Flow Effects on Endothelial Cell Glucose Metabolism: Glycolytic Flux and O-GlcNAcylation in Health and Disease

Cardiovascular disease, the leading global cause of death, is precipitated by endothelial cell (EC) dysfunction. When EC are exposed to unidirectional steady laminar flow (>12 dynes/cm2 shear stress), the cells assume a healthy, quiescent phenotype in which EC phosphorylate endothelial nitric oxide synthase (eNOS) to produce NO, a vasodilator that is also important in preventing vascular disease. However, when EC fail to adapt to flow, for example in areas of oscillating disturbed flow, they have impaired NO production. Areas of disturbed flow are linked to EC dysfunction and diseases such as atherosclerosis. Endothelial metabolism has recently emerged as a powerful tool to regulate the vasculature. Both cancer cells and ECs generate the majority of their energy through glycolysis even in the presence of oxygen (the Warburg effect). When EC glucose metabolism was decreased by PFKFB3 blockade, EC proliferation, angiogenic sprouting, and cancer metastasis were inhibited both in vitro and in vivo. Endothelial metabolism also decreases in chronic laminar flow, which put EC in a quiescent state and decreased angiogenesis. Little is known about how different blood flow regimes regulate EC metabolism and how this could impact macrovascular disease. The goal of this research was to elucidate how hemodynamics regulate EC glucose metabolism, and how EC glucose metabolism can be modulated to restore EC function in disturbed flow. We hypothesized that steady laminar flow (shear stress of 20 dynes/cm2) but not oscillating disturbed flow (shear stress of 4±6 dynes/cm2) reduces glycolytic flux and eNOS O-GlcNAcylation to promote a healthy endothelial phenotype. First, I adapted human EC to steady laminar and oscillating disturbed flow in a cone-and-plate device in vitro and quantified changes in (1) endothelial glycolytic activity and (2) eNOS O-GlcNAcylation. Stable isotope mass spectrometry and YSI Bioanalysis revealed an overall decrease in glucose uptake and glycolytic activity in EC exposed to steady laminar but not oscillating disturbed flow. HUVEC exposed to oscillating disturbed flow had over two times more glucose consumption compared to cells exposed to steady laminar flow. Glycolytic intermediate total pool sizes such as fructose-1,6-bisphosphate, 1,3-bisphosphoglyercate, and phosphoenolpyruvate were 60-70% lower in HUVEC exposed to steady laminar flow compared to cells exposed to oscillating disturbed flow. Next, I showed that eNOS O-GlcNAcylation was abolished in EC exposed to steady laminar (~75% lower) but not oscillating disturbed flow. Interestingly, there was no change in protein level, localization, or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, glycolysis inhibition via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation by 60%, thereby increasing eNOS phosphorylation by 20% and NO production by 65%. Finally, I investigated altered glucose metabolism in pulmonary arterial hypertension (PAH). Human pulmonary artery EC from PAH patients showed three times as much glucose uptake as healthy patients. Additionally, glycolytic intermediates such as fructose-1-6, bisphosphate and 3-phosphoglycerate showed 50-75% higher total pool levels in PAH patients compared to healthy patients. Furthermore, PAH patients showed reduced eNOS O-GlcNAcylation and NO bioavailability. My data demonstrate that steady laminar but not oscillating disturbed flow decreases glycolytic activity as well as HBP activity. Specifically, glycolytic flux controls eNOS O-GlcNAcylation and UDP-GlcNAc substrate availability, thus impacting eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli; relates flow-induced glycolytic flux changes to macrovascular disease; and highlights O-GlcNAcylation as a novel therapeutic target to restore eNOS activity and to prevent EC dysfunction in cardiovascular disease.Ph.D., Biomedical Engineering -- Drexel University, 201

Computational Models of Endothelial Cell Growth Factor Kinetics and Glucose Metabolism

Cardiovascular disease (CVD) is the leading cause of death worldwide, and endothelial dysfunction contributes to CVD progression. While experimental studies attribute endothelial dysfunction to changes in cell signaling, protein expression, and metabolism, it remains unclear how these individual changes interact within the complex endothelium to lead to the dysfunctional phenotype. Computational models provide an essential tool to understand the endothelium as a system. The dissertation objective was to use mass action kinetics and stoichiometric computational models to predict endothelial growth factor binding and glucose metabolic flux. I first created a mass action kinetics model to investigate fibroblast growth factor-2 (FGF2) binding to endothelial cells under laminar flow by incorporating flow-induced changes to the endothelial cell apical surface. FGF2 model dynamics were described by a set of ordinary differential equations and kinetic parameters from the literature. When the model included increased heparan sulfate proteoglycan production and binding site availability with shear stress, as well as increased FGF2 dissociation with shear stress, the model successfully predicted FGF2 biphasic binding response as a function of shear stress. Next, I created a carbon transition model to predict endothelial cell intracellular metabolic fluxes using isotope labeling experiments. The model traces carbon movement through glycolysis, the pentose phosphate pathway and the TCA cycle. The model was iteratively updated to reduce the error between the predicted and measured experimental data. The final model highlighted the importance of glutamine in reproducing TCA cycle intermediates and in feeding back into glycolysis. Finally, I developed a stoichiometric endothelial model to describe the metabolic state of a quiescent endothelial cell in vitro. A genome scale cell model was curated into an endothelial-specific model using publicly available transcriptomic data to remove reactions associated with proteins that were not expressed in endothelial cells. The carbon transition model fluxes were used to constrain the minimum and maximum fluxes in the stoichiometric endothelial model. I then used the model to explore possible endothelial metabolic objective functions, including minimizing oxidative stress or resource consumption. I concluded that no single metabolic objective function accurately predicted quiescent endothelial monolayer metabolism. Together these models provide valuable insight into FGF2 binding kinetics and steady state metabolic endothelial cell objectives. In addition, the mass action kinetic and stoichiometric endothelial models provide a systems-level analysis of endothelial function, enhancing our understanding of the complex interactions that contribute to endothelial dysfunction and eventual cardiovascular disease.Ph.D., Biomedical Engineering -- Drexel University, 201

Novel Magnetically Levitated Right Ventricular Assist Device for Pediatric and Adult Patients with Acquired or Congenital Heart Disease

Thousands of pediatric and adult patients in the U.S. are diagnosed with congestive heart failure (CHF) secondary to acquired or congenital heart disease. Symptoms of CHF include left-sided heart dysfunction, fluid retention and swelling in lower extremities, weakness, and shortness of breath. A substantial number of these patients also develop right-sided heart failure secondary to the left-sided failure or dysfunction. Strategies to address right ventricular heart failure include pharmacological treatments, which only slow the progression of heart disease, or complete heart transplants. With a lack of donor organs available, mechanical circulatory assist devices are alternative therapeutic options; however, there are few such devices for right ventricular failure in pediatric patients. To address this unmet clinical need, we are developing a new right ventricular assist device (RVAD) or blood pump as a novel treatment strategy for these patients. The axial flow RVAD utilizes third-generation drive technology by incorporating a magnetic suspension to levitate the axial impeller within the pump housing. This magnetically suspended configuration enables the pump to have clearances between rotating and stationary components that are much wider than afforded by conventional mechanical and fluid bearings, thus lowering shear stresses and risk of blood cell damage and clotting. The RVAD consists of five internal fluid domains and bladed regions: inducer, impeller, diffuser and flow straightener. It spans 30 mm in diameter by 60 mm in length. Acting to improve blood flow to the lungs, this RVAD is designed to achieve cardiovascular requirements by generating desired blood flow rates and increased pressures. Numerical simulations of the design were performed using ANSYS computer software to estimate the hydraulic performance of the RVAD. Pressure generation was analyzed over a physiologic blood flow range and varying rotational speeds. In addition to pressure generation, the fluid stress levels, axial and radial fluid forces on the impeller of the RVAD, and power consumption were evaluated. Sufficient pressure rises of 2-142 mmHg were attained for flow rates of 2-6 L/min at rotational speeds 7000-15000 RPM. It was observed that higher flow rates generate lower pressure rises for all speeds, and higher speeds generate increased pressure rises, which follows expected pump trends. For these operating conditions, axial forces were found to be less than 3 N and radial forces less than 1 N. Scalar stresses were less than 425 Pa with residence times less than 600 ms for operating speeds under 12000 RPM. Rotational speeds of 12000 RPM and higher generated high scalar stress values at the trailing edges of the impeller blades. Due to the strong computational data, a prototype of the RVAD was constructed and hydraulically tested. The prototype delivered sufficient pressure rises of 1-126 mmHg for flow rates of 0.1-5 L/min at rotational speeds 6000-14000 RPM with an average deviation of 48% from computational data. These results demonstrated strong pump performance for right ventricular assistance, thus supporting the continued development of this RVAD for pediatric patients with right-sided heart failure.M.S., Biomedical Engineering -- Drexel University, 201

Visual Art Journaling as a Mindful Practice with Patients and Professional Caregivers in Oncology Care

This paper describes the development and implementations of a method that combines the use of a visual art journal as a mindful practice to combat the effects of cancer on patients diagnosed and professional care providers. The development of the method required adaptations specifically to group membership and the structure used. Implementation of this method explored ways of using mindfulness and Visual Art Journaling to uncover, document, and creatively express areas of wellness each week of a seven-session process. To do this, group members journaled with both words and images the areas of wellness in their lives and ways of improving their coping mechanisms while undergoing cancer treatment and/or caring for others in their lives. This capstone discussion aimed to highlight ways in which group members developed purposeful awareness of thoughts and emotions, and as they occurred in a non-judgmental way, through the utilization of mindfulness-based Visual Art Journaling explorations, to address stress and anxiety within oncology treatment and care.M.A., Art Therapy and Counseling -- Drexel University, 201

Development of lectins for cancer-specific fucosylation

Changes in serum glycosylation have long been associated with hepatocellular carcinoma (HCC), and these modifications are often detected with sugar-binding proteins called lectins. HCC is associated with increased serum fucosylation, and this sugar is often detected with the Aleuria aurantia lectin (AAL). However, this lectin binds fucose in several forms, one of which is associated with cancer ([alpha]1-6 linked fucosylation) and some of which are not. Thus, its use in clinical and tissue analysis is limited. We have created a recombinant AAL, which contains a single amino acid mutation: N224Q (rAALN224Q), within the binding site 5, and this lectin has increased sensitivity to the cancer-specific fucosylated glycan. Our analysis of rAALN224Q has shown that the mutation within one specific fucose binding pocket had a major impact upon the mutated site as well as on sites that were not mutated and led to a lectin with altered binding specificities. This lectin is currently part of a diagnostic test under evaluation for the early detection of HCC. We have further explored the changes in N-linked glycosylation associated directly with HCC tissue through a method of N-linked glycan matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). MALDI IMS of N-linked glycan revealed inter- and intra-tumor heterogeneity shown by expression and spatial localization of glycans in the tumor. Tumors were associated with alterations in mannosylation, fucosylation, branching, and complex glycan formation. Importantly, all tumors were associated with some change in glycosylation, but there was significant glycan heterogeneity.Ph.D., Microbiology and Immunology -- Drexel University, 201

Exploring interactions between the parasite plasma membrane and the parasite vacuolar membrane of Plasmodium falciparum

Malaria poses a large health and economical threat to 40% of the world’s population. Treatment and prophylaxis with antimalarials remain a mainstay to control malaria. However, drug resistance remains a constant threat to these efforts. It is hoped that with the approval of new antimalarials and their proper distribution to endemic populations, the disease will stay contained and resistance to currently implemented drugs can be suppressed. Understanding the biology of the Plasmodium parasites that cause malaria infection will greatly aid in the hunt for potential antimalarials. Here, we study a series of compounds from the Medicines for Malaria Venture by monitoring their ability to induce lipid dysregulation within the parasites’ plasma membranes (PPM). Saponin sensitivity was measured in this newly developed assay via release of parasite cytosolic proteins after their exposure to drug. Proteins that contribute to lipid dynamic movement in P. falciparum are under investigation in our laboratory. Specifically, Niemann-Pick type C1-related protein (PfNCR1) and oxysterol-binding protein (PfOSBP) influence lipid movement across the PPM within an infected red blood cell. Though, we have shown PfOSBP not to be essential for asexual blood stage growth, this protein plays a notable role in sexual stage development and functionality. Additionally, [delta]PfOSBP transgenic parasites display altered PPM composition, which may link PfOSBP’s function with the lipid transfer pathway in P. falciparum as was shown with PfNCR1. We have proposed cholesterol to transfer from the parasite vacuolar membrane (PVM) inward, to the PPM upon drug exposure. The PPM and PVM are in proximity to one another, especially at the parasite’s cytostome which forms a site for lipid movement between membranes. To investigate proteins involved in cytostome development, we looked to analogous proteins found in a ciliate, Tetrahymena. A citrate synthase protein is involved in this ciliate’s oral apparatus formation. We show P. falciparum citrate synthase-like protein (PfCSL) to be an essential protein. In analogy with citrate synthase in Tetrahymena, we propose PfCSL to contribute to parasite cytostome function.Ph.D., Microbiology and Immunology -- Drexel University, 201

Retrospective Student Experiences: A Study of Living with Persistent Environmental Stress During High School

Home lives filled with stressors such as poverty, mental illness, substance abuse, and homelessness are some of the often-unrecognized realities students in schools across America face. These realities often put high-school students at risk for underperforming. Many current political policies are not suited to deal with the issues these students present, even though many of these policies provide guidelines for specific preventive measures, often in the form of student support programs embedded within high schools. However, little is known of the students' own perceptions of the intersection between their stressful home lives and high-school success. This study proposes to consider the experiences and stories of former high school students reflecting on their chronically stressful home environments and their experiences with supportive programs in high school. Understanding students' own perceptions will inform future practice within high schools. The findings of this study may be significant in redesigning student assistance programs across the state to provide supports that allow students to achieve academically and personally to their fullest potential. This phenomenological study is based on a conceptual framework with three main streams: struggles of students living with chronic stress, student support programs, and policy considerations. It also seeks to answer the following central question and three related sub-questions: 1. Central Question: How do former high-school students describe their chronically stressful home environments during high school, and how did the high school and supportive programming they may or may not have received at the time influence their home, social, or academic experiences? 2. Sub-Questions: a. What specific characteristics of high school do former high-school students identify as successfully supporting their ability to manage their chronically stressful environments? b. How did these characteristics allow for greater success during their high-school years? c. How do former high-school students who endured chronically stressful home environments describe their personal interactions with and attitudes toward school-facilitated services and support programs while in high school?Ed.D., Educational Leadership and Management -- Drexel University, 201

A Mixed Methods Case Study: Exploring Teacher and Principal Perceptions about Effective Principal Leadership Behaviors that Influence Student Academic Achievement in Philadelphia Area High Schools

In the Philadelphia area, there are too many high schools where students are not making adequate academic progress, as measured by the Pennsylvania Value Added Assessment System (PVAAS). As a result of this insufficient academic progress, there are many young adults lacking the minimal skills necessary to enroll in college or to enter into the workforce. The purpose of this mixed methods case study was to explore principal leadership behaviors and characteristics, and the extent in which they contribute to the achievement of students in urban high schools within the Philadelphia region. In every institution of learning, the overarching goal should be centered on providing students the necessary skills to assist them in growing academically, as well as socially. Principals as the leaders, of K-12 buildings, play an important role in ensuring that students receive the best education possible, while also maintaining and creating a safe environment for students and staff members. The research question for this investigational study was: What is the relationship between principal leadership behaviors and high school student achievement in urban districts within the Philadelphia area? The researcher used the following sub-questions to provide answers to the overarching question: (1) What leadership behaviors do teacher respondents perceive as positively impacting student achievement in urban high schools; (2) What leadership behaviors do principal respondents perceive as positively impacting student achievement in urban high schools; (3) How do principals describe the direct effects of their leadership behaviors on urban high school student achievement; and (4) How do principals describe the indirect effects of their leadership behaviors on urban high school student achievement? At two high schools in the Philadelphia region, 27 teachers and two principals participated in this mixed methods study. The high school teachers completed a survey using the well-known and validated Principal Instructional Management Rating Scale (PIMRS), designed for teachers by Dr. Philip Hallinger. The two principal participants took part in a semi-structured interview and completed the PIMRS survey designed for school leaders. The semi-formal interviews, scheduled for 30 minutes, were recorded using two digital devices and transcriptions were electronically and manually completed. Statistical Package for Social Sciences (SPSS) software was used to interpret and analyze quantitative data while InVivo and hand coding was used for qualitative data analysis. From the analysis of the qualitative data, three major themes emerged for principal behaviors: (a) data collection and analysis is essential, (b) curriculum and instruction must be monitored and (c) school culture is a factor. This study may inform current and future school leaders on the leadership behaviors essential to improving academic achievement. Considerations for further research in the area of principal leadership are offered.Ed.D., Educational Leadership and Management -- Drexel University, 201

A Qualitative Investigation of Independent School Educators' Lived Experiences with PreK-12 Accreditation

This study investigates how independent school educators perceive their role within accreditation review processes and associated organizational reform efforts. To fulfill this purpose, research presented here answered two questions: (1) How are educators incorporated into accreditation review processes established and administered by NAIS-certified accreditation agencies? (2) How do these educators perceive their role during accreditation review and how do these perceptions inform their beliefs about (a) the accreditation review process in general, and (b) specific education reform initiatives associated with accreditation specifically? To build context, this study drew on three literature streams. First, prior literature outlined the historical and philosophical development of independent school accreditation. Second, researchers analyzed beliefs surrounding prior school evaluation processes. Finally, this study outlined accreditation principles and requirements of NAIS, the chief certifier of independent school accreditation agencies. To answer each research questions, the researcher collecting public documents relevant to NAIS, its certified agencies, and specific institutions who agreed to participate. Moreover, surveys were distributed to faculty and staff at eight independent schools. In total, the researcher collected 156 valid surveys. Finally, the researcher conducted 30 to 45 minute semi-structured interviews with eight survey participants who completed the aforementioned survey. Findings revealed five common themes present across survey and interview data. (1) Accreditation drives and is driven by school leadership, (2) Accreditation-facilitated inclusivity, (3) Accreditation as an imperfect process, (4) System of continuous improvement, and (5) Institutional honesty. Four results emerged from these findings. Specifically, participants perceived that (1) independent schools and accreditation agencies recognized imperfection and encouraged growth; (2) the accreditation review process hinged on school leadership for independent schools and accreditation agencies; (3) despite being stressful, there was value in an inclusive accreditation review process; and (4) committee deliberation was the cornerstone of accreditation-related work. This study underscores the need for research in the practices of independent school accreditation agencies. It also stresses the need for accreditation agencies to continue its embrace for growth mindset principles by encouraging continual improvement. Finally, research accentuates the imperative of agencies to reform their evaluation practices to provide its member institutions with stronger assessments.Ed.D., Educational Leadership and Management -- Drexel University, 201

When Old Becomes New Again: The Emergence of Classic TV Broadcasting

This thesis focuses on the reemergence of decades-old television broadcasts and the growth in popularity of those classic shows in the United States over recent years. The research will showcase early television with a comparison to current television broadcasts. Answers to questions as they relate to how programming has evolved and how audiences influenced the change(s) are addressed to set a foundation for the current interest in previously aired broadcasts. The term ‘reruns’ and a brief explanation of ‘residuals’ (paid/unpaid) will be included as well. The information presented in this research has been located in several areas. The obvious place to begin was to investigate documentation that pertained to the emergence of new broadcasting entities such as MeTV (2010) and The Decades Channel (2015) that showcase what is now coined as Classic TV. Archival footage along with that documentation was found through such resources as The Museum of Broadcast Communications and The Broadcast Pioneers of Television. Additional information was also gathered from interviews of the staff of those stations and organizations as well as from veteran broadcast personalities who have witnessed the transition from early television to the reemergence of classic shows. Contact and interviews were made in person, by email and by phone conversation. Other areas of interest for research were discovered in viewer analytics and sociology as they relate to the changing world of media (how it is perceived, positive/negative impacts, the public's need for entertainment). It has been noted that (Keilbach, 2013) most current viewers initially disregard previously aired television programming as old or not interesting. This research shows how people considered the age of a program, in years, along with something they have not yet been exposed to when making the decision to watch vintage shows. The benefit of this research to professionals in the television industry would be to uncover ways to change the current assumption of the viewing audience surrounding vintage programming to be more of a positive experience through exposing these audiences to diverse entertainment. It may also be proven that exposure to less complicated and lighthearted programming may invite families to gather together in front of their favorite show as once was the custom. A final benefit would be geared toward the audience itself. Simply watching previously aired programming that concern subjects that relate to history, prominent personalities, technological advances, and others would increase general historical knowledge for the viewer(s) with little effort. Some studies suggest that more of what is learned/studied can be retained for a longer amount of time if a sense of interest or enjoyment is present.M.S., Television Management -- Drexel University, 201