Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
14 results
Search Results
Item PREPARATION OF A NANOSUSPENSION OF THE PHOTOSENSITIZER VERTEPORFIN FOR PHOTODYNAMIC AND LIGHT-INDEPENDENT THERAPY IN GLIOBLASTOMA(2024) Quinlan, John Andrew; Huang, Huang-Chiao; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photodynamic therapy (PDT) using verteporfin (VP) has treated ocular disease for over 20 years, but recent interest in VP’s light-independent properties has reignited interest in the drug, particularly in glioblastoma (GBM) (NCT04590664). Separate efforts to apply PDT to GBM using 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) have also garnered attention (NCT03048240), but, unfortunately, clinical trials using 5-ALA-induced PpIX-PDT have yet to yield a survival benefit. Previous studies have shown VP to be a superior PDT agent than 5-ALA-induced PpIX. Our lab has shown that 690 nm light activates VP up to 2 cm into the brain, while 635 nm light only activates PpIX at depths <1 cm into the brain. Additionally, VP is a more effective photosensitizer than PpIX because it has a higher singlet oxygen yield and is active in the vasculature as well as target tumor cells. However, the hydrophobicity of VP limits effective delivery of the drug to the brain for treatment of GBM.In this context, this thesis aims to re-evaluate the delivery method for VP. VP traditionally requires lipids for delivery as Visudyne. Recent shortages of Visudyne and potential drawbacks of liposomal carriers motivated our development of a carrier-free nanosuspension of VP, termed NanoVP. Previous work has shown that cellular uptake of VP is greater when delivered as NanoVP rather than liposomal VP, resulting in improved cell killing after light activation. This thesis builds on this previous work by (1) evaluating synthesis and storage parameters for NanoVP, (2) determining the pharmacokinetics, biodistribution, and brain bioavailability of NanoVP, and (3) evaluating the potential efficacy of NanoVP as a PDT and a chemotherapy agent, and by supporting development of a zebrafish model of the blood-brain barrier (BBB) for mechanistic studies of improved drug delivery to the brain.Item The effects of mechanical confinement on cancer cell growth and migration mechanisms(2021) Moriarty, Rebecca; Stroka, Kimberly M; Mili, Stavroula; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cancer is a disease in which cell growth proceeds unchecked and cells accumulate mutations to adopt an invasive and migratory phenotype that promotes metastasis. Once a cancer becomes metastatic, survival rates plummet, and vast tumor heterogony and lesion formation leave current therapeutics and treatments unable to maintain pace. Therefore, there exists a clinical need to gain a more complex understanding of factors that promote and encourage metastasis. Cancer cells are subjected to mechanical forces in vivo that influence their behavior. Mechanical cues are transmitted through the cell from the membrane to the cytoskeleton, and ultimately to the nucleus where gene expression and subsequently protein output can be altered. In this dissertation, we probed the effects of mechanical confinement, a restrictive force present at various stages during the metastatic cascade, on (1) cancer cell growth and cell cycle progression, (2) global mRNA translational and its relationship to cell migration, and (3) mRNA localization mechanisms for use in confined cell migration. We modeled confinement in vitro through fabrication of microfluidic microchannel devices. We show here that mechanical confinement halts sarcoma cell cycle progression and division and leads to an increase in abnormal divisions. We explored the connection between mRNA translation and cell migration and found that global mRNA translation is spatially altered in confinement and that it is necessary for confined cell migration. We explored this idea further, investigating a subset of mRNAs that are known to influence cell migration in unconfined spaces and show that their regulation in confinement is cell type dependent, but that it primarily relies on cell mechanoactivity. Together, this work contributes a detailed understanding of key cell behaviors that are altered in confined environments and emphasizes the importance of studying mechanical cues that cells experience in vivo, in the context of understanding and treating cancer progression and metastasis.Item Mechanical Adaptability of Ovarian Cancer Tumor Spheroids(2021) Conrad, Christina Barber; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A major obstacle in ovarian cancer treatment is the onset of ascites, an abnormal build-up of fluid in the peritoneal cavity. Using in vitro perfusion models, ascitic flow has been shown to drive epithelial-mesenchymal-transition (EMT) biomarker expression, promote epidermal growth factor receptor (EGFR) downstream signaling, and upregulate chemoresistance. Given the close ties between cell mechanics and behaviors, it is of interest to establish if mechanotransduction serves a role in cell signaling dysfunction. Here, we identified the mechanical behavior of tumor spheroids subjected to flow using Brillouin confocal microscopy, a non-contact optical method based on the interaction between incident light and microscopic mechanical waves within matter. We validated this technique by establishing a relationship with the traditionally derived Young’s modulus measured using atomic force microscopy and a parallel-plate compression device. Following characterization, we used Brillouin confocal microscopy to map mechanical properties of tumor spheroids embedded in a microfluidic chip and found that continuous flow for 7 days caused a decreased Brillouin shift (i.e., stiffness) compared to tumors in a static condition. Another physical phenomenon related to ascites is dysregulated osmolality. Maintaining cell water homeostasis is driven by the transport of water to balance solute concentration and can have direct consequences on mechanics and biochemical signaling in cells. Recently, it was demonstrated in single cells that cell volume correlated with mechanical properties; but the effects in tumor spheroids which exhibit multi-cellular interfaces has remained unclear. Here, we derived relationships between osmolality and nuclear volume, tumor cell density, and Young’s modulus, and found the correlations in spheroids resembled single cell relationships previously described in literature. Additionally, we looked at the impact of osmotic shocks on E-cadherin junctions and found aggregates formed with a unique timescale compared to morphology. Lastly, we observed reversibility of the mechanical, morphological, and molecular properties which showed the tumor’s dynamic ability to respond to physical cues. Altogether, this work demonstrated how flow and osmosis associated with ovarian cancer ascites can trigger phenotype transformations. These findings warrant future investigations into how the regulation of mechanotransduction pathways can be harnessed to prevent chemoresistance and signaling dysfunction.Item Bio-Derived Microscale Containers for Disease Treatment and Diagnostics(2017) Liu, James; Raghavan, Srinivasa R; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Micro-erythrosomes (mERs) are microscale containers (3 to 5 µm in diameter) derived from red blood cells (RBCs, also called erythrocytes). They are prepared by removing hemoglobin from RBCs and resuspending the empty structures in buffer. In this work, we focus on adding new functionalities to mERs, with both therapeutics and diagnostics in mind. In our main study, we demonstrate the use of mERs as “Killer Cells” to attack cancer. mERs are loaded with the enzyme glucose oxidase (GOx) and then incubated in vitro with a strain of head and neck cancer cells (15B). In the presence of glucose from external media, the Killer Cells generate hydrogen peroxide (H2O2). H2O2 is a reactive oxygen species (ROS) which induces the cancer cells to undergo apoptosis (programmed cell death). We find a reduction in 15B cell viability of over 80%. In ancillary studies, we explore strategies for the long-term retention of solutes in mERS. Specifically, the cationic biopolymer chitosan is adsorbed to the surfaces of mERs, and the anionic biopolymer alginate is encapsulated in their cores. Both strategies are able to extend the diffusion time for loaded solutes. Additionally, we have attempted to adapt mERs for use as MRI contrast agents by incorporating lipids containing gadolinium into the membrane. These studies lay the foundation for many mER applications and demonstrate their versatility.Item COMPUTATIONAL METHODS IN MISSENSE MUTATION ANALYSIS: PHENOTYPES, PATHOGENICITY, AND PROTEIN ENGINEERING(2017) Yin, Yizhou; Moult, John; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding the molecular, phenotypic, and pathogenic effects of mutations is of enormous importance in human disease research and protein engineering. Both create a demand for computational methods to leverage the explosion of new sequence data and to explore the vast space of possible protein modifications and designs. My study in this dissertation demonstrates the value of computational methods in these areas. First, I developed a new ensemble method to predict continuous phenotype values as well as binary pathogenicity and objectively tested it in CAGI (Critical Assessment of Genome Interpretation). In two recent CAGI challenges, the method was ranked third in predicting the enzyme activity of missense mutations in NAGLU (N-Acetyl-Alpha-Glucosaminidase) and second in predicting the relative growth rate of mutated human SUMO-ligase in a yeast complementation assay. I also demonstrated the effectiveness of the new ensemble method for addressing a key problem limiting the use of current mutation interpretation methods in the clinic – identifying which mutations can be assigned a pathogenic or benign status with high confidence. Next, I characterized and compared missense variants in monogenic disease and in cancer. The study revealed a number of properties of mutations in these two types of diseases, including: (a) methods based on sequence conservation properties are as effective for identifying cancer driver mutations as they are for monogenic disease mutations; (b) mutations in disordered regions of protein structure play a relatively small role in both classes of disease; (c) oncogenic mutations tend to be on the protein surface while tumor suppressors are concentrated in the core; (d) a large fraction of tumor suppressors act by destabilizing protein structure and (e) mutations in passenger genes display a surprisingly high level of deleteriousness. Finally, I applied computational methods to screen for appropriate mutations to enhance the thermostability of a catalytic domain of PlyC. This bacteriophage-derived endolysin has been demonstrated to have antimicrobial potential but its potential use is limited by its inherent thermosuseptibility. To prioritize stabilizing mutations, I conducted a rapid exhaustive survey of point mutations followed by validation using protein modeling and expert knowledge. The approach yielded three stabilizing mutants experimentally verified by our collaborators, with one particularly successful in terms of both thermal denaturation temperature and kinetic stability.Item Biodegradable Prussian blue nanoparticles for photothermal immunotherapy of advanced cancers(2015) Cano-Mejia, Juliana; Fernandes, Rohan; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Multifunctional nanoparticles represent a class of materials with diverse therapy and imaging properties that can be exploited for the treatment of cancers that have significantly progressed or advanced, which are associated with a poor patient prognosis. Here, we describe the use of biodegradable Prussian blue nanoparticles (PBNPs) in combination with anti-CTLA-4 checkpoint blockade immunotherapy for the treatment of advanced cancers. Our nanoparticle synthesis scheme yields PBNPs that possess pH-dependent intratumoral stability and photothermal therapy (PTT) properties, and degrade under mildly alkaline conditions mimicking the blood and lymph. Studies using PBNPs for PTT in a mouse model of neuroblastoma, a hard-to-treat cancer, demonstrate that PTT causes rapid reduction of tumor burden and growth rates, but results in incomplete responses to therapy and tumor relapse. Studies to elucidate the underlying immunological responses demonstrate that PTT causes increased tumor infiltration of lymphocytes and T cells and a systemic activation of T cells against re-exposed tumor cells in a subset of treated mice. PBNP-based PTT in combination with anti-CTLA-4 immunotherapy results in complete tumor regression and long-term survival in 55.5% of neuroblastoma tumor-bearing mice compared to only 12.5% survival in mice treated with anti-CTLA-4 alone and 0% survival both in mice treated with PTT alone, or remaining untreated. Further, all of the combination therapy-treated mice exhibit protection against tumor rechallenge indicating the development of antitumor immunity as a consequence of therapy. Our studies indicate the immense potential of our combination photothermal immunotherapy in improving the prognosis and outlook for patients with advanced cancers.Item Folate intake and biomarkers and risk of chronic disease(2014) Hu, Jing; Sahyoun, Nadine R; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Background: Folate status of the U.S. population significantly improved after folic acid fortification of enriched cereal-grain products in 1998. Recent evidence suggests that the increased folate levels may have impacts on the risk of chronic disease. The kidneys are known to be highly involved in folate metabolism. Reduced renal function may affect folate metabolism and play a role in the associations between folate and chronic disease. Objectives: The purpose of this study was to review key events regulating folate homeostasis along folate metabolic pathway. In addition, we examined the associations between folate intake and biomarker levels and the incidence of cancer, stroke and cardiovascular disease (CVD) and between folate biomarker levels and renal function among older adults in post-fortification years. Design: The Key Events Dose-Response Framework was used to review key steps of folate metabolism. Data of adult participants of the National Health and Nutrition Examination Survey 1999-2002 were used as the baseline data. Incidence of cancer, stroke and CVD were obtained from the linked Medicare and mortality files. The associations between folate intake and biomarker levels and incidence of cancer, stroke and CVD, and the associations between estimated glomerular filtration rate (eGFR) and folate biomarkers, serum unmetabolized folic acid (UMFA) and plasma homocysteine levels were assessed using Cox proportional hazards regression models and multivariable regression models, respectively. Results: The saturation of dihydrofolate reductase in the liver is the determining point regulating the release of UMFA in circulation. Lower red blood cell (RBC) folate levels and intake of dietary folate equivalents were associated with a higher cancer incidence. Lower RBC folate and serum folate levels were associated with a higher stroke incidence. No significant associations between folate and CVD were observed. In addition, reduced renal function was associated with higher RBC folate and plasma homocysteine levels among men and women, and higher prevalence of UMFA in blood among women. Conclusion: High intake of folate may disturb folate metabolism by overwhelming folate regulation mechanisms. Folate may play a protective role against cancer and stroke even at high levels in post-fortification years. Reduced renal function may be implicated in the increased blood folate concentrations.Item RACIAL AND ETHNIC DIFFERENCES IN ACCESSING TIMELY CANCER SCREENING AND TREATMENT SERVICES: A QUANTITATIVE ANALYSIS(2013) King, Christopher Jerome; Thomas, Stephen B.; Chen, Jie; Health Services Administration; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research is organized into three integrated studies that explored differences in screening and treatment services across the cancer care continuum by race and ethnicity. The Andersen Behavioral Model of Health Services Use and the Five Dimensions of Access were used as conceptual frameworks. In the first study (Chapter 2), data from the Medical Expenditure Panel Survey were used to examine breast and cervical cancer screening rates before and during the Great Recession (2007-2009). The interaction terms of recession and race and ethnicity were controlled to examine whether minorities exhibited different utilization patterns under economic shock compared to Whites. In Chapter 3, data from the National Health Interview Survey (NHIS) from 2006-2010 were used to identify adult cancer survivors and adults without a history of cancer. Multivariate logistic regressions were applied to examine the prevalence of cost, organizational and transportation barriers between survivors and the general population. The likelihood of experiencing barriers was explored by race and ethnicity. In Chapter 4, differences in the likelihood of experiencing access barriers among survivors by race and ethnicity was explored. Data were merged from the 2000-2011 (NHIS) to identify adult cancer survivors who reported cost, organizational and transportation barriers. Logistic regressions were applied to determine the likelihood of reporting each type of barrier, while controlling for demographic and socioeconomic variables. The Fairlie decomposition technique was applied to identify contributing factors that explained differences in accessing care based by race and ethnicity. Overall, results of the investigations demonstrate that: (1) breast and cervical screening rates declined most among White women during the recession period, while rates increased among Hispanic women during the same period; (2) minority cancer survivors were significantly more likely to experience access-to-care barriers than Whites; and (3) insurance, comorbidity, perceived health and nativity were leading factors that contributed to racial and ethnic differences in timely receipt of cancer screening and treatment services. As provisions of the Affordable Care Act take effect, findings provide insight into practices, policies, and future research that will help achieve Healthy People 2020 screening objectives and reduce racial and ethnic disparities in accessing timely cancer care.Item CHARACTERIZATION OF SINGLE RESIDUE VARIATIONS IN THE HUMAN POPULATION AND IN DISEASE: FUNCTIONAL IMPACT, STRUCTURAL IMPACT, AND DISTRIBUTION PATTERN(2011) Shi, Zhen; Moult, John; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We have investigated the properties of three sets of human missense genetic variations: cancer somatic mutations, monogenic disease causing mutations, and population SNPs, from the point of view of their impact on molecular function, distribution propensity in different protein structure environments, and disease mechanism. Cancer genome sequencing projects have identified a large number of somatic missense mutations in cancers. We have used two analysis methods in the SNPs3D software package to assess the impact of these variants on protein function in vivo. One method identifies those mutations that significantly destabilize three dimensional protein structure, and the other detects all types of effect on protein function, utilizing sequence conservation. Data from a set of breast and colorectal tumors were analyzed. In known cancer genes, approaching 100% of missense mutations are found to impact protein function, supporting the view that these methods are appropriate for identifying driver mutations. Overall, we estimate that 50% to 60% of all somatic missense mutations have a high impact on structure stability or more generally affect the function of the corresponding proteins. This fraction is similar to the fraction of all possible missense mutations that have high impact, and much higher than the corresponding one for human population SNPs, at about 30%. We found that the majority of mutations in tumor suppressors destabilize protein structure, while mutations in oncogenes operate in more varied ways, including destabilization of the less active conformational states. A set of possible drivers with high impact is suggested. We also studied a set of germline missense variants in phenylalanine hydroxylase, found in phenylketonuria (PKU) patients. With the aid of SNPs3D, we reinforced the previous finding that a high proportion of disease missense mutations affect protein stability, rather than other aspects of protein structure and function. We then focused on the relationship between the presence of these stability damaging missense mutations and the corresponding experimental data for the level and activity of the PAH protein product present under `in vivo' like conditions. We found that, overall, destabilizing mutations result in substantially lower protein levels, but with the maintenance of wild type like specific activity. The overall agreement between predicted stability impact and experimental evidence for lower protein levels is high, and in accordance with the previous estimates of error rates for the methods. We next investigated the involvement of missense single base variants in the interface between two interacting proteins and their role in disease. This work consisted of three steps: first, mapping of variants onto the protein structure and identification of those in the interaction interfaces; second, distribution enrichment analysis in three structure locations (protein interior, surface, and interface); and third, impact analysis with SNPs3D. Nearly a quarter of disease causing mutations are mapped onto protein interfaces, with a strong propensity for the heteromeric interfaces, indicating that interruption of functional contacts between proteins is a significant disease mechanism. We found the enrichment propensity in the interfaces is intermediate between protein surface and interior for all three types of variants considered, namely SNPs, inter-species variants, and disease mutations. We also found missense SNPs and inter-species variants share the same enrichment pattern, with a relatively high density on the protein surface and depletion in the interior. In contrast, the disease mutations display the reverse pattern, with interior and interface the most susceptible places.Item Mathematical modeling of drug resistance and cancer stem cells dynamics(2010) Tomasetti, Cristian; Levy, Doron; Dolgopyat, Dmitry; Applied Mathematics and Scientific Computation; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation we consider the dynamics of drug resistance in cancer and the related issue of the dynamics of cancer stem cells. Our focus is only on resistance which is caused by random genetic point mutations. A very simple system of ordinary differential equations allows us to obtain results that are comparable to those found in the literature with one important difference. We show that the amount of resistance that is generated before the beginning of the treatment, and which is present at some given time afterward, always depends on the turnover rate, no matter how many drugs are used. Previous work in the literature indicated no dependence on the turnover rate in the single drug case while a strong dependence in the multi-drug case. We develop a new methodology in order to derive an estimate of the probability of developing resistance to drugs by the time a tumor is diagnosed and the expected number of drug-resistant cells found at detection if resistance is present at detection. Our modeling methodology may be seen as more general than previous approaches, in the sense that at least for the wild-type population we make assumptions only on their averaged behavior (no Markov property for example). Importantly, the heterogeneity of the cancer population is taken into account. Moreover, in the case of chronic myeloid leukemia (CML), which is a cancer of the white blood cells, we are able to infer the preferred mode of division of the hematopoietic cancer stem cells, predicting a large shift from asymmetric division to symmetric renewal. We extend our results by relaxing the assumption on the average growth of the tumor, thus going beyond the standard exponential case, and showing that our results may be a good approximation also for much more general forms of tumor growth models. Finally, after reviewing the basic modeling assumptions and main results found in the mathematical modeling literature on chronic myeloid leukemia (CML), we formulate a new hypothesis on the effects that the drug Imatinib has on leukemic stem cells. Based on this hypothesis, we obtain new insights on the dynamics of the development of drug resistance in CML.