Theses and Dissertations from UMD
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Item EXAMINING QUINONE CONTRIBUTION TO THE OPTICAL PROPERTIES OF CHROMOPHORIC DISSOLVED ORGANIC MATTER(2024) Ashmore, Rachel; Blough, Neil; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chromophoric Dissolved Organic Matter (CDOM) in natural waters is largely responsiblefor absorption of light and photochemistry in the water, impacting environmental reactions and aquatic life. The composition of CDOM is greatly varied based on source, photochemical reactions, and natural cycles. The impact of quinone moieties on this structure and photochemical and redox reactions involving CDOM remains the subject of controversy. To investigate the impact of quinone structure on optical properties, model quinone compounds were thoroughly characterized by their optical properties and reactions with sodium dithionite and sodium sulfite. A series of methyl-substituted p-benzoquinones, a methoxy p-benzoquinone, and a range of napthoquinones and anthraquinones were investigated. These model compounds were characterized according to their quinone and hydroquinone molar absorptivities and fluorescence quantum yields. Sodium dithionite reduction of quinones and the impact of structure on the products of this reaction was investigated by reducing the quinones with both sodium dithionite and sodium sulfite and comparing the optical properties of the products to those of the quinone and hydroquinone. The spectra of dithionite reduced p-benzoquinones and napthoquinones suggested the presence of products other than the hydroquinone. Sulfite is produced in solution as a result of dithionite reduction of quinones. Model quinones were therefore also reduced with sodium sulfite to investigate the impact of this side reaction on the dithionite reduction products. High performance liquid chromatography (HPLC) was used to further investigate and quantify the products of dithionite reduction of quinones and the importance of sulfite interference. Although some of the model quinones react with sulfite to form a proposed sulfonated hydroquinone product, based on the observed extent of this reaction in dithionite reductions, the structures of quinones likely to be found in CDOM, and their relatively small contribution to CDOM optical properties, the sulfite reaction was determined to not significantly impact the study of quinone moieties in CDOM. Dithionite selectively reduces quinones, while borohydride reduces ketones, aldehydes, and quinones. Therefore, in CDOM samples, dithionite can be used to isolate the effects of quinone moieties on the optical properties. Dithionite reduction was used to analyse CDOM standards and natural water extracts from the North Pacific Ocean and the Chesapeake Bay to investigate quinone contribution to their optical properties. These results are compared to borohydride reduction results from Cartisano and McDonnell to compare the contribution of quinones to that of ketones and aldehydes. (1, 2) Dithionite reduction showed small impacts on absorbance and fluorescence, whereas significant changes in both were observed for borohydride reduction. Therefore, the optical changes observed under borohydride reduction are attributed to primarily ketones and aldehydes rather than quinones. Model quinones showed significant changes in fluorescence intensity due to dithionite reduction, which are largely not observed for CDOM standards and natural water extracts, further supporting the conclusion that their role in CDOM optical properties is small.Item 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.Item New method for kinetic isotope effect measurements(2023) Kljaic, Teodora; Poulin, Myles B; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Kinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precision. In this thesis I described the development of a novel competitive KIE measurement method using MALDI-TOF-MS and the investigation of the transition state of glycosyltransferase enzyme BshA from B. subtilis. We developed a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). This approach enabled quantitative measurements of both relative isotope abundance of an analyte and fractional conversion F in single measurements without the need for purification prior to analysis. The application of this MALDI-TOF MS approach has demonstrated the precision of KIE measurements comparable to those obtained using competitive radioisotope labelling, and NMR based approaches while requiring smaller amounts of stable isotope labelled substrates. Using two chemoenzymatic approaches, we then synthesized 5 substrates for the application of our method to investigate the transition state of BshA: UDP-GlcNAc (3.1), [1''-13C]UDP-GlcNAc (3.2), [2''-13C]UDP-GlcNAc (3.3), [13C6]UDP-GlcNAc (3.4) and [2''-2H]UDP-GlcNAc (3.5). Finally, we have begun to work on the synthesis of [1''-18O]UDP-GlcNAc and describe an approach to prepare this substrate that is currently underway in the lab. Application of the quantitative whole molecule MALDI-TOF MS approach enabled us to determine multiple competitive KIEs for the enzymatic reaction catalyzed by BshA. While previous studies suggested a front-face SNi (DNAN) TS for the conjugation of UDP-GlcNAc and L-malate, our KIE results show that a stepwise mechanism resulting in the formation of a discrete, though likely short lived, oxocarbenium ion intermediate is more likely. Our method be applied to study other glycosyltransferases whose mechanisms still remain to be elucidated and to design TS based inhibitors for enzymes involved in different bacterial infections. Future work on automation of this method would simplify the KIE measurement process and increase reproducibility making the measurement of KIEs for TS analysis a more experimentally accessible technique for the broader enzymology research community.Item 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.Item Enhanced Throughput Single-Cell Capillary Electrophoresis Mass Spectrometry(2023) Mendis, John Udara; Nemes, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mass spectrometry (MS) has allowed for the analysis of small molecules and metabolites with high specificity and sensitivity. Capillary Electrophoresis mass spectrometry (CE-MS) is an ultrasensitive analytical technique to process amount-limited samples. Robust high-throughput ultrasensitive CE-MS methods and technologies are needed to be developed to comprehensively study the metabolome or proteome of a sample with a limited amount of material. In this study, we developed an enhanced-throughput multi field amplified sample stacking (M-FASS) method. The resulting approach has a sample processing throughput of 5–10 times that of conventional CE methods. FASS voltage duration and strength were optimized for peak area and peak resolution. The M-FASS CE-MS method was then applied for single cell analysis (SCA) of metabolic differences and gradients in the developing embryo of Xenopus Laevis. The statistical analysis: PCA and Fuzzy-c means clustering analysis revealed cell-to-cell differences among D11, V11, D12, and V12 cells and uncovered 6 distinct metabolite gradients between the four cells in X. Laevis 16-cell embryos. The findings showcase inherent metabolic gradients in the developing embryo.Item INVESTIGATION OF THE PRODUCTION AND DECAY PATHWAYS OF SUPEROXIDE BY CHROMOPHORIC DISSOLVED ORGANIC MATTER(2022) Le Roux, Danielle Marie; Blough, Neil; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chromophoric dissolved organic matter (CDOM) in natural waters absorbs sunlight which leads to the production of a suite of reactive intermediates and reactive oxygen species (ROS) such as superoxide (O2⦁-) and hydrogen peroxide (H2O2). A significant amount of research over the years has investigated the sources and sinks of these two ROS. The currently accepted sequence of reactions for their production involves photochemically produced one-electron reductants (OER) within CDOM reacting with dissolved oxygen to form O2⦁-, which undergoes self-dismutation to produce H2O2. A previously used method to detect radical species with CDOM has been modified herein to be conducted simply using a fluorometer. Production rates of OER and H2O2 were measured for a variety of samples and correlations between the rates and optical/structural properties of the samples indicate that lower molecular weight species produce more OER and H2O2. Based on the stoichiometry of the mechanism above, the ratio of the production rate of OER to that of H2O2 should be two. However, ratios from five to sixteen were obtained, which suggests that O2⦁- undergoes oxidative reactions that compete with dismutation. The possibility of a light-dependent pathway for O2⦁- decay has been proposed but had yet to be explicitly demonstrated. Herein this sink is directly shown through O2⦁- spiking experiments. Rapid consumption of the O2⦁- spike occurs if injected into a sample during irradiation, as compared to a spike introduced into the sample in the dark, suggesting the presence of a light-dependent sink. Extensive data analysis and kinetic modeling of the O2⦁- decay data has allowed for approximations as to the extent of the sink and its decay rate constant. O2⦁- and H2O2 are environmentally important species, and a significant amount of work has been done on modeling their concentrations in natural waters. Based on the work here, O2⦁- is produced at higher concentrations than previously believed, which has implications on the modeling of O2⦁- and H2O2 in natural waters. Additionally, the light-dependent oxidative sink of O2⦁- could be with moieties within CDOM, providing further insight to the photochemical transformation of DOM during transit from terrestrial sources to marine waters.Item UNDERSTANDING THE SURFACE CHEMISTRY OF GAS PHASE ORGANOPHOSPHORUS CHEMICAL WARFARE AGENTS WITH SORBENT MATERIALS(2019) Holdren, Scott; Zachariah, Michael R.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chemical warfare agents (CWAs) pose a serious threat to civilians and warfighters due to their high toxicity and usage in recent attacks. Although existing filtration media (ASZM-TEDA) used in gas mask devices absorbs and decomposes a wide spectrum of CWAs, the filtration performance of this material can be compromised in the battlefield due to poorly understood mechanisms. The high toxicity of CWAs remains a barrier for most research institutions to study these compounds experimentally which hinders the search for improved filtration materials. To overcome this issue, studies are performed using relatively benign simulant compounds that have similar adsorption and decomposition properties as toxic CWAs. In this work, a report of experimental findings will be presented regarding how dimethyl methylphosphonate (DMMP), an organophosphorus CWA simulant, will adsorb and decompose on components that makeup ASZM-TEDA. The work presented in this dissertation deconstructs the components that makeup ASZM-TEDA in order to identify the role of specific metal oxides and the carbon support. This approach was facilitated using different analytical techniques including TGA, FTIR spectroscopy, and DFT modeling to gain a molecular understanding of how DMMP interacts with porous carbon (Chapter 3) and metal oxide nanoparticles/surfaces (Chapters 4 and 5). Lastly, a new method is described (Chapter 6) that overcomes many of the difficulties encountered in conventional measurements that monitor gas phase DMMP adsorption/desorption processes on sorbent materials. This method can be used to obtain reliable quantitative measurements and parameters (e.g. adsorption capacities, ∆Hads, and kads) of low vapor pressure adsorbate/sorbent systems making it particularly useful for CWAs/CWA simulants and new filtration materials (e.g. DMMP and porous carbon).Item CHROMOPHORIC DISSOLVED ORGANIC MATTER (CDOM) IN THE OPEN OCEAN: OPTICAL AND CHEMICAL PROPERTIES AND THEIR RELATION TO CDOM STRUCTURE AND SOURCES.(2019) Cartisano, Carmen Marie; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The carbon contained as dissolved organic matter (DOM) in the Earth’s oceans is an important factor in the global carbon cycle, but studying and tracking DOM in the aquatic environment can be challenging. However, the light-absorbing and emitting subcomponents of DOM, called chromophoric dissolved organic matter (CDOM) and fluorescent dissolved organic matter (FDOM) can be directly probed using absorption and fluorescence spectroscopy, respectively. Detailed studies on CDOM from the open oceans are limited with many of the existing studies having very limited data sets (only select wavelengths or indices). To address this, the optical properties of CDOM from a variety of geographic locations (North Pacific Ocean: NPO, Equatorial Atlantic Ocean: EAO, Middle Atlantic Bight: MAB, Delaware River and Delaware Bay) were compared, and chemical tests performed (sodium borohydride (NaBH4) reductions and pH titrations). The responses to the chemical tests along with similarities and differences in the optical properties were examined to compare the structures present in terrestrial, coastal and open ocean samples. A long-pathlength capillary waveguide spectrometer was used to characterize open ocean CDOM samples, with the need for a calibration and validated protocol addressed prior to use. The optical properties of the NPO samples did not vary significantly at depths from ~300-4500 meters with only the surface samples showing significant differences. Solid phase extraction of the natural waters did remove unique absorbing and emitting bands in the UV region that could be marine in origin, while enriching the “humic-like” fraction. The open ocean samples showed similarities to the coastal and riverine samples including: 1) monotonically decreasing and unstructured absorbance with increasing wavelength; 2) loss of absorption upon NaBH4 reduction at all wavelength, with the largest percent loss in the visible; 3) enhanced absorption with increasing pH with spectral changes that occurred over the same pH ranges as the pKas of carboxylic acids and phenols; 4) attenuation of absorption enhancement with increasing pH following reduction at most wavelengths. These similarities not only suggest that there are structural similarities throughout all samples, but also indicate that there may be a terrestrial source of CDOM in the open ocean.Item THE APPLICATION OF MICRODEVICES FOR INVESTIGATING BIOLOGICAL SYSTEMS(2018) Shang, Wu; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The gastrointestinal (GI) tract is a complex ecosystem with cells from different kingdoms organized within dynamically-changing structures and engaged in complex communication through a network of molecular signaling pathways. One challenge for researchers is that the GI tract is largely inaccessible to experimental investigation. Even animal models have limited capabilities for revealing the rich spatiotemporal variation in the intestine and fail to predict human responses due to genetic variation. Exciting recent advances in in vitro organ model (i.e., organ-on- chips (OOC)) based on microfluidics are offering new hope that these experimental systems may be capable of recapitulating the complexities in structure and context inherent to the intestine. A current limitation to OOC systems is that while they can recapitulate structure and context, they do not yet offer capabilities to observe or engage in the molecular based signaling integral to the functioning of this complex biological system. This dissertation focuses on developing microfluidic tools that provide access to interrogating signaling events amongst populations in the GI tract (e.g., microbes and enterocytes). First, a membrane-based gradient generator is built to establish linear and stable chemical gradients for investigating gradient-mediated behaviors of bacteria. Specifically, this platform enables the study of bacterial chemotaxis and potentially facilitates the development of genetically rewired lesion-targeted probiotics. Second, “electrobiofabrication” is coupled with microelectronics, for the first time, to create molecular-to-electronic (i.e., “molectronic”) sensors to observe and report the dynamic exchange of biochemical information in OOC systems. Last, to address the issue of poor compatibility between OOCs and sensors, we assemble OOCs with molectronic sensors in a modular format. The concept of modularity greatly reduces the system complexity and enables sensors to be built immediately before applications, avoiding functional decay of active biorecognition components after long-term device storage and use. We envision this work will “open” OOC systems for molecular measurement and interrogation, which, in turn, will expand the in vitro toolbox that researchers can use to design, build and test for the investigation of GI disease and drug discovery.Item Characterization of Organic Compounds in Hydraulic Fracturing Fluid(2017) Luek, Jenna Lynn; Gonsior, Michael; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Over the past decade, hydraulic fracturing combined with horizontal drilling has become the dominant technique for extracting shale gas in the US, and is increasingly important globally. A complex mixture of chemicals is used in hydraulic fracturing fluid to stimulate natural gas production, but federal regulations have exempted operators from reporting the specific chemicals used in any given well. Recent state-by-state regulations and voluntary disclosures have increased our understanding of these fluids, but knowing this list of chemicals going into a well is just the tip of the iceberg. Once these chemicals are injected, they mix with fluids and naturally occurring chemicals originating in the shale formation and can undergo physical, chemical, and biological transformations. In Chapter 2, I reviewed the literature to date regarding the characterization of organic compounds in injected hydraulic fracturing fluids and waste fluids returning to the surface. I identified a substantial knowledge gap in our understanding of organic compounds in these fluids, particularly non-volatile compounds, and potential transformations within the organic compound pool over the lifetime of the well. I analyzed a number of different shale gas wastewaters using ultrahigh resolution mass spectrometry and identified halogenated organic compounds in these fluids (Ch. 3), suggesting that these compounds were transformation products. Using a time series of shale gas fluids (Ch. 4), I was able to track changes in halogenated organic compounds and find evidence for both biological and chemical transformation pathways. Hierarchical cluster analyses helped identify sulfur-containing transformation products (Ch. 4), and I then determined that sulfur-containing molecules may be useful tracers of shale gas wastewaters in the environment (Ch. 6). In Chapter 5, I used toxicological tests and photoirradiation experiments to track the fate of organic compounds in shale gas wastewaters. Using a primarily non-targeted approach, I have been able to identify a number of organic compounds that are indicative of biological and chemical transformations occurring within hydraulic fracturing fluids and wastewaters. Understanding how these fluids change within the well and during storage and disposal provides critical information for engineering the safe and effective operation of wells, wastewater treatment techniques, and environmental impacts.