UMD Theses and Dissertations
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Item INTERACTIONS BETWEEN NITROGEN AND TEMPERATURE ON THE METABOLISM OF THE RED-TIDE MIXOTROPHIC DINOFLAGELLATE KARENIA SPP. IN SUPPORT OF PREDICTIVE MODELS: IMPLICATIONS FOR BLOOM DYNAMICS ON THE WEST FLORIDA SHELF(2023) Ahn, So Hyun; Glibert, Patricia; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The toxic mixotrophic dinoflagellate Karenia spp. forms blooms almost annually in the Gulf of Mexico, especially on the West Florida Shelf (WFS). Blooms typically initiate in early fall but can persist from months to years. Daily, Karenia vertically migrates to the surface water during the day, possibly experiencing changes in temperature, light, nitrogen (N), and prey type and availability. Therefore, this dissertation aimed to examine the interplay between Karenia’s photo-autotrophic and phago-mixotrophic metabolism and the short-term fluctuations in environmental conditions to understand how these factors may relate to the conditions under which Karenia spp. are found in the WFS.Title of Dissertation: INTERACTIONS BETWEEN NITROGEN AND TEMPERATURE ON THE METABOLISM OF THE RED-TIDE MIXOTROPHIC DINOFLAGELLATE KARENIA SPP. IN SUPPORT OF PREDICTIVE MODELS: IMPLICATIONS FOR BLOOM DYNAMICS ON THE WEST FLORIDA SHELF So Hyun (Sophia) Ahn, Doctor of Philosophy, 2023 Dissertation directed by: Professor Patricia M. Glibert, Marine Estuarine Environment Sciences A culture of K. mikimotoi balanced photon flux pressure (light availability) with consumption in overall metabolism when pulsed with 15N-NO3-, 15N-NH4+, or 15N-urea over the range of 15-25°C as shown by photosynthetic fluorescence. However, when shifted to 30°C, cells were significantly stressed, but urea-enriched cells showed a smaller decline in fluorescence, implying that urea might induce a photoprotective mechanism by increasing metabolic “pull.” Studies conducted with natural K. brevis winter and summer populations during 2021 showed that thermal history played a critical role. Unusually, summer blooms had higher biomass but were stressed photosynthetically and nutritionally. However, 15N-urea enriched summer cells had higher uptake rates as well as carbon (C) and N cell-1, especially in warmer waters, showing differential thermal responses based on N forms. Mixotrophy grazing measurements showed that K. brevis grazed both the picoplankter Synechococcus as well as the cryptophyte Rhodomonas. Grazing did not selectively target specific qualities of Synechococcus (based on differing N and P of the prey growth media), but ingestion rates were a function of prey-to-grazer ratios (R2=0.76) as well as prey amounts (R2=0.71). NanoSIMS confirmed 15N incorporation from Synechococcus in K. brevis. In natural communities of K. brevis, ingestion rates were also significantly related to prey-to-grazer ratios (p < 0.01) and by temperatures (p < 0.05) to a lesser degree (R2= 0.75) when incubated at ambient (24°C) and ambient temperature ± 5°C (19, 29°C). The grazer effects on the photosynthetic performance of grazer and prey were also examined. Grazing on Synechococcus indirectly reduce the photosynthetic performance of prey, especially at warmer temperatures but had little or no effect on the photosynthesis of K. brevis itself.Item A DETAILED OPTICAL ANALYSIS OF CHROMOPHORIC DISSOLVED ORGANIC MATTER AND C18 EXTRACTED ORGANIC MATTER IN THE CHESAPEAKE BAY(2023) McDonnell, Shannon Marie; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chromophoric dissolved organic matter (CDOM) is a large portion of the open ocean dissolved matter pool which contributes largely to ocean color. The composition and distribution of CDOM is essentially controlled by in-situ biological production, terrestrial inputs, photochemical degradation, and microbial consumption. Estuarine environments contain particularly diverse CDOM composition due to their large variety of inputs and shoreline land usage in addition to the mixing of freshwater and salt water. Developing a further understanding of CDOM variation and composition will help develop and improve satellite remote sensing algorithms, help us understand CDOM’s role in the global carbon, nitrogen, and oxygen cycles, and may help to prioritize in-situ sampling for water quality monitoring in areas of concern. The use of inherent optical properties combined with pH titration and chemical reduction with sodium borohydride (NaBH4), helps to probe the molecular composition of CDOM and its spatial variability. Detailed studies of CDOM from the Chesapeake Bay are limited with many studies only investigating the main channel of the Bay and neglecting the various tributaries. Also, there is a lack of studies which specifically probe the molecular composition of the CDOM samples. To address this, an in-depth analysis of the optical properties of CDOM and C18 extracted organic matter (C18-OM) from the Chesapeake Bay, focusing on various inputs, was performed. Chemical reduction with NaBH4 and pH titration were employed to probe the presence of specific functional groups and their contribution to overall optical properties, and how they vary between locations. Spectral slope (S300-700), E2:E3 absorption ratio, fluorescence intensity, and apparent quantum yield of fluorescence (AQY) were used to analyze 170 samples from various tributaries in the Chesapeake Bay. Overall, this study suggested 1) there may be multiple inputs of CDOM within the Chesapeake Bay 2) the Top of the Bay and central channel of the Bay are impacted by the heavy terrestrial input from the Susquehanna River 3) A lack of correlation between phytoplankton fluorescence and CDOM absorption suggest phytoplankton are not an immediate source of CDOM within the Chesapeake Bay and 4) removal of protein and phytoplankton fluorescence after sample filtration indicates these species must exist in aggregates >0.2 µm. Optical analysis combined with pH titration and NaBH4 reduction investigated the variation between 9 C18-OM extracts from various regions in the Chesapeake Bay and a humic material standard Suwannee River Fulvic Acid (SRFA). Additionally, this study investigated the validity of the Charge-Transfer (CT) model using the optical properties of model compounds. This study suggested 1) certain absorbing and emitting species are lost during C18 extraction but extracts are still representative of their CDOM 2) nearly identical optical responses to pH titration and NaBH4 reduction suggest similar chromophore content throughout the Chesapeake and 3) CT interactions leading to long wavelength absorption are more prevalent in Suwannee River Fulvic Acid (SRFA) than they are in the Chesapeake. To compare the molecular and optical properties of the Chesapeake Bay to other locales, these extracts were compared to extracts from the Delaware Bay (DEL), Equatorial Atlantic Ocean (EAO) and North Pacific Ocean (NPO) in addition to reference materials Suwannee River Fulvic Acid (SRFA), Pony Lake Fulvic Acid (PLFA), and Elliott Soil Humic Acid (ESHA). This study showed 1) composition of deprotonatable and reducible chromophores within the Chesapeake and Delaware Bays is nearly identical but different from the oceans 2) despite being estuaries and containing a mixture of fresh and ocean water, CDOM within both Bays looks terrestrially dominated 3) deep ocean extracts from the Atlantic and Pacific exhibit similar optical response to pH titration, NaBH4 reduction, and NaBH4 reduction combined with pH titration suggesting the similarity of deep ocean waters from both ocean basins.Item Development of Fluorescent Imaging Methods and Systems to Determine Photodynamic Potential and Inform Cancer Treatment Efficacy(2022) Gaitan, Brandon; Huang, Huang-Chiao; Chen, Yu; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photodynamic therapy (PDT) is a treatment modality that has gained rapid popularity in both research and clinical settings over the past 20 years. PDT involves harmless red/near-infrared light excitation of non-toxic photosensitizers to generate reactive molecular species (RMS) that can induce tissue damage and/or cell death. In addition, the fluorescence signal generated from the photosensitizer can also be used for optical imaging. These effects have been harnessed for image-guided treatment of cancer and other diseases. As PDT gains popularity, it is crucial to understand and monitor different factors that could impact overall treatment efficacy. These factors include, but are not limited to, the RMS yield of photosensitizers, the distribution of photosensitizers in tissue, and the PDT activation depth in tissues. Our work focused on developing methodologies and devices to characterize and improve PDT treatment. In collaboration with the FDA, we developed a cell-free assay to rapidly and more quantitatively determine the potential phototoxicity of fluorescent probes through the measurement of singlet oxygen. We also developed a method to compare the maximal PDT activation depth of FDA-approved photosensitizers (BPD and PpIX) in the brain. We found that BPD can be activated 50% deeper into brain tissues compared to PpIX at the same radiant exposure. Next, we tested the ability of a 3D imaging system, Fluorescence Laminar Optical Tomography (FLOT), to image the distribution of photosensitizers in the rodent brain. We demonstrated that FLOT could accurately map the photosensitizer distribution up to 0.5 mm in tissues. Lastly, we developed an autofluorescent-based endoscopic imaging system to measure the metabolic impact of PDT on cancer and normal tissues, finding that PDT leads to significant changes in tissue metabolism immediately after treatment. In summary, we have developed a series of systems that can aid in PDT treatment optimization in three major ways:1) rapidly quantifying the singlet oxygen production of photosensitizers, 2) more accurately measuring a photosensitizers localization and activatable depth, and 3) developing the ability to measure a tissues response to PDT in real-time.Item The Yin and Yang of Amyloids: Insights from alpha-Synuclein and the Pmel17 Repeat Domain(2011) Pfefferkorn, Candace Marie; Lee, Jennifer C; Thirumalai, Devarajan; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While amyloidogenic proteins are commonly associated with human diseases such as Alzheimer's and Parkinson's disease, it is intriguing that amyloid fibrils also are utilized for essential biological processes. A key question then is why many amyloids are harmful whereas some serve essential functional roles. To begin to address this question, the environmental factors regulating the conformational changes in the Parkinson's disease-related protein, alpha-synuclein (alpha-syn), and a critical polypeptide fragment, the repeat domain (RPT) of Pmel17, the protein required for melanin formation are examined. The role of membranes in modulating alpha-syn conformation is investigated because membranes are ubiquitous in vivo and affect alpha-syn aggregation in vitro. Using single tryptophan-containing alpha-syn variants (F4W, Y39W, F94W, Y125W) as site-specific fluorescent probes, distinct phospholipid vesicle and SDS micelle interactions have been identified and membrane binding equilibria measured. The role of specific N-terminal residues in membrane binding also has been assessed. Specifically, environments of the highly sensitive Trp4 probe in alpha-syn polypeptide fragments (residues: 1 - 4, 1 - 6, 1 - 10, and 1 - 15) upon membrane binding were characterized using steady-state fluorescence and time-resolved anisotropy. The penetration depths of alpha-syn and N-terminal peptides into the lipid bilayer also were determined using brominated lipids as heavy-atom quenchers. To simultaneously monitor alpha-syn and bilayer structure, neutron reflectometry (NR) and a sparsely-tethered bilayer lipid membrane (stBLM) were employed. Using NR and an stBLM, alpha-syn concentration dependent effects on both protein structure and membrane properties were measured. To begin to address biophysical and biochemical differences between pathological and functional amyloid, a systematic investigation of the effects of solution pH (4→7) on RPT aggregation was performed since melanosomes, acidic organelles where Pmel17 fibrils are formed, change pH during maturation. Using intrinsic tryptophan fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, local, secondary, and fibril morphological structure were monitored, respectively. Notably, RPT fibril morphology can be transformed directly by changing solution pH, suggesting that pH is a natural regulatory mechanism for Pmel17 amyloid formation and its subsequent dissolution in vivo.Item Portable Hyperspectral Imaging Device for Surface Sanitation Verification in the Produce Industry(2011) Wiederoder, Michael; Lo, Y. Martin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Produce processors must clean and sanitize surfaces before production to reduce the risk of foodborne illness. Current surface hygiene verification methods require direct surface sub-sampling at selected locations and a wait time. To augment these methods, a portable hyperspectral imaging device was developed to find potential contaminants in real-time and increase sub-sampling effectiveness. Analysis of hyperspectral fluorescence images showed that fresh-cut produce processing exudates in the regions of 460-540 and 670-680 nm are detectable from background materials, while select cleaning agents are not. The portable single operator imaging system includes a charge coupled device (CCD) camera, tunable optical filter, laptop, light emitting diodes (LED's) for fluorescence excitation, and a touchscreen display. Within a commercial plant, fluorescence imaging identified produce processing residuals following routine cleaning procedures that were not readily visible to the naked eye. These tests demonstrate the system's potential to enhance post-cleaning inspection, and helped improve routine cleaning procedures.Item CHARACTERIZATION OF FLUORESCENCE FROM QUANTUM DOTS ON NANOSTRUCTURED METAL SURFACES(2011) Hwang, Ehren; Davis, Christopher C; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The behavior of fluorescent materials coupled to surface plasmon supporting surfaces and structures is an area of active research due to their fluorescence enhancing properties. The inherent field enhancements present near structures and interfaces where surface plasmons are excited provide great potential for increasing the response of many optical interactions. While many studies focus on the application of plasmonic nanoparticles or finite metallic structures the use of dielectric structures on a continuous metallic film has received little attention. A comprehensive experimental study using dielectric gratings on gold films is presented illustrating the fundamental properties of fluorescence enhancement on such structures. A process for fabrication of samples using Electron Beam Lithography is demonstrated and comparisons between various quantum dot deposition methods are made to determine the best conditions for surface coating. Conditions for optimization of the fluorescence enhancement phenomena for practical application are explored for gratings with square function profile illustrating the influence of gratings on fluorescence behavior and identifying conditions for optimal enhancement. Complementing these results, an understanding of the underlying physical phenomena is developed by differentiation between enhanced emission and enhanced absorption effects using measurements of fluorescence decay lifetime and emission spectra. Using these observations a thorough description of these systems and the requirements for their practical application is illustrated.Item Differentiating a Landscape of Lac Repressor Mediated DNA Loops Using FRET(2011) Haeusler, Aaron Raymond; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The variety of DNA loops formed by a protein such as Lac Repressor (LacI), predicted to populate multiple loop topologies and geometries, increases the uncertainty of models that have not been experimentally verified. We created a method for evaluating the multitude of LacI-DNA looped states using FRET with fluorophore position variants (FPVs) on a landscape DNA constructs with dual operator phasing variants bracketing an intrinsically curved DNA sequence. The FRET signature for each FPV corresponds to a specific LacI-DNA loop topology, and when applied to the landscape of constructs we could systematically explore DNA sequence dependent LacI mediated DNA looping. The FRET efficiencies calculated for each FPV landscape revealed the first unambiguous detection of antiparallel (A1 and A2) and distributions among parallel loop (P1) and antiparallel loops. The FRET efficiency maximum of each FPV landscape was used to calculate the loop population distribution for each topology assuming a uniform population at the peaks. The differences in the observed peak FRET among FPV landscapes led to an unavoidable estimate of loop populations with an extended LacI-DNA loop geometry, and also slightly extended LacI-DNA loop in the cases of antiparallel loops. The addition of saturating inducer to the FPV landscape demonstrated that IPTG-LacI-DNA loops have different properties than uninduced loops: they have increased instability, increased competition among previously energetically unfavorable states, and presumably have increased switching between specific and nonspecific LacI-DNA interactions. This multivariate data set confirms that rod mechanics models of DNA looping should also consider protein flexibility, and also demonstrates the importance of protein flexibility in modeling genetic regulatory circuits that are similar to the "hydrogen atom of gene regulation", the lac operon.Item INTERFACIAL SOLVATION AND EXCITED STATE PHOTOPHYSICAL PROPERTIES OF 7-AMINOCOUMARINS AT SILICA/LIQUID INTERFACES(2010) Roy, Debjani; Walker, Robert A; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The properties of solutes adsorbed at interfaces can be very different compared to bulk solution limits. This thesis examines how polar, hydrophilic silica surfaces and different solvents systematically change a solute's equilibrium and dynamic solvation environment at solid/liquid interfaces. The primary tools used in these studies are steady state fluorescence spectroscopy and time correlated single photon counting (TCSPC) -a fluorescence method capable resolving fluorescence emission on the picosecond timescale. To sample adsorbed solutes, TCSPC experiments were carried out in total internal reflection (TIR) geometry. These studies used total of six different 7 aminocoumarin dyes to isolate the effects of molecular and electronic structure on solute photophysical behavior. Fluorescence lifetimes measured in the TIR geometry are compared to the lifetimes of coumarins in bulk solution using different solvents to infer interfacial polarity and excited state solute conformation and dynamics. Steady state emission experiments measuring the behavior of the coumarins adsorbed at silica surfaces from bulk methanol solutions show that all coumarins had a similar affinity &delta G ads &sim &minus 25-30 kJ/mole. Despite these similar adsorption energetics solute structure had a very pronounced effect on the tendency of solutes to aggregate and form multilayers. Our finding suggests that hydrogen bonding donating properties of the silica surface plays a dominant role in determining the interfacial behavior of these solutes. The silica surface also had pronounced effects on the time dependent emission of some solutes. In particular, the strong hydrogen bond donating properties of the silica surface inhibit formation of a planar, charge transfer state through hydrogen bond donation to the solute's amine group. A consequence of this interaction is that the time dependent emission from solutes adsorbed at the surface appears to be more similar to emission from solutes in nonpolar solvation environments. To test the role of solvent identity on the photophysical properties of adsorbed solutes, additional experiments were carried out with a nonpolar solvent (decane), a moderately polar solvent (n decanol) and a polar aprotic solvent (acetonitrile). The results from these studies demonstrated that interfacial solvation depends sensitively on a balance of competing forces including those between the solute and substrate, the solute and solvent and the surface and adjacent solvent.Item A Novel Pre-fluorescent Nitroxide Probe for the Highly Sensitive Determination of Peroxyl and Other Radical Oxidants(2009) Jia, Min; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Peroxyl and other radical oxidants react with stable cyclic nitroxides, such as the piperidinyl and pyrrolidinyl nitroxides to form initially the one electron oxidation product, the oxoammonium cation. For most of the nitroxides studied thus far, the oxoammonium cation can in part be regenerated to the nitroxide through reduction by solution constituents. The reaction mechanisms, however, remain a matter of debate. Further, the highly-sensitive, quantitative determination of peroxyl and other radical oxidants has yet to be achieved, posing a major hurdle to a further understanding of the impact of peroxyl radicals in many biological and environmental processes. A unique, amino-pyrrolidinyl nitroxide, 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) is shown to undergo an irreversible reaction with peroxyl radicals and other radical oxidants to generate a diamagnetic product. When a fluorophore, fluorescamine is covalently linked through the amino group on the nitroxide, the resulting compound (3-apf, or I) has very low fluorescence quantum yield. Upon reaction with peroxyl and other radical oxidants, the quantum yield of the product increases dramatically (~100 fold), and thus 3-ap or 3-apf can be used as a highly sensitive and versatile probe to determine oxidant production optically, either by monitoring the changes in fluorescence intensity using a spectrofluorometer, by HPLC analysis with fluorescence detection, or by a combination of both approaches. By changing the [O2]/[nitroxide] ratio, it is shown that peroxyl radicals can be detected and quantified preferentially in the presence of other radical oxidants, such as *NO2 and CO3*-. When decreasing the [O2]/[nitroxide] ratio, the oxidation product decreases, with a concomitant increase of the alkoxylamine product resulting from reaction of 3-ap (3-apf) with carbon centered radicals. Preliminary studies suggest that the reactions of 3-ap and 3-apf with peroxyl radical produce different final products. High resolution mass spectrometry and NMR studies indicate that 3-ap is oxidized to form a cyclic peroxide structure, while 3-apf is oxidized to form a cyclic -NH-O- structure, with this difference resulting possibly from the presence of the fluorescamine moiety in 3-apf. Detection of photochemically produced peroxyl radicals is achieved by employing 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) alone, followed by derivatization with fluorescamine, while detection of thermally-generated peroxyl radicals employs 3-apf. Preliminary applications include the detection of peroxyl radicals generated thermally in soybean phosphatidylcholine liposomes by 3-apf and produced photochemically in tap water by 3-ap.