UMD Theses and Dissertations
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Item Strategies for small RNA loading into extracellular vesicles(2022) Pottash, Alex; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Small RNAs are an exciting class of therapeutics with significant untapped therapeutic potential, due to their ability to affect cell behavior at the RNA level. However, delivery of RNA is a challenge due to its size and labile nature. Extracellular vesicles (EVs) are promising as delivery vehicles due to their natural role as physiological intercellular microRNA transporters, and research has shown that EVs have significant advantages compared to competing technologies such as lipid nanoparticles. Specifically, EVs more readily transport through biological barriers, deliver RNA more efficiently, and are less immunogenic. However, intrinsic microRNA content in EVs is low and thus active small RNA loading strategies are needed to enable therapeutic use. Consequently, a variety of small RNA loading methods for EVs have been developed. These include endogenous and exogenous approaches. Exogenous approaches, in which EVs are loaded directly, have been shown to enable loading of hundreds to thousands of small RNAs per EV, but they are not readily amenable to scalable production processes. Endogenous approaches, in which EVs are loaded by upstream manipulation of the producer cell, are compatible with large scale EV production, but loading by these approaches is inconsistent and has scarcely been quantitatively analyzed. The work in this dissertation is focused on enabling small RNA therapeutics via EV delivery. The lack of an ideal small RNA loading approach for EVs is addressed by tackling important issues of both endogenous and exogenous loading. First, the loading capacity of several common endogenous loading methods was optimized and quantitatively analyzed. Additionally, new approaches to endogenous small RNA loading involving genetic manipulation of the RNA structure and the microRNA cellular processing pathway were developed and evaluated. Finally, exogenous loading via sonication was applied to enable delivery of a novel microRNA combination that was identified via a rational selection process. This combination of miR-146a, miR-155, and miR-223 was found to have potentially synergistic anti-inflammatory activity, and EV-mediated delivery of the combination opens the possibility for therapeutic application in inflammatory diseases and conditions such as sepsis. Overall, this work both improves understanding of current techniques for small RNA loading into EVs and opens new opportunities for advanced strategies, bringing EV-based small RNA therapeutics closer to clinical application.Item THE ENDOGENOUS REGULATION OF THE HUMAN MACROPHAGE ACTIVATION RESPONSE(2020) Hamidzadeh, Kajal; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are innate immune cells that participate in host defense to invading pathogens. They are powerful producers of cytokines and inflammatory mediators due to their efficient recognition of pathogen associated molecular patterns (PAMPs) via toll like receptors (TLRs). We and others have shown that the activation response to PAMPs is transient. In the present work, we demonstrate that stimulated macrophages produce adenosine and prostaglandin E2, which function as regulators of the macrophage activation response. Macrophages also upregulate receptors for these regulators to terminate inflammation and promote wound healing. We performed high throughput RNA sequencing to characterize the transcriptomes of human monocyte-derived macrophages in response to stimulation with LPS + Adenosine or LPS + PGE2. These cells exhibited a decrease in inflammatory transcripts and an increase in transcripts associated with cell growth and repair when compared to cells stimulated in the absence of these regulators. Macrophages can be generated from precursor cells in response to two different growth factors; M-CSF (macrophage colony stimulating factor) and GM-CSF (granulocyte-macrophage colony stimulating factor). M-CSF is expressed constitutively in a variety of tissues, while GM-CSF is expressed primarily in the lung, but can be induced in other tissues under inflammatory conditions. We demonstrate that human macrophages differentiated in M-CSF readily adopt an anti-inflammatory, growth promoting phenotype in response to LPS + Adenosine or LPS + PGE2, while macrophages differentiated in GM-CSF do not. This observation suggests that M-CSF derived human macrophages may be better able to alter their activation state in response to surrounding signals in order to maintain homeostasis. GM-CSF derived macrophages, in contrast, may undergo a more prominent activation response that is associated with inflammation and tissue destruction due to their inability to efficiently respond to resolving molecules.Item RACIAL DIFFERENCES IN VASCULAR FUNCTION FOLLOWING INDUCED ACUTE INFLAMMATION(2020) Chesney, Catalina Anne; Ranadive, Sushant M; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)African-Americans (AAs) have higher rates of cardiovascular disease (CVD), including hypertension and stroke, as compared to their Caucasian-American (CA) counterparts. High resting concentrations of systemic inflammatory biomarkers contribute to vascular dysfunction and are predictive of future cardiovascular events; differential resting levels of inflammatory markers between groups may reveal increased potential for CVD in at-risk groups. Additionally, impaired endothelial function and increased arterial stiffness, subclinical measures of CVD progression, have been reported in AA groups. The purpose of this study was to examine race differences between young, healthy AA and CA adults after a systemic inflammatory stimulus and subsequent endothelial responses to inflammation. Endothelial function, arterial stiffness, and hemodynamic variables were measured. The results suggest there were no race differences in vascular function or hemodynamic responses following an acute inflammatory stimulus.Item HIGH PERFORMANCE AGENT-BASED MODELS WITH REAL-TIME IN SITU VISUALIZATION OF INFLAMMATORY AND HEALING RESPONSES IN INJURED VOCAL FOLDS(2019) Seekhao, Nuttiiya; JaJa, Joseph; Li-Jessen, Nicole Y. K.; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The introduction of clusters of multi-core and many-core processors has played a major role in recent advances in tackling a wide range of new challenging applications and in enabling new frontiers in BigData. However, as the computing power increases, the programming complexity to take optimal advantage of the machine's resources has significantly increased. High-performance computing (HPC) techniques are crucial in realizing the full potential of parallel computing. This research is an interdisciplinary effort focusing on two major directions. The first involves the introduction of HPC techniques to substantially improve the performance of complex biological agent-based models (ABM) simulations, more specifically simulations that are related to the inflammatory and healing responses of vocal folds at the physiological scale in mammals. The second direction involves improvements and extensions of the existing state-of-the-art vocal fold repair models. These improvements and extensions include comprehensive visualization of large data sets generated by the model and a significant increase in user-simulation interactivity. We developed a highly-interactive remote simulation and visualization framework for vocal fold (VF) agent-based modeling (ABM). The 3D VF ABM was verified through comparisons with empirical vocal fold data. Representative trends of biomarker predictions in surgically injured vocal folds were observed. The physiologically representative human VF ABM consisted of more than 15 million mobile biological cells. The model maintained and generated 1.7 billion signaling and extracellular matrix (ECM) protein data points in each iteration. The VF ABM employed HPC techniques to optimize its performance by concurrently utilizing the power of multi-core CPU and multiple GPUs. The optimization techniques included the minimization of data transfer between the CPU host and the rendering GPU. These transfer minimization techniques also reduced transfers between peer GPUs in multi-GPU setups. The data transfer minimization techniques were executed with a scheduling scheme that aims to achieve load balancing, maximum overlap of computation and communication, and a high degree of interactivity. This scheduling scheme achieved optimal interactivity by hyper-tasking the available GPUs (GHT). In comparison to the original serial implementation on a popular ABM framework, NetLogo, these schemes have shown substantial performance improvements of 400x and 800x for the 2D and 3D model, respectively. Furthermore, the combination of data footprint and data transfer reduction techniques with GHT achieved high-interactivity visualization with an average framerate of 42.8 fps. This performance enabled the users to perform real-time data exploration on large simulated outputs and steer the course of their simulation as needed.Item ASSOCIATIONS BETWEEN AMBIENT PARTICULATE MATTER EXPOSURES, STROKE, AND MARKERS OF CARDIOVASCULAR INFLAMMATION(2017) Fisher, Jared A.; Puett, Robin C; Epidemiology and Biostatistics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Stroke is a leading cause of morbidity and mortality in the United States with 795,000 people experiencing a new or recurrent stroke every year. Identifying modifiable risk factors for stroke should therefore be considered a research priority. While associations between ambient exposure to air pollution and other cardiovascular diseases are well established in the literature, the evidence linking particulate matter (PM) air pollution exposures to the risk of ischemic or hemorrhagic stroke remains equivocal. Furthermore, the exact pathophysiologic mechanisms by which exposure to PM may lead to cerebrovascular events are not yet fully understood. Hypothesized pathways include the mediation of effects through a combination of inflammatory responses, autonomic dysregulation, and/or vascular endothelial disturbances. This dissertation addresses existing gaps in the literature in three separate studies. Two time-stratified case-crossover studies examined the association between short-term PM exposures and stroke risk, one in the Health Professionals Follow-up Study (HPFS) and the other among a large database of Maryland stroke hospitalizations. Conditional logistic regression models were used to examine associations by stroke subtype, population subgroups, and clinically-relevant variables. Our third study took place within the Nurses’ Health Study cohort. Multivariable linear regression models were used to examine the associations between PM and residential distance to road exposures and four inflammatory biomarkers (CRP, IL-6, fibrinogen, and ICAM-1). We found positive significant associations between PM10 and ischemic stroke events in the HPFS cohort, and associations were elevated for nonsmokers, aspirin nonusers, and those without a history of high cholesterol. Concentrations were elevated for both CRP and IL-6 among participants who lived close to a major roadway, but no significant results were found by estimated PM exposure. This work provides additional evidence that PM exposure is associated with ischemic stroke and adds to the current literature that those not currently taking aspirin and those without a history of high cholesterol may be at elevated risk. Although the direct role of inflammatory processes requires more investigation, this work does provide additional evidence that proximity to traffic may influence cardiovascular-related inflammation.Item Investigating genetic and health factors related to AA amyloidosis prevalence in captive cheetahs (Acinonyx jubatus): implications for population management(2014) Franklin, Ashley Danielle; Porter, Tom E; Crosier, Adrienne E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Systemic amyloid A (AA) amyloidosis is an increasingly important cause of morbidity and mortality among captive cheetahs, yet wild cheetahs are virtually unaffected, suggesting the phenomenon is a result of the captive condition. The self-aggregating AA protein responsible for this disease, is a byproduct of serum amyloid A (SAA) protein degradation, an acute phase protein highly upregulated during inflammation. The objective of this study was to identify the relationship between genetics, stress, and inflammation with serum concentrations of the SAA protein and the incidence of AA amyloidosis in captive cheetahs. Fecal and serum samples collected from cheetahs held at the Smithsonian (NZP-SCBI) and Cheetah Conservation Fund (CCF) facilities, as well as wild, free-ranging cheetahs, were examined. Enzyme-linked immunosorbent assays were used to measure SAA protein and proinflammatory cytokine concentrations in serum samples and cortisol concentrations in feces. Additionally, cheetahs were genotyped for the SAA1A-97delG single nucleotide polymorphism (SNP) in the promoter region of the SAA1 gene. This study was the first to demonstrate that serum concentrations of the SAA protein in cheetahs are affected by the SAA1A-97delG SNP (P=0.0453). However, the high prevalence of AA amyloidosis observed among captive cheetahs is not attributable to genetic differences at this locus, but rather appears to be related to stress and/or inflammation, as captive cheetahs at NZP-SCBI have significantly higher SAA protein concentrations in serum compared to captive cheetahs at CCF, regardless of genotype (P=0.0003). Captive cheetahs at NZP-SCBI show levels of stress (fecal cortisol concentrations) greater than their captive counterparts at CCF in Namibia. Interestingly, wild cheetahs and captive cheetahs at CCF in Namibia had significantly higher proinflammatory cytokine concentrations (TNF-α and IL-1β) in serum compared to cheetahs at NZP-SCBI (P<0.0001). It is possible that chronic stress may be suppressing the production of proinflammatory cytokines in the NZP-SCBI cheetah population. Controlling the currently high SAA protein concentrations associated with AA amyloidosis is the best strategy to decreasing the diseases prevalence among captive cheetahs. Promoting management practices that reduce stress could help re-establish proper immune system homeostasis and mitigate the overproduction of SAA protein, decreasing the probability of developing AA amyloidosis.Item THE IDENTIFICATION AND CHARACTERIZATION OF AN INTRINSIC CD39/A2R-BASED REGULATORY MECHANISM THAT GOVERNS MACROPHAGE ACTIVATION RESPONSES(2014) Cohen, Heather Bloom; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are acutely sensitive to changes within their environment and can readily develop into a variety of activation states important for both the progression and resolution of inflammation. In response to immunological threats, macrophages must be able to effectively clear infections without sacrificing the integrity of the affected tissue. Thus, these cells must successfully balance their activation responses in order to preserve tissue function and the overall health of the host. The failure to properly regulate macrophage activation responses often manifests in the clinic in a variety of disease scenarios including sepsis, chronic inflammatory disorders, and cutaneous Leishmaniasis. While many factors that drive the initiation of macrophage activation are known, it remains unclear what governs the transition to an immunosuppressive state. This study reveals that macrophages can control their own activation status through the coordination between the ecto-ATPase, CD39, and the adenosine 2a and 2b receptors (A2Rs). The first part of this work shows that soon after toll-like receptor (TLR) stimulation, macrophages secrete and convert ATP into immunosuppressive adenosine via CD39. Moreover, we show that CD39 on macrophages is necessary to induce regulatory macrophage development and prevent severe immunopathology in a mouse model of septic shock. The next sets of data demonstrate that TLR activation also enhances A2bR expression, thus completing the CD39-initiated autoregulatory circuit to limit inflammatory macrophage responses. The second part of this work demonstrates that the chronic inflammatory disease-asociated cytokine, IFN-gamma, prevents TLR-induced A2bR expression and consequently promotes the hyper-production of inflammatory cytokines by macrophages thereby revealing a novel mechanism by which IFN-gamma; maintains overactive macrophages. The final chapter illustrates that while the A2bR is the dominant adenosine receptor mediating the inhibition of inflammatory cytokine production, A2aR signaling inhibits nitric oxide generation and that its expression may be hijacked by intracellular parasites to evade innate host defense mechanisms. Thus, this study demonstrates that inflammatory macrophage activation is inherently transient and that macrophages can reprogram themselves. These results culminate in the discovery of a novel immunomodulatory mechanism reliant on macrophage purinergic signaling and offer new targets and strategies to more effectively treat myriad inflammatory and infectious diseases.Item CD23 MEDIATED IGE TRANSCYTOSIS IN AIRWAY INFLAMMATION(2012) Palaniyandi, Senthilkumar; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)CD23 (FceRII), a C-type lectin type II membrane glycoprotein, plays an important role in IgE homeostasis and development of allergic inflammation. I showed that CD23 was constitutively expressed in the established or primary human airway epithelial cells and its expression was significantly up-regulated by IL-4 stimulation. In a transcytosis assay, human IgE or IgE derived immune complex was transported and enhanced by IL-4 stimulation across a polarized Calu-3 monolayer. A CD23 specific antibody or soluble CD23 significantly reduced the transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed antigen-IgE complexes were competent in inducing degranulation of the cultured human mast cells. This study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation. To verify the above results in a mouse model, CD23 expression was detected in epithelial cells lining mouse airway and enhanced by IL-4 exposure as well as in ovalbumin (OVA) sensitized mouse. I showed that CD23 transported IgE and OVA-IgE derived immune complex across airway epithelial cells in wild-type, but not CD23 knockout (KO), mice. The chimeric CD23KO mice repopulated with wild-type myeloid cells, sensitized and challenged with OVA showed significant reduction in siglec-F+ cells, eosinophils, macrophages and IL-4 in bronchoalveolar lavage fluid recovered 24 hours later compared to the wild-type mice. Our finding of CD23-mediated IgE transport in airway epithelial cells suggest a possibility of CD23 transporting an IgE Fc-fused protein for immunotherapy. CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) which competitively binds CD80 and CD86 expressed on antigen presenting cells and inhibits CD28 mediated co-stimulation of T cell activation. A CTLA4-Fc (IgE) fusion protein produced in Chinese hamster ovary cells was intranasally administrated into mouse airway for assessing its specific transport by CD23. The effect of this fusion protein on the development of allergic inflammation is being fully investigated in wild-type, CD23-KO, and chimeric mouse model.Item Of Mice and Math: A Systems Biology Model for Alzheimer's disease(2011) Kyrtsos, Christina Rose; Baras, John S; Lee, Hey-Kyoung; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder in the US, affecting over 1 in 8 people over the age of 65. There are several well-known pathological changes in the brains of AD patients, namely: the presence of diffuse beta amyloid plaques derived from the amyloid precursor protein (APP), hyper-phosphorylated tau protein, neuroinflammation and mitochondrial dysfunction. Recent studies have shown that cholesterol levels in both the plasma and the brain may play a role in disease pathogenesis, however, this exact role is not well understood. Additional proteins of interest have also been identified (ApoE, LRP-1, IL-1) as possible contributors to AD pathogenesis. To help understand these roles better, a systems biology mathematical model was developed. Basic principles from graph theory and control analysis were used to study the effect of altered cholesterol, ApoE, LRP and APP on the system as a whole. Negative feedback regulation and the rate of cholesterol transfer between astrocytes and neurons were identified as key modulators in the level of beta amyloid. Experiments were run concurrently to test whether decreasing plasma and brain cholesterol levels with simvastatin altered the expression levels of beta amyloid, ApoE, and LRP-1, to ascertain the edge directions in the network model and to better understand whether statin treatment served as a viable treatment option for AD patients. The work completed herein represents the first attempt to create a systems-level mathematical model to study AD that looks at intercellular interactions, as well as interactions between metabolic and inflammatory pathways.Item Defining Critical Parameters for Producing and Modulating Inflammation Caused by Cell Encapsulating Alginate Microspheres(2007-09-11) Breger, Joyce Catherine; Wang, Nam Sun; Lyle, Dan B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Minimizing induced inflammation, particularly nitric oxide (NO) production, is critical to optimal function or failure of implanted encapsulated cells. The purpose of this study is to define critical factors that affect toxic NO production from the macrophage cell line RAW264.7 in response to alginate microcapsules. The effects of the following were determined: 1) concentration of endotoxin (LPS) contamination; 2) presence of interferon-gamma (IFN-γ); 3) bead diameter and alginate volume; and 4) anti-inflammatory drugs in the alginate. A higher concentration (5 X) of LPS was required in alginate to produce the effect seen by LPS free in medium, sensitivity was enhanced by IFN-γ, bead diameter was inversely proportional to NO2 under low inflammatory conditions, and parthenolide in alginate significantly reduced inflammation. These results suggest that survival of implanted encapsulated cells may be improved by using highly purified alginate, avoiding ancillary inflammation, controlling surface area presentation, and incorporating anti-inflammatory drugs into the capsule matrix.