producing a disproportionate inflammatory response. Thus, we hypothesize that SLE lymphocytes will have altered FOXO1 localization, reflecting altered lymphocyte activation. To address this hypothesis, we first developed a method of examining dynamic native FOXO1 localization in human peripheral lymphocyte subsets using imaging flow cytometry (IFC). IFC combines the quantitative power of flow cytometry with the qualitative images of microscopy and can be performed with many fewer cells than are needed for the more traditional methods. We demonstrated that we can visualize native FOXO1 and detect significant kinetic differences in localization within user-defined subsets of HuT102 cells, a human CD4 T cell line with baseline nuclear FOXO1, as well as primary peripheral human T and B cells. We then used IFC to compare FOXO1 localization in SLE and healthy donor lymphocytes. Interestingly, we found that most T and B cell subsets have nuclear FOXO1 localization in both health and SLE. However, FOXO1 is significantly more cytoplasmic in SLE double negative (DN) atypical memory B cells. Based on our findings, we propose a model by which these DN B cells are highly active in disease flares and may serve as a death-resistant reservoir of autoreactive cells. Future experiments will be aimed at elucidating at how these cells persist in the periphery.
  • Steps Towards an Intervention: Exploring Correlates and Measurement of Fatigue in Osteoarthritis

    Hackney, Alisha Jean; Klinedinst, N. Jennifer; 0000-0002-0928-0366 (2018)
    Background: Fatigue affects up to 90% of adults with osteoarthritis and contributes to disability and reduced quality of life. Treatment options are non-specific and limited to self-management. These limitations are due to least two gaps in current research: the lack of a standardized, reliable, and valid fatigue measure, and the lack of mechanistic insight. Purpose: To begin to address these limitations, the purposes of this three-manuscript dissertation were: 1) to examine standardized, valid, and reliable measures of osteoarthritis fatigue and 2) to explore correlates of fatigue to provide mechanistic insight. Methods: The first manuscript is a narrative literature review of osteoarthritis fatigue correlates. The second and third manuscripts analyze data from cross-sectional, retrospective studies. Analyzing pilot study data in SPSS and WINSTEPS, the second manuscript examines psychometrics of the standardized PROMIS Fatigue Short Forms 8a and 7a in osteoarthritis. The third manuscript uses data from the 2007-2010 National Health and Nutrition Examination Survey (NHANES) to examine fatigue correlates. Using SPSS complex samples analysis, adjusted logistic regression models were generated to predict odds of osteoarthritis fatigue as a function of a biological correlate (i.e., systemic inflammation: c-reactive protein [CRP] and white blood cell count [WBCC]). Results: Correlates of osteoarthritis fatigue include age, gender, medications, comorbidities, anxiety, depression, joint pain, physical activity, physical exercise, physical function, sleep quality, and systemic inflammation. The 8a and 7a were reliable (α =.86-.93) in adults with osteoarthritis. Differences existed in 8a, but not 7a, total scores, between adults with (N=20) and without osteoarthritis (t29=-2.8, p<.001; N=11). From the NHANES data, with every 1 mg/dL increase in CRP, adults with osteoarthritis had 3.19 times higher odds of fatigue (95% CI 1.11-9.19, p=.03) when controlling for age, pain, depression, sleep quantity, sleep disturbances, and body mass index. Conclusion: These findings have begun to fill the gaps that hindered development and testing of targeted interventions. Future research is necessary to gain more understanding of the use of the 7a and 8a in osteoarthritis and to delineate the relationship between other correlates, including additional systemic inflammatory markers, and fatigue in osteoarthritis. This will propel development and testing of targeted interventions.
  • Structure and Synthesis of Lipid A of Rickettsia Species

    Guillotte, Mark; Azad, Abdu F.; 0000-0002-1826-8879 (2018)
    Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In mouse infection models, control of rickettsial burden and disease resolution depends upon inflammatory cytokine production that is driven, in part, by Toll-like receptor (TLR) activation. The lipid A component of Gram-negative lipopolysaccharide (LPS) is the classical agonist of TLR4, however lipid A structure of rickettsial LPS and its inflammatory potential are unknown. Here we report the structure of lipid A from several species of Rickettsia including fatty acid analysis of Rickettsia rickettsii str. Sheila Smith, the most virulent species and the etiological agent of Rocky Mountain Spotted Fever. Furthermore, we have identified and characterized a new member of the recently discovered LpxJ family, a late-acyltransferase in Rickettsia typhi, the etiological agent of murine typhus, and R. rickettsii. Our results demonstrate that this enzyme, LpxJ, catalyzes the addition of a secondary acyl chain (C16) to the 3'-linked primary acyl chain of the lipid A moiety in the last step of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial outer-membrane integrity, we believe LpxJ is important in maintaining ideal membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work promises to reveal novel insights into Rickettsia pathogenesis and contribute greatly to our understanding of rickettsial physiology.
  • Utilization and Cost of the Biologic Disease Modifying Anti-rheumatic Drugs among Medicare Beneficiaries with Rheumatoid Arthritis

    Gaitonde, Priyanka; Shaya, Fadia T. (2018)
    Background: Disease modifying anti-rheumatic drugs (DMARDs) are essential for symptom control among rheumatoid arthritis (RA) patients. Biologic DMARDs are expensive and typically used among moderate to severe RA patients. The prevalence of RA is higher among Medicare beneficiaries compared to the rest of the population in the U.S (2% vs. 0.6%). The coverage rules of Medicare, in addition to access factors and patient preferences, may influence the use of facility-administered, infusible biologics (Part B covered) and home-administered self-injectable/oral biologics (Part D covered). However, there is limited information about utilization patterns of biologic DMARDs by route of administration and their impact on Medicare spending overall. The goals of this dissertation were to identify patient factors and healthcare expenditure associated with biologic DMARD use by route of administration among Medicare beneficiaries with RA. Methods: The study population consisted of Medicare beneficiaries with RA from the 5% random sample of the Chronic Conditions Warehouse database from 2006-2015. First, the study analyzed patient-level factors associated with biologic DMARD use by route of administration using generalized estimating equations. Second, adherence (PDC>80%), discontinuation, and switching patterns for biologic DMARDs were measured, accounting for patient level-factors , using logistic regression, Cox proportional hazards models, and chi-square analyses, respectively. Third, the study compared annualized average healthcare costs of patients who were adherent to versus non-adherent to biologic DMARDs. Results: Among Medicare beneficiaries diagnosed with RA who received DMARD treatment (n=46,002), 71.8% (n=33,028) used traditional DMARDs, and among biologic DMARD users (n=12,931), twice as many used infusible biologics (18.3%, n=8,436) compared to self-injectable/oral (9.9%, n=4,538). Beneficiaries who were low-income subsidy (LIS) recipients i.e. who had lower out-of-pocket costs for using biologics, (compared to non-LIS) had 4.54 times higher odds of using self-injectable/oral biologics (95% CI: 4.2 - 5.0) and 5% lower odds of discontinuing biologic DMARDs (OR=0.94, 95% CI: 0.91-0.97). The total healthcare cost was lower among adherent compared to non-adherent infusible biologic DMARD users ($33,797 vs. $90,181; p<0.001) and among adherent vs. non-adherent self-injectable/oral biologic DMARD users ($64,977 vs. $80.908; p<0.05). Conclusions: Adherence and cost savings generated, as a result, varied by the biologic DMARD route of administration. Additionally, beneficiaries' LIS status was associated with the route of administration used and the discontinuation rates, indicating an association with their out-of-pocket costs. These findings are relevant to the discussion about the proposed transition of Part B covered infusible medications under the Part D which is projected to increase the beneficiary out-of-pocket cost. The evidence on adherence can also be used for value-based insurance design involving RA biologics. Future research could leverage the findings from this study to additionally explore how biosimilar products may impact overall biologic utilization and RA treatment spending.
  • Biological Insight from Mass Spectrometry Through Novel Computational Approaches

    Fondrie, William Ellis; Strickland, Dudley K.; Goodlett, David Robinson, 1960-; 0000-0002-1554-3716 (2018)
    Mass spectrometry is a powerful tool to identify, quantify, and characterize diverse biological molecules. As a result, it has become a mainstay in modern biology enabling studies ranging from the structural characterization of a single lipid, to the identification and quantification of complete proteomes. With the increasing abundance of high-throughput mass spectrometry experiments, the major challenge has shifted from acquisition to interpretation. In most cases, these high-throughput experiments require inference beyond the directly measured characteristics of the analyte to draw insightful biological conclusions. However, this can be difficult, particularly when the outcomes of an experiment are not clearly defined. To address this challenge, we developed novel experimental and computational methods that utilize high-throughput mass spectrometry techniques to answer specific biological questions. We first describe a method called serial dilution-affinity purification-mass spectrometry (SDAP-MS) to identify and characterize protein-protein interactions. When investigating interactions to a protein of interest, researchers must decide between high-throughput screening methods that merely yield binary results, or low-throughput approaches that yield insight into the biochemical properties of these interactions. The SDAP-MS approach alleviates this burden by providing a high-throughput screening method capable of estimating equilibrium binding constants, which we demonstrate using two LDL receptor family members, LRP1 and LRP1B. We then describe a technique to discern the protein cargo of exosomes from contaminants co-isolated during purification. Exosomes are microvesicles and potential carriers of biomarkers and, as a result, it is vital to ensure that the proteins attributed to the exosome are not artifacts from the isolation strategy. To assess this, we present an approach that uses proteomics and machine learning to investigate the enrichment of proteins across multiple stages of exosome isolation, culminating in a score that indicates the confidence of each protein being of exosomal origin. Finally, we develop a machine learning approach to identify microbial pathogens from glycolipid mass spectra, enabling their rapid diagnosis. We show that Acinetobacter baumannii and Klebsiella pneumoniae can be identified from a library containing 48 additional organisms in both isolate and polymicrobal specimens. Together, these studies present methods that result in valuable insight beyond analyte identification and quantification by mass spectrometry.
  • Intracellular Stability of Precursor IL-33 is Maintained Through Interaction with Importin-5

    Clerman, Andrew; Atamas, Sergei P. (2018)
    Interleukin (IL)-33 is a member of the IL-1 family and a potent regulator of immunity. The precursor, or full-length (FLIL33), form behaves differently from the mature (MIL33) cytokine. MIL33 is a potent pro-Th2 cytokine acting through the T1/ST2 receptor, whereas FLIL33 is constitutively expressed and resides predominantly in the nucleus of epithelial and endothelial cells, as well as fibroblasts. Increasing evidence suggests that this intracellular FLIL33 can mediate inflammatory responses and wound healing in a non-Th2 and receptor-independent manner. Additionally, nuclear retention of FLIL33 is essential to maintain homeostasis and prevent unintentional sterile inflammation. FLIL33 has been shown to bind histones and chromatin, but the mechanism of nuclear localization remains elusive. Though FLIL33 contains a conserved bipartite nuclear localization sequence (NLS), this is not required for nuclear localization. Importins are a family of proteins that act as chaperones for import of proteins through the nuclear pore complex (NPC). Here, importin-5 (IPO5) is identified as a unique intracellular binding partner of IL-33 based on co-immunoprecipitation of HA-tagged IL-33 from normal human lung fibroblasts (NHLF). It was hypothesized that IPO5 binds the N-terminal domain of FLIL33 and that nuclear localization of FLIL33 is dependent on IPO5. To test this hypothesis, HA-tagged FLIL33 and HA-tagged peptide fragments of FLIL33 were expressed in cell culture and immunoprecipitated. We show that FLIL33, but not MIL33, co-immunoprecipitates with IPO5 and that this interaction localizes to the N-terminal domain of FLIL33. Knockdown of IPO5 in NHLFs using RNA interference did not prevent nuclear localization of FLIL33. Instead, attenuation of IPO5 led to a significant decrease in the intracellular protein levels of overexpressed FLIL33, which was reversed by treatment of cells with bortezomib, a proteasome inhibitor. Furthermore, FLIL33 mutant proteins deficient in IPO5-binding remained intra-nuclear. These mutants also had reduced protein levels that were restored by proteasome inhibition with bortezomib. These data indicate that the interaction between FLIL33 and IPO5 localizes to specific segments of the FLIL33 protein, is not required for nuclear localization of FLIL33, and results in its protection from proteasome-dependent degradation.
  • The Association of Integrated Management of Childhood Illness' Asthma Management Protocol and Asthma Control in Omani Children 2-5 years Old

    Al Amri, Warda; Ogbolu, Yolanda; Johantgen, Mary E. (2018)
    Background: Oman had the highest asthma prevalence and severity in children among Eastern Mediterranean countries. Despite national implementation of Integrated Management of Childhood Illness (IMCI) asthma protocol, reports show no improvement in asthma-related hospital admissions among children aged 1-4 years. IMCI asthma treatment protocol implementation and factors affecting asthma control are understudied. Purpose: Describe characteristics of IMCI asthma management protocol implementation (medication, counselling, and specialist referral); explore multilevel factors associated with asthma control in children attending IMCI clinics; and analyze trends in acute care visits (emergency department [ED] and hospital admissions) by asthmatic children and their characteristics during one year follow-up. Methods: Retrospective cohort study of Omani children aged 2-5 years in Muscat was conducted using electronic health data (2012-2015). Asthma control was measured by number of exacerbations requiring visits to the health center, emergency care or hospitalization within one year of asthma index diagnosis. Generalized linear mixed-effects modelling was used to account for the clustering of children within health centers. Results: Mean age was 2.8 years, predominantly male (63.2%) and originally from Muscat (96.2%). On index diagnosis day, 61.2% were given reliever medications, and on discharge 47.1% were prescribed controller medications. Most (74%) children had well-controlled asthma that was highly associated with being treated in a health center with higher proportion of IMCI training (Adj. OR= 3.0; 95% CI =1.34, 6.73; p <0.01), and receiving short acting β2-agonists for acute management at index diagnosis (Adj. OR=2.4; 95% CI=1.38-4.09). There was limited data for specialist referrals and counselling. Majority of children with ED visits were infrequent attenders (94.1%), however, a small percentage had high acute care utilization (e.g. 21 visits/year). Conclusion: IMCI is designed to improve health outcomes of young children. A high proportion of children treated in IMCI clinics achieved well-controlled asthma. Training of physicians improved asthma outcomes, but specific components in the IMCI asthma management protocol need more reinforcement and further exploration. Utilizing prevention quality indicators to screen for implementation of IMCI components is recommend. Longitudinal investigation of multilevel factors (patient, family, practice, and health care system) associated with childhood asthma control in Oman is needed.
  • Longitudinal Patterns of Early Mental Health Service Utilization in a Medicaid-insured Birth Cohort and the Impact of Continuity of Care on the Quality of Pediatric Mental Health Treatment

    Pennap, Dinci; Zito, Julie Magno (2018)
    Background: The prevalence of pediatric mental health (MH) diagnosis and treatment have expanded in the U.S. We assessed the longitudinal patterns of incident diagnosis and new psychotropic medication use in a Medicaid-insured birth cohort. Additionally, continuity and quality of MH service utilization were assessed in a publicly-insured pediatric population. Quality care was defined by the 2009 Children's Health Insurance Program Reauthorization Act (CHIPRA) mandated children's health care quality measures. Methods: We applied longitudinal designs to Medicaid claims data from a Mid-Atlantic state (2007-2014). Using Kaplan-Meier estimators we assessed the cumulative incidence of MH service use in a cohort of newborns (aim 1). We assessed the association between relational patient-provider continuity of care and: 1) emergency department (ED) visits or hospitalizations in the 12 months following first MH diagnosis among 3-16 year olds (aim 2); and 2) the quality of follow-up care among 6-12 year old new users of ADHD medications (aim 3), using logistic regression models. Quality was defined as having ≥1 follow-up outpatient visit in the 30 days following medication initiation and ≥2 follow-up visits in the 270 days after the first follow-up visit, with a total medication supply of ≥210 days. Results: By age 8, 19.7% and 10.2% of the birth cohort (n=35,244) had received a MH diagnosis or psychotropic medication, respectively. Among medication users, 80.5% received monotherapy, 16.4% received 2 medication classes, and 4.3% received ≥3 medication classes concomitantly for ≥60 days. Compared to children with high CoC, the odds of ED visits was significantly higher among youths with low CoC [Odds Ratio(OR)=1.27; 95% CI=1.13-1.41] and low CoC was associated with greater odds of hospitalization [OR=1.17; 95% CI=1.06-1.29]. Compared to those with low CoC, children with higher continuity of care had greater odds of meeting CHIPRA initiation- [OR=1.41; 95% CI=1.25-1.60] and continuation-phase [OR=1.45; 95% CI=1.29-1.64] visit-based measures. Conclusions: Early exposure to psychotropic medications and prolonged duration of use have implications for long-term safety, highlighting the need for safety and outcomes research in pediatric populations. Our findings suggest a need for more research in the areas of quality assessment and continuity of care among youths with mental health conditions.
  • Rapid Diagnosis of Microbial Infection via Mass Spectrometric Phenotyping

    Liang, Tao; Goodlett, David Robinson, 1960-; 0000-0001-9883-3999 (2018)
    Microbial infection is a perpetual public threat, causing more than 15 million annual deaths worldwide. Clinical microbiology laboratories currently rely on bacterial culture-based methods for microbial diagnosis to identify causative pathogens, which is time-consuming and labor-intensive. This drives the development of novel diagnostics to identify pathogens accurately and more rapidly. Mass spectrometry (MS) now plays a vital role in clinical diagnosis due to its high accuracy, high specificity, rapidity and high- throughput capability. In this thesis, we present a multi-faceted mass spectrometry approach for rapid microbial infection diagnosis. We first used a novel sample transfer technique, surface acoustic wave nebulization (SAWN) for bacterial membrane lipid analysis, specifically lipid A. Analytical performance of different SAWN chips was characterized, and the optimized SAWN chip was used for bacterial phenotyping. Results showed that lipid A mass spectra from different bacterial species can be differentiated by dot product analysis, in turn, demonstrating feasibility of using SAWN for rapid bacterial identification. We next developed a rapid sodium acetate (SA) based method for lipid extraction, which greatly improved our lipid-based library for pathogen identification by reducing the process time to less than an hour. Importantly, the novel SA method maintained the key components of the reported lipid library method for bacterial identification. Namely, these were the ability to detect 1) antibiotic resistance, 2) microbes direct from biological fluids without culture, and 3) single microbes in polymicrobial samples. This platform can be a complementary approach to the commercialized protein-based systems to improve patient outcomes. The last objective of this thesis is to understand the proteome change in response to lipopolysaccharide stimulus in the context of sepsis, which will facilitate the discovery of new biomarkers for sepsis diagnosis. Shotgun label-free quantification proteomics results showed that 27 new sepsis-related proteins were found among 182 significantly changed proteins in the septic mouse group. A longitudinal, but not pair-wise, data analysis strategy overcame inherent heterogeneity detected twice as many significant changes using each mouse's data as its own control sample. Overall, the advances made in this thesis have broad implications in MS-based rapid diagnosis of microbial infections.

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