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

Recent Submissions

  • 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.
  • 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.
  • 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.
  • 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.
  • The Impact of Medicare Contracting Reform on Metastatic Colon Cancer Treatment, Survival, and Costs

    Vandigo, Joseph Edward; Mullins, C. Daniel (2018)
    Background: Receipt of metastatic colon cancer (mCC) treatment for Medicare beneficiaries varies across geography, potentially due to claims processors. Medicare contracting reform consolidated legacy claims processors, known as fiscal intermediaries (Part A) and carriers (Part B) into larger entities known as Medicare Administrative Contractors (MACs), responsible for both Part A and B claims. Methods: This retrospective study used Surveillance, Epidemiology and End Results (SEER) and Medicare claims data to examine the effect of Medicare contracting reform using a difference-in-differences approach. Outcomes included the receipt of guideline-recommended chemotherapy and biologic therapy, overall survival, and mean costs for Medicare beneficiaries over age 66, diagnosed with mCC between 2005 and 2009. A generalized linear model estimated receipt of therapy and mean costs. An inverse-probability of treatment weighted Cox proportional-hazards regression modeled adjusted hazard ratios for predictors of mortality. Results: Among 4,030 mCC patients, there was no statistically significant association between MAC implementation and patient receipt of guideline chemotherapy (Odds Ratio [OR]: 1.00; 95% Confidence Interval [CI] 0.65, 1.56) or biologic therapy (OR: 0.90; 95% CI: 0.57, 1.39). No impact of MAC implementation was observed on overall survival (Hazard Ratio: 1.14; 95% CI: 0.92, 1.41) or mean total costs, regardless of number of treatment lines received. Patients diagnosed in regions where the MAC was a new entity were more likely to receive biologic therapy and had higher costs as compared to patients diagnosed in regions where a legacy contractor became the MAC. Findings were robust to changing assumptions regarding timing of MAC implementation. Conclusions: Receipt of treatment and total costs varied by geographic region in both the pre-implementation and post-implementation periods. However, Medicare contracting reform did not impact mCC patients' access to guideline therapy, survival, or total costs. Future research on geographic variation should focus on later stages of contractor consolidation and disease states vulnerable to local coverage decisions.
  • The Role of the Constitutive Androstane Receptor in Cyclophosphamide-based Treatment of Lymphomas

    Hedrich, William Dominic; Wang, Hongbing, Ph.D.; Hassan, Hazem (2018)
    Cyclophosphamide (CPA) is an alkylating prodrug which has been utilized extensively in combination chemotherapies for the treatment of cancers and autoimmune disorders since its introduction to the market in the late 1950s. The metabolic conversion of CPA to its pharmacologically active metabolite 4-OH-CPA is catalyzed primarily by cytochrome P450 (CYP) 2B6. CPA is also subject to metabolism by CYP3A4 to an inactive metabolite, N-dechoroethyl-CPA (N-DCE-CPA), and a neurotoxic byproduct, chloroacetaldehyde. CPA is the backbone of the frontline chemotherapeutic regimen used for the treatment of non-Hodgkin lymphoma which combines the CHOP (CPA-doxorubicin-vincristine-prednisone) regimen with rituximab, a monoclonal CD20 antibody. The constitutive androstane receptor (CAR, NR1I3), an orphan nuclear receptor, is recognized as the key mediator of xenobiotic-induced expression of CYP2B6, Importantly, mounting evidence suggests that activation of hCAR leads to preferential induction of CYP2B6 over CYP3A4 which suggests that selective hCAR activation may enhance CPA bioactivation and enhance the efficacy:toxicity ratio of CHOP chemotherapy for NHL. CHOP chemotherapy has been associated with severe and cumulative cardiotoxicity arising from the doxorubicin component of the regimen and it is recommended that lymphoma patients with existing heart conditions avoid treatment with the full CHOP combination. Recently, it has been demonstrated that Nrf2 (nuclear factor (erythroid-derived 2))-like 2, NFE2L2) plays a key role in governing doxorubicin-induced cardiotoxicity. Nrf2 regulates the expression of important antioxidant genes and proteins which protect tissues from damage due to oxidative stress and inflammation. It has been shown both that insufficient Nrf2 expression results in hypersensitivity to doxorubicin cardiotoxicity and that stimulation of Nrf2 by small molecule activators can provide protection from doxorubicin-mediated toxicity. Our hypothesis was that hCAR activation will increase hepatic expression of CYP2B6 while having a negligible impact on other genes responsible for the disposition of CHOP drugs. Further, activation of Nrf2 in cardiac tissue may provide protection against cardiotoxicity induced by the doxorubicin component of CHOP. Together, these gene expression alterations will lead to augmented antineoplastic activity of CHOP in target lymphoma cells while alleviating the untoward cardiotoxicity associated with this regimen. This hypothesis was tested with a variety of methods including a novel hepatocyte-lymphoma-cardiomyocyte cell co-culture system as an in vitro model for studying the biotransformation of CPA and therapeutic effects of CHOP as well as the off-target toxicity in healthy tissues in an environment that closely resembles the in vivo condition. Using this system we successfully demonstrated that activation of hCAR with small molecule activators can significantly increase the anticancer activity of the CHOP regimen in lymphoma cells. Additionally, activation of Nrf2 in cardiomyocytes in co-culture significantly reduced the doxorubicin-induced cardiotoxicity of CHOP. Utilizing a hCAR-transgenic mouse model, we were able to show in vivo that the combination of a selective hCAR activator alongside CHOP significantly increases the anticancer activity of CHOP in a lymphoma tumor xenograft study. Taken together, these results implicate hCAR and Nrf2 as drug targets for facilitating CHOP-based treatment of lymphomas. We were able to identify several compounds from the NIH Chemical Genomics Center Pharmaceutical Collection which activate both hCAR and Nrf2 and have provided preliminary evidence for their utility in CHOP-based lymphoma treatment.
  • Evaluation of In Vitro/In Vivo Correlations for Transdermal Delivery Systems by In Vitro Permeation Testing and Human Pharmacokinetic Studies, With and Without a Transient Heat Application

    Shin, Soo Hyeon; Stinchcomb, Audra L.; Hassan, Hazem; 0000-0001-5091-8870 (2018)
    An in vitro model that exhibits in vitro/in vivo correlations (IVIVC) is a powerful tool in biopharmaceutical drug development because it can efficiently predict drug product performance in vivo. While the concept of IVIVC has been utilized mostly for oral dosage forms, demonstrations of IVIVC with in vitro permeation testing (IVPT) for transdermal delivery systems (TDS) are emerging. The objective of this work was to evaluate IVIVC for TDS using two model drugs, nicotine and fentanyl, with different physicochemical characteristics (e.g. log P). Additionally, the effect of heat exposure (42 ± 2 °C) on the rate and extent of TDS drug delivery was evaluated. IVPT studies using excised human skin and in vivo pharmacokinetic (PK) studies in human subjects were conducted under harmonized study conditions and designs to evaluate IVIVC. The correlations were evaluated in multiple ways, including a single point comparison of parameters such as steady-state concentration and heat-induced increase in partial AUCs, as well as a point-to-point correlation (Level A IVIVC). Level A IVIVC was examined using multiple approaches. A strong IVIVC was consistently observed for nicotine TDS in presence and absence of heat, suggesting the utility of IVPT as a tool to evaluate and predict in vivo performance of nicotine TDS. The IVIVC results for fentanyl were relatively weaker, especially when IVIVC for heat effects were examined, with greater in vivo heat effects observed compared to the in vitro heat effects. A separate study evaluating IVIVC for fentanyl TDS without a heat exposure component and utilization of some PK parameters obtained directly from study subjects yielded improved IVIVC results. The findings from the present research work suggest that IVPT data generally shows good predictability of in vivo performance of TDS at normal temperature conditions. However, the usefulness of IVPT for assessing and predicting external factors such as heat, especially for lipophilic drug molecules, may have some limitations that could be further improved.
  • Budgetary Impact of TRICARE Antidiabetic Drug Formulary Changes

    Hung, Anna; Mullins, C. Daniel (2018)
    Background: There is guidance on how to conduct budget impact analyses (BIA) from the International Society of Pharmacoeconomics and Outcomes Research (ISPOR). However, there is a growing need to ensure that budget impact models are valid, accurate, and usable to payers. In 2016, the Defense Health Agency implemented antidiabetic formulary changes to the TRICARE pharmacy benefit. The goal of this dissertation is to predict and validate the budget impact of these formulary changes. Objective: The specific objectives of this study were to: i) estimate the annual financial consequences of antidiabetic formulary changes from the TRICARE payer perspective using TRICARE claims data over three years; ii) assess the face validity, internal validity, and predictive validity of the model; and iii) identify and compare cost drivers of both the budget and the budget impact identified through the model versus the empirical analysis. Methods: Following the ISPOR BIA guidance, a budget impact model was created in Microsoft Excel®. The counterfactual was predicted using autoregressive integrated moving average models. One year after the formulary changes, actual utilization was used to determine the realized budget and compare this to what was predicted in the budget impact model. Cost drivers were determined through one-way sensitivity analyses, subgroup analyses, and the comparison of utilization versus price in the model versus the empirical analysis. Results: In the year after the formulary changes, the model predicted a lower budget impact than what was realized ($24 million in savings versus $49 million in savings). Meanwhile, the model predicted a higher annual budget than what was realized ($686 million versus $609 million). The higher-than-predicted savings was largely due to lower utilization seen in the empirical analysis compared to the model. The antidiabetic drug classes that contributed most to these savings were the dipeptidyl peptidase-4 inhibitors, glucagon-like receptor-1 agonists, and sodium-glucose cotransporter 2 inhibitors. Conclusion: Future budget impact models should be validated by waiting at least one year and comparing model predictions to what is realized. The end user of the model should also be involved in the process of creating the model.
  • Mechanisms of PrrF-Mediated Iron regulation in Pseudomonas aeruginosa

    Djapgne, Louise; Oglesby-Sherrouse, Amanda G. (2018)
    Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that infects people with compromised immune systems. P. aeruginosa is highly resistant to multiple antibiotics, in part due to its ability to organize into robust biofilm communities. Iron is required for P. aeruginosa virulence and biofilm formation, but iron can also be toxic to the bacteria. Therefore, iron acquisition and utilization are tightly regulated in response to iron availability. Iron homeostasis is maintained in part by the production of two small RNAs (sRNAs), PrrF1 and PrrF2, which repress the expression of iron containing proteins in iron-depleted conditions. PrrF1 and PrrF2 are encoded in tandem on the P. aeruginosa genome, allowing the expression of a longer heme-regulated sRNA named PrrH. Due to the unique sequence of this sRNA, PrrH is hypothesized to regulate distinct mRNAs involved in heme metabolism. Our lab previously showed that the locus for the PrrF and PrrH sRNAs is required for virulence in an acute murine lung infection model, yet the mechanisms guiding PrrF and PrrH regulation of virulence have not yet been determined. In this work, we identified Hfq, an RNA-binding protein that stabilizes many bacterial sRNAs, as a potential PrrF-and PrrH-binding protein. Using gel-shift assays, I showed that Hfq has a strong binding affinity for PrrF1 and PrrF2. Moreover, I showed that Hfq increases the annealing rate of PrrF with one of its mRNA targets, antR. I identified nucleotides required for PrrF interaction with antR in vitro and in vivo, and I showed that PrrF1 and PrrF2 regulation of antR is redundant. PrrF regulation of antR was previously shown to promote the production of key virulence-related metabolites, including the Pseudomonas quinolone signal (PQS). Accordingly, we showed that the PrrF1 and PrrF2 sRNAs have redundant function in the production of PQS and structurally related metabolites. I further showed that Hfq binds to the PrrH sRNA in vitro, and I performed preliminary in vitro analysis of the PrrF sRNAs with additional mRNA targets involved in iron metabolism and biofilm formation. Lastly, I demonstrated that iron and PrrF allow for increased P. aeruginosa biofilm formation in the presence of certain antibiotics. Altogether, these studies established the mechanisms of PrrF regulation of a key virulence trait, and they provided the basis for future work into the biochemical and genetic basis of PrrF-mediated virulence in P. aeruginosa.
  • Applications of Quantitative Proteomics and Phosphoproteomics to Study the Development of Resistance to Targeted Therapy in Cancer

    Awasthi, Shivangi; Shapiro, Paul, Ph.D.; Goodlett, David Robinson, 1960-; Guha, Udayan (2018)
    Targeted inhibition of protein kinases is a major approach to treat cancer. However, the effectiveness of kinase inhibitors is limited due to intrinsic and acquired resistance mechanisms that promote the progression and survival of cancer cells. The objective of this dissertation is to use liquid chromatography coupled to mass spectrometry (LC MS) based quantitative proteomics to identify potential biomarkers of resistance and response to molecularly targeted therapies in cutaneous melanoma and lung adenocarcinoma in vitro. For the first part of this thesis, I conducted a proteomic analysis of the acquired drug resistance to extracellular signal-regulated kinase (ERK1/2) pathway inhibitors in a melanoma cell line model. A combination of immunoblot assays, global label-free bottom-up proteomics, phosphoproteomics and pathway analysis was used to characterize the differential protein expression in drug resistant melanoma cells. Examination of the quantitative data pointed to an invasive and metastatic phenotypic signature in the resistant cells. We also identified and verified the overexpression of β-catenin and Caveolin-1 (CAV-1) in MEK1/2 and ERK1/2 inhibitor resistant cells. These findings suggest that these proteins have a role in the development of resistance and may represent novel targets for co-therapy. For the second part of this thesis, I have utilized a multiple reaction monitoring (MRM) based targeted proteomic technique to verify previously identified potential biomarkers of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) response in lung adenocarcinoma. Published global phosphoproteomic data were used to select a list of phosphotyrosine peptides (pY) and MRM based relative and absolute quantitative methods were developed to measure their expression in TKI sensitive and resistant lung adenocarcinoma cells. Modified immuno-MRM assays were optimized using heavy labelled synthetic peptide standards which identified the targets with good reproducibility and repeatability. The results indicated that of the 11 chosen sites, EGFR-pY1197 can be used as potential biomarker of EGFR TKI sensitivity, regardless of the EGFR TKI used. Overall these data advance our understanding of the mechanisms of targeted therapy resistance and highlight candidate biomarkers of resistance and sensitivity.
  • Learn and Apply Paradigm to Optimize Pharmacotherapy in Neonatal Abstinence Syndrome Using Pharmacometrics

    Liu, Tao; Gobburu, Jogarao; 0000-0002-9943-2131 (2017)
    Every one hour a baby with neonatal abstinence syndrome (NAS) is born in the United States. NAS is a clinical syndrome of opiate withdrawal in infants exposed to drugs either prenatally in the form of maternal use (non-iatrogenic), or postnatally in the form of medical therapy (iatrogenic). The syndrome is comprised of a combination of central nervous system, digestive system and autonomic system abnormalities that result from uninhibited excitatory neurotransmitter release from the neurons. Between 2000 and 2009, a 3-fold increase in the use of opiate drugs among pregnant women led to an increase in NAS and associated higher health care costs. Currently, morphine is the first line pharmacotherapy for NAS. Pharmaceutical companies have no incentive to invest in therapy optimization for NAS, and the current dosing strategies vary from hospital to hospital. This research is based on a virtual consortium between the Center for Translational Medicine at the University of Maryland Baltimore, Johns Hopkins Medical Institute and Thomas Jefferson University Hospital. The purpose of this research is to optimize the morphine dosing strategy in NAS patients who require pharmacotherapy by using a pharmacometrics approach with the "learn and apply" philosophy. First, a comprehensive morphine pharmacokinetic model that accounts for prognostic factors, such as body size and age, was developed in neonates. The results suggested a uridine diphosphate glucuronic acid dependent morphine clearance during the first week of life. Second, an exposure-response (ER) relationship between morphine plasma concentrations and modified Finnegan scores was built, and the model prediction was evaluated for the primary and secondary clinical outcomes, such time-on-treatment and total morphine dose. Lastly, different morphine dosing strategies were simulated based on the ER relationship and then optimized dosing strategies were proposed. The proposed dosing strategies will minimize suffering due to the withdrawal symptoms and ultimately lead to an earlier hospital discharge.
  • Exploring Protein Dynamics and Folding with Constant pH Molecular Dynamics Simulations

    Yue, Zhi; Shen, Jana; 0000-0002-4231-7474 (2017)
    Solution acidity or pH is a key environmental regulator with profound impact on biological processes such as protein dynamics and functions. Over the past decade, our research group has developed a set of tools to explicitly account for solution pH in molecular dynamics simulations. In this dissertation we demonstrate the new application areas of the constant-pH molecular dynamics tools. First we use constant-pH MD alongside fixed-protonation-state simulations to explore the dynamics of a cytoplasmic heme-binding protein (PhuS) in Pseudomonas aeruginosa, a notorious opportunistic pathogen. Our results provide atomic-level information on how heme binding affects PhuS dynamics which suggests an induced-fit mechanism, in support of the recent hydrogen-deuterium exchange data. Secondly, we apply constant-pH MD to investigate the acid-induced unfolding of BBL, a small component of the ubiquitous pyruvate dehydrogenase multienzyme complex involved in carbohydrate metabolism inside mitochondria. Our data support that BBL is a barrier-limited two-state folder, an issue that has drawn intense debate among experimental groups. More importantly, our simulations reveal that acid-induced unfolding of BBL is triggered by sequential protonation of His166 and Asp162 and thereby offering atomic details unattainable via experimental means. This work is the first time the constant-pH MD in explicit solvent has been applied to protein folding studies. Next, we employ the membrane-enabled constant-pH MD to understand how proton release drives the conformational transition of the transmembrane multi-drug efflux pump AcrB, which is crucial for the intrinsic resistance of E. coli to clinically important antibiotics. Our data address the controversy regarding the proton/drug stoichiometry and reveal the details of how deprotonation of a single residue leads to a global conformational transition in AcrB. This work paves the way for understanding the complete cycle of drug transport in AcrB and validates the membrane-enabled constant-pH MD technique for mechanistic studies of proton-coupled transporters. Lastly, we benchmark the accuracy of the all-atom constant-pH MD with charge-leveling co-ion using titration simulations of five proteins (HP36, BBL, NTL9, HEWL and SNase). The average and maximum absolute errors between the calculated and experimental pKa values based on 10-ns pH-based replica-exchange simulations are 0.7 and 0.9 units, respectively. Detailed analyses indicate that limited sampling is a major source of error. This work demonstrates the all-atom constant-pH MD method a practical tool for accurate prediction of pKa's and atomically detailed studies of proton-dependent conformational dynamics. In summary, my studies offered new mechanistic insights into the various roles of protons in protein dynamics and folding that were previously not well understood. My work further established constant-pH MD as a powerful tool for revealing atomic details of proton-coupled dynamic processes.
  • Molecular Mechanisms of Osteocyte Mechanotransduction

    Lyons, James Stephen; Stains, Joseph P. (2017)
    Diseases of skeletal fragility affect >200 million people worldwide and contribute to ~9 million factures annually. Preventing bone loss and/or restoring lost bone mass is of vital importance to limiting the personal and economic impact of these diseases. The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely through regulating the secretion of sclerostin, an inhibitor of bone formation. Osteocytes sense mechanical load in the form of fluid shear stress (FSS), and respond by reducing expression of sclerostin leading to "de-repression" of osteoblastogenesis and stimulation of de novo bone formation. However, key mechanistic details of how osteocytes sense mechanical load, transduce these signals to biologic effectors, the identity of these effectors and how sclerostin bioavailability is regulated remain unclear. A widely accepted technique for mechanically stimulating cells in culture is the introduction of FSS on cell monolayers. Here, we describe a novel, multifunctional fluid flow device for exposing cells to FSS. We validated the device using the biologic response of UMR-106 cells in comparison to a commercially available system of FSS. Utilizing this FSS device we show that the microtubule (MT) network plays a critical role in how osteocytes sense and respond to FSS. We define a microtubule-dependent mechanotransduction pathway that links FSS to the generation of react ROS and Ca2+ signals, leading to reductions in sclerostin in osteocytes. In Ocy454 osteocyte-like cells, we demonstrate that an intact MT network is required for FSS-induced Ca2+-influx, calcium calmodulin-dependent protein kinase II phosphorylation, and reduction in sclerostin. Further, the abundance of detyrosinated Glu-tubulin dictates the cytoskeletal stiffness of these cells. By tuning the abundance of Glu-tubulin/cytoskeletal stiffness, we demonstrate that Glu-tubulin regulates the mechano-responsive range at which FSS elicits a Ca2+ response in osteocytes. Further, we determined that the FSS-induced reduction in sclerostin requires activation of a signaling cascade that includes production of Nox2-activated ROS, which stimulates Ca2+-influx through the cation-permeable channel TRPV4 and the subsequent activation of CamKII. By developing a better understanding of this fundamental aspect of skeletal physiology, we will raise the possibility of outlining new drug targets to combat diseases of skeletal fragility.
  • Novel Lipid A Structures for Adjuvant Discovery and Development

    Gregg, Kelsey; Ernst, Robert K.; 0000-0002-1483-735X (2017)
    There is an urgent need to develop effective immunizations against infectious diseases that continue to be a major cause of morbidity and mortality worldwide. Modern subunit vaccines require immunopotentiators or adjuvants to strengthen the protective immune response. Adjuvants in human licensed vaccines include aluminum hydroxide, a TH2-biased adjuvant, and monophosphoryl lipid A (MPLA), a TH1-biased, non-toxic Toll-like receptor 4 (TLR4) agonist. MPLA is chemically derived from Salmonella minnesota R595 lipopolysaccharide and displays reduced endotoxicity. Phosphorylated hexacyl disaccharide (PHAD) is a similar, but synthetically produced, monophosphoryl lipid A adjuvant. We hypothesized that functionally diverse lipid A adjuvant structures could be rationally designed by reprogramming the lipid A biosynthetic pathway by adding or removing lipid A modifying enzymes in a process called bacterial enzymatic combinatorial chemistry (BECC). BECC lipid A molecules were engineered in an avirulent Yersinia pestis strain, purified, and screened in immortalized cell lines for TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) responses. BECC-derived structures exhibit dose-dependent TLR4-driven NF-?B activation with both human and murine TLR4 signaling complexes. Structures that displayed reduced NF-?B activity were screened to identify BECC molecules that induced cytokine secretion patterns similar to PHAD in murine splenocytes and human primary blood mononuclear cells (PBMCs), and induced similar activation markers on human monocyte-derived dendritic cells (DCs). Selected BECC molecules were tested in vivo for their ability to adjuvant the recombinant fusion protein, rF1-V, in a murine subunit vaccination model against Y. pestis, the causative agent of plague. BECC-adjuvanted vaccines elicited strong rF1-V-specific antibody titers in C57BL/6J mice with increased titers of the TH1-associated immunoglobulin, IgG2c. The BECC adjuvant groups' titers were similar to or higher than the aluminum hydroxide and PHAD control adjuvant groups. The BECC-adjuvanted and control adjuvant vaccines were protective against a lethal, intraperitoneal Y. pestis CO92- challenge, and were more protective than the unadjuvanted rF1-V vaccine control. These data demonstrate that BECC can be used to generate functionally diverse TLR4 ligands with potential for use as TH1-biasing vaccine adjuvants.
  • Evaluation of the Use of Hydroxypropyl methylcellulose acetate succinate (HPMCAS) in Enteric Coating of Tablets and Solubility Enhancement of BCS Class II Compounds

    Deshpande, Tanvi Mahesh; Hoag, Stephen W. (2017)
    Hydroxypropyl methylcellulose acetate succinate (HPMCAS), an anionic polymer, demonstrates wide applicability in drug delivery. The goal of this dissertation is twofold: Applicability of HPMCAS in aqueous enteric coating of tablets and in solubility enhancement of BCS Class II compounds. HPMCAS used for aqueous enteric coating has a tendency to aggregate and clog the spray-nozzle during the coating process. This limitation motivated us to design and develop a stable aqueous enteric coating formulation that minimizes aggregation and spray-nozzle clogging, and maintains the enteric coating profile of tablets. Results show that elevated processing temperature activates polymer particle coalescence in plasticized dispersions, forming large aggregates that clog the spray-nozzle. We successfully developed a stable formulation containing PEG 4000, sodium lauryl sulfate (SLS), and Aerosil® R972 Pharma that formed hydrogen bonds with HPMCAS (steric repulsion), provided electrostatic stabilization, and provided hydrophobicity to the aqueous coating respectively, which in turn prevented nozzle clogging and maintained the tablet's enteric profile. The second application of HPMCAS focused on spray dried dispersions (SDDs), which is one of the most successful solubility enhancement techniques for BCS Class II compounds (e.g. Itraconazole (ITZ), a model drug for this study). Selection of suitable drug carriers (polymers) was found to be crucial for solubility enhancement and for ensuring the stability of ITZ. Incorporation of surfactants with polymers could further enhance ITZ solubility by micellar solubilization mechanism. The project goal was to develop a screening methodology for selecting polymer/surfactant combinations for enhancing solubility and kinetic stability of ITZ spray-dried dispersions (SDD). 1H NMR and fluorescence spectroscopy aided in screening polymer/surfactant combinations by determining critical micelle concentration (CMC) of the system. Observations indicated that stronger surfactant/polymer interactions (inferred by the decrease in CMC) show limited solubility and kinetic stability enhancement (due to their unavailability to interact with ITZ). Further screening of excipients for SDD preparation was performed based on the supersaturation ratio and the precipitation induction time of ITZ in the presence of different surfactant/polymer combinations. ITZ/HPMCAS-HF SDD was found to be most efficient since it enhanced and maintained the solubility of ITZ by 60-fold for up to 24 h in simulated intestinal fluid.
  • Molecular Insight into the Structure, Function, and Regulation of Bile Acid Transport

    Czuba, Lindsay Christine; Swaan, Peter W.; 0000-0001-9142-5706 (2017)
    The human Apical Sodium-dependent Bile Acid Transporter (SLC10A2), also known as hASBT, plays an integral role in the enterohepatic circulation of bile acid and cholesterol homeostasis. As a member of the solute carrier family of membrane transporters, it uses the established Na+ electrochemical gradient as an energy source to reclaim bile salts from the ileum. hASBT has been identified as a promising target for the management of hypercholesterolemia, cholestatic pruritis, and as a prodrug-targeting approach for improved bioavailability of drugs. Limiting the development of such therapeutics, is an incomplete understanding of hASBT's structure. Extensive biochemical and mutagenesis studies for hASBT support a seven transmembrane model. Yet conflicting structures have emerged with the elucidation of the crystal structures of two putative homologues from Neisseria meningitidis (nmAsbt) and Yersinia frederiksenii (yfAsbt). In the absence of a physiological context, the use of their structure as models of the human transporter is limited. In addition to the discrepancy in hASBT's fold, there is limited information regarding the specific proteoforms that are relevant to the functional expression of hASBT and in its regulation. In this work we provide novel molecular insight into the structure, function, and regulation of human ASBT. We contrasted the biochemical, inhibitory, and evolutionary attributes of nmAsbt, yfAsbt, and hASBT and identified their critical differences. The fundamental differences in ion dependency, substrate specificity, and evolutionary context imply divergent structure-function relationships and negate the use of the bacterial transporters as suitable models for hASBT. Additionally, we characterized the role of tyrosine phosphorylation in regulating the functional expression and stability of hASBT. We identified Src family kinases as critical modulators and provide support for hASBT's regulation by phosphatases. As the clinical relevance of PTMs is growing, so too are the number of FDA-approved therapeutics that target these modifications. In this regard, we have made critical advances and gained valuable insight into hASBT's regulation. Finally, we have optimized the biological sample preparation methods and have significantly increased the purity of hASBT samples. When coupled with mass spectrometry analysis, these methods will identify critical proteoforms of hASBT and facilitate a global understanding of its structure-function relationship.
  • Mechanistic Evaluation of Polyethylene Oxide for Physical Barrier Type Abuse Deterrent Formulations: Techniques and In vitro methods

    Boyce, Heather J.; Hoag, Stephen W.; 0000-0001-9817-7784 (2017)
    Abuse deterrent formulations (ADF) are designed to mitigate misuse and abuse of prescription narcotics. One mechanism of an ADF is to increase the tablet strength to reduce ease of tablet comminution while increasing the difficulty of drug extraction. Polyethylene oxide (PEO) is a popular polymer used in these complex opioid products. PEO provides these abuse resistance properties while providing extended release of the opioid drug. Evaluation of the manufacturing process and material attributes of this polymer is important to improve upon the next generation of abuse deterrent products. In addition, methods to assess abuse deterrence of these products in vitro is challenging, but important for product development and generic abuse deterrent product approvals. Thus, this dissertation seeks to determine the best evaluation methods for these products and establishes an in vitro method to assess a formulation's ability to prolong nasal absorption when nasally insufflated. Key results of this study show that heating tablets with PEO will significantly increase the strength of the tablet and key variables such as PEO composition, particle size distribution, high initial tablet solid fraction, and 1 h sinter time were found to be the optimal sintering conditions. It was also demonstrated that this sintering process could be monitored by near infrared spectroscopy to predict sintering end points. Finally, the in vitro nasal insufflation studies demonstrated that for each type of physical manipulation employed (i.e. cutting, grinding, milling), discreet particle size distributions were formed regardless of formulation. One exception occurred with milling where one drug product resulted in a much greater particle size distribution than the other milled tablets. A vertical diffusion cell was then used to assess release rate of drug from the comminuted dosage form. It was demonstrated that the VDC method was discriminant with respect to particle sizes of comminuted particles and formulation variables such as molecular weight of PEO used.
  • Towards Targeted Anti-Neoplastics: The Disruption of Aberrant Protein-Protein Interactions with Low Molecular-Weight Proteomimetics

    Lanning, Maryanna Elizabeth; Fletcher, Steven; 0000-0002-9511-4435 (2017)
    Protein-protein interactions (PPIs) play pivotal roles in a range of cellular processes including proliferation, differentiation, metabolism and apoptosis. Dysregulations of certain PPIs can lead to the development and progression of human cancers. In particular, the overexpression of the anti-apoptotic BCL-2 family members, specifically Mcl-1, have been linked to pancreatic, colorectal and lung cancers as well as leukemia and lymphoma. When over-expressed, MCL-1 prevents cell death by binding and sequestering the BH3 "death" domain of its pro-apoptotic counterpart, such as Bim. Mcl-1 has become an important target for the development of novel antineoplastics. As with many, helix-mediated PPIs, several key residues are hydrophobic and located on one face of the BH3 α-helix, specifically at the i, i + 3/4, i + 7 residues. In addition to exhibiting a hydrophobic face, Asp67 on the "other" face of the Bim-BH3 helix forms a salt bridge with the protein (Arg263). In an improved effort to develop more potent and more selective agents to disrupt the MCL-1-BH3 PPI, we used structure-based design, and developed two complementary strategies: synthetic α-helix (purine based) and BH3 mimetics (naphthoate based). Both scaffolds yielded molecules that disrupted the MCL-1-BIM PPI consistently, and subsequent studies were undertaken towards second-generation molecules. The designed molecules were subjected to biological assays and further structure-activity relationship (SAR) studies to increase affinity and potency in an effort to translate in vitro activity to on target cell activity. It is expected anticipated that the SAR developed in the present study will facilitate the development of novel therapeutics capable of inhibiting Mcl-1 will be identified, which can be advanced to preclinical evaluation.

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