Theses and Dissertations from UMD

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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.

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Now showing 1 - 5 of 5
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    Spatiotemporal proteomic approaches for investigating patterning during embryonic development
    (2024) Pade, Leena Rajendra; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Characterization of molecular events as embryonic cells give rise to tissues and organs raises a potential to better understand normal development and design remedies for diseases. In this work, I integrated bioanalytical chemistry with neurodevelopmental biology to uncover mechanisms underlying tissue induction in a developing embryo. Specifically, I developed ultrasensitive proteomic approaches to study the remodeling of the proteome as embryonic cells differentiate in space and time to induce tissue formation. This dissertation discusses the design and development of proteomic strategies to deepen proteomic coverage from limited embryonic tissues. A novel sample preparation workflow and detection strategy was developed to address the challenge of interference from abundant proteins such as yolk in Xenopus tissues which in turn boosts the sensitivity of detecting low abundant proteins from complex limited amounts of tissues. The refined analytical workflow was implemented to study the development of critical signaling centers and stem cell populations and the tissues they induce to form in developing embryos.
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    Next-generation Mass Spectrometry With Multi-omics For Discoveries In Cell And Neurodevelopmental Biology
    (2022) Li, Jie; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Understanding tissue formation advances our understanding of the causes of disease and the obtained knowledge can be potentially applied to develop personalized interventions. However, to explore the underlying mechanisms that govern tissue formation, there is a high and unmet need to develop new technologies to characterize different types of biomolecules from early-stage embryonic precursor cells and their descendent cells during development. This dissertation discusses new technological advancements to facilitate multi-omic (proteomic and metabolomic) analysis to explore cell-to-cell differences and uncover mechanisms underlying tissue formation. The work presented herein illustrates the development of in vivo microsampling and single-cell mass spectrometry (MS) to uncover cell heterogeneity among embryonic cells. Additionally, this dissertation work studies the biological role of metabolites in cell fate determination by exploring the mechanisms underlying metabolite-induced cell fate change. Moreover, this work introduces a novel technique called MagCar developed to track and isolate tissue-specific cells at later stages, which enables studying temporal molecular changes to gain new information about tissue formation.
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    DEVELOPMENT OF SINGLE-CELL MASS SPECTROMETRY TOOLS TO INVESTIGATE METABOLIC REORGANIZATION DURING EARLY EMBRYOGENESIS
    (2020) Portero, Erika Paola; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Measurement of metabolism in single cells holds the potential to advance our understanding of fundamental biological processes during cell differentiation and development. However, to characterize the metabolic state of single cells, further technological advances are still required. This dissertation discusses the development and application of single-cell mass spectrometry (MS) technologies to investigate metabolism and its role during tissue induction in the early developing vertebrate (frog) embryo. The work presented herein illustrates the strategies devised to advance single-cell analysis using capillary electrophoresis (CE)-MS. Additionally, this work features several contributions to our understanding of cell heterogeneity and the role of small molecules during tissue specification in the vertebrate embryo, providing new information to advance cell and developmental biology.Chapter 1 overviews the current state of metabolomics for cell and developmental biology, as well as the research significance and motivations. Chapter 2 describes the fundamental concepts of CE and the current state of single-cell metabolomics by CE-MS. This chapter also discusses the development of a minimally invasive microprobe sampling technique designed for the Xenopus laevis embryo. Chapter 3 presents the development of a CE-MS approach that enables dual cationic and anionic analysis of metabolites from the same single embryonic cell to deepen the detectable coverage of metabolism. Chapter 4 discusses a stable-isotope labeling strategy and single-cell CE-MS to uncover metabolic pathways involved in cell differentiation. Chapter 5 details the application of our custom-built microprobe sampling technique to investigate spatial cell heterogeneity in the same vertebrate embryo. This chapter examines cell-to-cell communication and small molecule transport between adjacent cells. Moreover, dual-fluorescent cell lineage tracing reveals cell fate changes induced by small molecule transport. Chapter 6 summarizes the results generated from this dissertation work and reflects on technical challenges and potential advancements needed to drive the field of MS-based single-cell metabolomics forward.
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    Development of Magnetic Nanoparticle-based Enrichment Techniques and Mass Spectrometry Methods for Quantification of the Clinical Biomarker Cardiac Troponin I
    (2016) Schneck, Nicole; Lee, Sang Bok; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Human cardiac troponin I (cTnI) in serum is a well-known clinical biomarker for cardiac tissue damage and is used for diagnosing myocardial infarction. Unfortunately, commercial cTnI immunoassays from different manufacturers can produce significantly different measurement results for the same sample. In order to improve the comparability of these measurements, clinical cTnI immunoassays need to be standardized. Ultimately, the goal of this work was to develop an isotope dilution-liquid chromatography-mass spectrometry (ID-LC-MS/MS) reference measurement procedure that could be used to assign concentration values to cTnI reference materials. However, given that most serum protein biomarkers are low abundant, enrichment was mandatory to successfully quantify cTnI at clinically significant concentrations (≈ 1-10 ng/mL). As such, the specific aim of this work was to develop enrichment techniques and an ID-LC-MS/MS method to quantify cTnI in patient serum samples. In order to achieved the required LC-MS/MS sensitivity, novel enrichment strategies were investigated to selectively isolate cTnI from serum and plasma. Silica coated magnetic nanoparticles were synthesized and conjugated with antibodies to act as immunoaffinity carriers. Magnetic nanoparticles were selected due to their variable surface modifications, high binding capacity, and the fact that they can be easily isolated using a magnet. After optimizing the enrichment and digestion procedures, isotopically labeled cTnI proteins were used as an internal standard for ID-LC-MS/MS analysis of cTnI to compensate for variations in the sample preparation. Finally, the developed LC-MS/MS-based assay was applied to measure cTnI concentrations in patient plasma samples. Effective enrichment methods proved to be crucial for achieving quantification of cTnI by ID-LC-MS/MS. To this end, a complementary ID-LC-MS/MS method was also developed to evaluate different antibody immobilization strategies and magnetic particle types as part of the method optimization. Overall, this work demonstrates significant improvements in magnetic particle enrichment techniques and LC-MS/MS detection for the analysis of cTnI in patient samples.
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    Alterations in the Primary Structures of Ribosomal Proteins in Acquired Drug Resistance
    (2012) Lohnes, Karen Lynn; Fenselau, Catherine C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Acquired drug resistance is a multifactorial process that is one of the major causes for cancer treatment failure. The anticancer drug, mitoxantrone, was recently determined to inhibit ribosome biogenesis. Changes in ribosomal protein composition and efficiency with which the ribosomes incorporate 35S-methionine has been noted in a mitoxantrone resistant MCF7 cell line when compared with a drug-susceptible parental cell line. This dissertation evaluated three proteomic workflows in order to successfully characterize the changes in the primary structures of cytoplasmic ribosomal proteins isolated from a mitoxantrone resistant breast cancer cell line that could serve some functional significance to the resistance when compared with a parental drug-susceptible cell line. A combination of the data from the three workflows allowed for the identification of 76 of the 79 human ribosomal proteins with an average sequence coverage of 76%. The N-terminal ends of 52 of the ribosomal proteins were identified using bottom-up and middle-down mass spectrometric approaches. An additional 7 N-terminal fragments were identified by top-down high resolution mass spectrometric analysis. Forty of the 52 N-terminal peptides were observed to have lost their N-terminal methionine and 19 were acetylated. Identification of the N-terminal peptides was most successful using the middle-down approach. Internal acetylations (on lysine) and phosphorylations were only noted with trypsin in-gel digestion and HPLC fraction analysis. Gel arrays of the two ribosomal populations illustrated differences in the protein compositions. Comparative densitometry imaging software indicated the presence of two novel protein spots in the drug resistant cell line as well six additional spots with increased and decreased abundances. High coverage bottom-up mass spectrometric analysis allowed for these protein spots to be assigned as isoform pairs of RPS3, RPS10, RPL11 and RPL23A. Molecular masses and top-down analyses were used to define the alterations in the ribosomal proteins in conjunction with high coverage bottom up and middle-down analyses. The change in the primary structures of these four ribosomal proteins is believed to alter access to the mRNA tunnel in the ribosome. This suggests that these ribosomes may participate in differential selective translation to allow for the cell to produce the necessary proteins during cellular stress.