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Title: Electrochemistry of nanostructured carbon materials in aqueous electrolytes and room temperature ionic liquids
Creator: Jarosova, Romana
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Carbon is one of the most plentiful elements on the planet. From a materials perspective, carbon is unique because of the microstructurally distinct allotropes it forms. These include single and polycrystalline diamond, diamond-like carbon, glassy carbon, and graphite. All of these carbon materials are commonly used in electroanalysis, energy storage and conversion, separation, and chemical analysis, due to numbers of reasons, including low cost, high mechanical strength, wide usable...
Show moreCarbon is one of the most plentiful elements on the planet. From a materials perspective, carbon is unique because of the microstructurally distinct allotropes it forms. These include single and polycrystalline diamond, diamond-like carbon, glassy carbon, and graphite. All of these carbon materials are commonly used in electroanalysis, energy storage and conversion, separation, and chemical analysis, due to numbers of reasons, including low cost, high mechanical strength, wide usable potential range, rich surface chemistry, chemical inertness, and compatibility with a variety of solvents and electrolytes. For the optimal usage of carbon electrodes in electrochemistry, it is critical to fully understand and control the variables that impact background voltammetric current, capacitance, and heterogeneous electron-transfer kinetics at these materials. Over the years of carbon electrode usage in electrochemistry, much knowledge has been gained about the structure-function relationship at sp2- and sp3- bonded carbon electrodes. Nevertheless, as most of this knowledge pertains to aqueous electrolyte solution, there is still a significant gap about the properties of the electric double layer and the transport processes near the electrode interface in room temperature ionic liquids. The room temperature ionic liquids are solvent-free medium, composed purely of ions, with a melting point near or below room temperature. In electrochemistry, they are appreciated for several of their excellent properties, such as wide working potential window, moderate electrical conductivity, high thermal and chemical stability, negligible vapor pressure etc. As the RTILs does not contain any solvent, their interfacial structure at an electrified interface is significantly distinguished from the conventional Gouy-Chapman-Stern model describing the double layer in aqueous solutions. Additionally, also the redox analyte environment in RTILs is expected to different compared to those in aqueous solutions. The work in this dissertation thesis is focused on the electrochemical performance of microstructurally different carbon electrodes in aqueous electrolytes and room temperature ionic liquids. The physical, chemical and electronic properties of glassy carbon, boron-doped diamond, and tetrahedral amorphous carbon electrodes are discussed. Furthermore, the microstructure of carbon electrodes is correlated to the heterogeneous electron transfer rate constants of soluble inorganic and organic redox couples in aqueous electrolytes and room temperature ionic liquids. The attention was primarily focused on tetrahedral amorphous carbon electrode that can be doped with nitrogen, resulting in significant physical, chemical an electrochemical properties. Moreover, the electrode surface chemistry was alternated by an oxygen plasma modification and its effect on the electrochemical performance (background voltammetric current, capacitance and electron transfer kinetics), as well as potential surface damage was studied. Lastly, as it is believed that the nitrogen incorporated tetrahedral amorphous material possesses an equally superb properties compared to the boron doped diamond, both electrode materials were used for determination of endocrine disruption compounds, specifically estriol, estradiol and estrone, using high-pressure liquid chromatography with electrochemical detection. The detection figures of merit for both boron doped diamond and nitrogen-incorporated tetrahedral amorphous carbon thin-film electrodes were determined.
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Title: Characterization of molecular interactions and organization in solution and interfacial systems
Creator: Hay, Christine Ella
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Liquid solutions and liquid-liquid interfaces are the media for numerous chemical reactions, where the local environments are conducive to interactions and exchanges. As a means of broadening our understanding of the molecular organization in liquid solutions and in proximity to phase boundaries, we report on the rotational diffusion dynamics of probe molecules in increasingly complex systems. Neat liquids represent the simplest systems interrogated. In our first study, we measure the induced...
Show moreLiquid solutions and liquid-liquid interfaces are the media for numerous chemical reactions, where the local environments are conducive to interactions and exchanges. As a means of broadening our understanding of the molecular organization in liquid solutions and in proximity to phase boundaries, we report on the rotational diffusion dynamics of probe molecules in increasingly complex systems. Neat liquids represent the simplest systems interrogated. In our first study, we measure the induced orientational anisotropy function, R(t), using time-correlated single photon counting of two chromophores, resorufin and 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBDHA) in water and in N-octyl-2-pyrrolidone (NOP). Our data show distinctly different anisotropy decays in aqueous solution compared to NOP solutions, and taken in the context of the Chuang and Eisenthal model, this indicates a change in effective rotor shape swept out by the solute's rotation. The similarity in the behavior of the two different chromophores in these solvent systems points to solvent-solvent interactions and local organization as the dominant factors in mediating motional dynamics. By adding varying concentrations of electrolyte to aqueous and NOP systems, the rotational diffusion dynamics of the anionic chromophore, resorufin, are altered as a result of interaction with electrolyte cations. In aqueous solutions containing lithium perchlorate (LiClO4), resorufin exhibits a single exponential anisotropy decay as was the case in pure water, however, reorientation times are dependent on electrolyte concentration. In contrast to the observed behavior of resorufin in pure NOP, where bi-exponential decay occurs, we observe a single exponential anisotropy decay for resorufin in NOP with the addition of non-aqueous electrolyte. For resorufin in NOP, the reorientation time constant increases with increasing electrolyte concentration, consistent with formation of a complex between the resorufin anion and the electrolyte cation. Elucidating the local organization near phase boundaries compared to bulk solutions represents a more complex problem to interrogate. We use a TCSPC confocal imaging instrument to obtain depth-resolved fluorescence lifetime and anisotropy decay data for lissamine rhodamine B sulfonyl chloride (LRSC) in NOP saturated with water and in ethylene glycol (EG) supported on a glass surface. The fluorescence anisotropy data of LRSC in NOP and EG as a function of distance from NOP|glass and EG|glass interfaces reveal a gradient spanning tens of micrometers from the NOP|glass interface into the NOP phase, and no corresponding gradient in EG. From these data, we assert there is a compositional heterogeneity in the form of water nano-droplets in the NOP phase, where water molecules solvate LRSC. NOP is miscible with small amounts of water allowing for the presence of water inclusions in solution, while phase separation occurs for sufficiently high water concentrations. By forming a liquid-liquid interface composed of water and NOP supported on glass, we observe both compositional and dielectric gradients. We report on spatially resolved fluorescence lifetime and anisotropy decay time constant data of LRSC in the NOP phase near the liquid-liquid (NOP|water) interface and the liquid-solid (NOP|glass) interface. These data reveal a micron-scale position-dependent molecular environment characterized by an anisotropy decay gradient as a function of distance from the NOP|glass interface. In addition, the presence of a fluorescence lifetime gradient normal to the NOP|water interface indicates the existence of a gradient in the concentration of water nano-droplets.
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Title: Protease-containing membranes for rapid, controlled antibody digestion prior to mass spectrometry analysis
Creator: Pang, Yongle
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Monoclonal antibodies are the fastest growing class of therapeutic drugs because of their high specificities to targeQt cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. Conventional antibody characterization workflows contain an in-solution digestion step, which is labor-intensive and time-consuming. Protease-containing membranes...
Show more"Monoclonal antibodies are the fastest growing class of therapeutic drugs because of their high specificities to targeQt cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. Conventional antibody characterization workflows contain an in-solution digestion step, which is labor-intensive and time-consuming. Protease-containing membranes are an attractive alternative platform for protein digestion because of their high local enzyme concentrations, short radial diffusion distances, rapid convection in pores, simple fabrication and low cost. Additionally, variation of protein residence time in the membrane gives control over the size of proteolytic peptides. This research focuses on developing workflows for monoclonal antibody characterization using functionalized porous membranes. Sequential adsorption of poly (styrene sulfonate) and pepsin in a porous nylon membrane forms a pepsin membrane reactor. Pepsin is inexpensive and catalyzes proteolysis in acidic solutions, which avoids the need to alkylate cysteine residues and limits antibody deamidation. Variation of the residence times (3 ms to 3 s) of antibody solutions in pepsin-containing membranes yields "bottom-up" (1-2 kDa) to 'middle-down' (5-15 kDa) peptides in less than 10 min. These peptic peptides cover the entire sequences of Herceptin and a WatersTM antibody. Compared with the performance of bottom-up (in-solution tryptic digestion) and top-down (intact protein fragmentation) analysis of an antibody light chain, middle-down (in-membrane peptic digestion) analysis gives the highest bond cleavage (99%). In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation and glycosylation. Recently developed protease-containing spin membranes provide an excellent platform for rapid, membrane-based protein digestion prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100-200 æL of pretreated protein solutions through the pepsin- or trypsin-containing membranes takes less than 1 min and gives nearly 100% coverage of the protein sequences in subsequent direct infusion MS analysis of digests of apomyoglobin and four commercial monoclonal antibodies (Herceptin, Avastin, Rituxan and Vectibix). MS analysis of peptic and tryptic peptides also reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal Lysine clipping and glycosylation. Liquid chromatography coupled to tandem mass spectrometry analysis of tryptic spin digests and subsequent MaxQuant data searching show 100% sequence coverage of all four antibody light chains, and 75.1%-98.4% coverage of the heavy chains. Compared to in-solution tryptic digestion of mAbs, spin digestion yields higher sequence coverage and a larger number of unique peptides. In-membrane digestion also facilitates protein sequence comparison. Rapid peptic in-membrane digestion of two antibodies with direct infusion MS analysis accurately reveals the antibody modification site in less than 1 h. Overall, membrane-based protein digestion uses minimal sample preparation time and yields high peptide and sequence coverages for identification of protein PTMs."--Page ii-iii.
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Title: Design and assessment of flow-through catalytic system using palladium nanoparticle catalyst confined on a porous silica support
Creator: Zhang, Xiaoran
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Catalysis has the potential to play a significant role in the converting biomass feedstock to commodity chemicals and fuels. There are a few requirements for the catalysts to fulfill during the biorefinery process: optimal reactivity and high selectivity under relatively mild reaction conditions. These properties can be optimized through the choice of catalyst and the morphology of the catalyst support, immobilization chemistry, solvent system for the reaction, and reaction conditions. The...
Show more"Catalysis has the potential to play a significant role in the converting biomass feedstock to commodity chemicals and fuels. There are a few requirements for the catalysts to fulfill during the biorefinery process: optimal reactivity and high selectivity under relatively mild reaction conditions. These properties can be optimized through the choice of catalyst and the morphology of the catalyst support, immobilization chemistry, solvent system for the reaction, and reaction conditions. The purpose of this work is to create and demonstrate a flow-through catalytic reaction system that enhances the target heterogeneous reaction under mild reaction conditions, and to assess the performance of catalyst reactivity and selectivity. To design the flow-through catalytic system, we created a silica-polymer composite inverse opal structure with relatively low defect density to be used as a catalyst support. The construction of the inverse opal structures involved the self-assembly of a template with colloidal polystyrene nanospheres, deposition of the poly(ethylene glycol)-silica sol gel composite precursors in the interstitial spaces between the colloidal spheres, and removal of the template nanospheres by dissolution. The addition of PEG into the silica sol gel modifies the morphology of inverse opal structures to reduce defect density. The resulting inverse opals were characterized by scanning electron microscopy (SEM). By optimizing the composition and molecular weight of polymer, the composite inverse opal structure was improved to reduce the number of defects compared to a silica sol gel inverse opal. The inverse opal structure was replaced by a porous glass frit because of the improved structural integrity of the latter as well as its reduced resistance to flow when used in a flow-through catalytic format. Using Glucose Oxidase (GOx) as a catalyst, a porous glass frit was shown to be a practical, functional catalyst support that compared favorably to the inverse opal support. We chose porous glass frits as catalyst supports for further studies with flow-through catalytic reactions. Using a porous glass frit support, the catalytic performance of palladium nanoparticles was evaluated in a flow-through reaction format. The reactivity and selectivity of the catalyst was assessed by heterogeneous hydrogenation of p-coumaric acid and cinnamaldehyde under mild conditions and in different solvents. A microelectric actuator control system was used to maintain H2(g) level in the reaction stream. The results of this work demonstrate high efficiency and the requisite selectivity for the reactions used. This work represents an initial step in the development of a catalysis system for biomass conversion and biorefinery processing."--Pages ii-iii.
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Title: Expression, purification and functional characterization of influenza hemagglutinin (HA2) membrane fusion protein
Creator: Ratnayake, Punsisi Upeka
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Influenza virus causes substantial public health risk worldwide. Influenza is an enveloped virus and hemagglutinin membrane protein present in the viral membrane plays an important role in the viral infection process. Hemagglutinin protein is composed of two subunits called HA1 and HA2. Binding of the virus to the host cell is governed by HA1 subunit, and HA2 subunit is responsible for the fusion of viral membrane and endosomal membrane.My research has focused on production and...
Show moreInfluenza virus causes substantial public health risk worldwide. Influenza is an enveloped virus and hemagglutinin membrane protein present in the viral membrane plays an important role in the viral infection process. Hemagglutinin protein is composed of two subunits called HA1 and HA2. Binding of the virus to the host cell is governed by HA1 subunit, and HA2 subunit is responsible for the fusion of viral membrane and endosomal membrane.My research has focused on production and characterization of several protein constructs containing different domains (with or without FP or with or without TM) and the characterization of the full length HA2. Biophysical comparisons between full-length HA2 and shorter constructs including SHA2, FHA2, and SHA2-TM were performed. Biophysical characterization techniques such as CD spectroscopy, SEC, crosslinking experiments, mass spectrometry and vesicle fusion assays were used in this study. Physiologically relevant oligomeric states of these hemagglutinin constructs were identified. At pH 7.4, the physiological pH, these constructs are trimeric helical molecules in detergents. The melting temperature for full length HA2 is > 90 oC in decylmaltoside (DM) representing a highly thermostable structure. All constructs are positively charged at pH 5.0 and induce vesicle fusion with negatively-charged vesicles. However, fusion with negatively charged vesicles at pH 7.4 was negligible. With positively charged vesicles, pH-dependence was reversed leading to conclude that attractive protein/vesicle electrostatics play a role in fusion between vesicles and hemagglutinin constructs.My work will help future scientists set up crystallography experiments since this is the first time that full length HA2 has been produced in mg quantities using bacterial expression system. Furthermore, it is hypothesized that in the final stage of the fusion of full length HA2 constructs as well as full length HIV gp41 constructs there is a formation of complex in between FP and TM. But, still there is no experimental evidence for support this hypothesis. HDX experiment completed and proposed in this dissertation will help to investigate this hypothesis.
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Title: Functionalized membranes for protein purification and proteolysis prior to mass spectrometry analysis
Creator: Ning, Wenjing
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Protein isolation and digestion are often vital steps in studies of protein structures, interactions and post-translational modifications. Porous membranes present an attractive platform for rapid proteolysis and protein purification because convective flow through pores quickly transports proteins or reagents to functional sites. This dissertation demonstrates functionalization of porous membranes with metal-chelating polyelectrolytes, peptide ligands, and enzymes, to create methods for fast...
Show moreProtein isolation and digestion are often vital steps in studies of protein structures, interactions and post-translational modifications. Porous membranes present an attractive platform for rapid proteolysis and protein purification because convective flow through pores quickly transports proteins or reagents to functional sites. This dissertation demonstrates functionalization of porous membranes with metal-chelating polyelectrolytes, peptide ligands, and enzymes, to create methods for fast protein purification, affinity tag removal and protein digestion. Additionally, attachment of functionalized membranes to pipette tips enables especially rapid and convenient protein digestion or isolation. Development of high-capacity affinity membranes for protein isolation requires membrane pores coated with thin films that bind multilayers of proteins. To prepare membranes that selectively capture polyhistidine-tagged (His-tagged) proteins, this work explores layer-by-layer adsorption of polyelectrolytes containing chelating groups that form Ni2+ complexes. Sequential adsorption of protonated poly(allylamine) (PAH) and carboxymethylated branched polyethyleneimine (CMPEI) leads to membranes that bind Ni2+ and capture ~60 mg of His-tagged ubiquitin per mL of membrane. Both binding capacity and metal-ion leaching are similar to values seen with high-binding commercial beads, but membranes should facilitate protein isolation in minutes.After purification, fusion tag removal is often an essential step prior to protein characterization. Removal of small ubiquitin-like modifier (SUMO) tags in SUMO-protease-containing membranes served as a proof-of-concept demonstration for in-membrane tag removal. The time required for tag removal is similar with dissolved and immobilized His-tagged SUMO protease, but the membrane is reusable, and immobilized proteases retain much of their activity after three uses.Membranes are also convenient substrates for trypsin immobilization and subsequent proteolysis. Passage of protein solutions through 100-μm thick trypsin-modified membranes enables reaction residence times as short as milliseconds to limit digestion and provide large peptides for mass spectrometry (MS) analysis. Large peptides can both enhance protein sequence coverage and help identify flexible regions in a protein. With either cytochrome c or apomyoglobin, in-membrane trypsinolysis cleaves the protein after lysine residues in highly flexible regions to generate two large peptides that cover the entire protein sequence. Further combining membrane techniques with pipette tips yields a convenient platform for rapid protein purification and digestion. Pushing a protein-containing solution through a trypsin-modified membrane at the end of a pipette tip digests proteins in <30 s, and enables tryptic digestion without alkylation of cysteine residues. Similarly, when membranes contain Ni2+ complexes, pipetting aqueous His-tagged protein through the membrane and subsequent rinsing and elution yield purified protein in 2 min. These applications demonstrate the potential of functional membranes for rapid proteolysis and protein isolation.
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Title: Profiles of volatile compounds as microbial markers in applications of biosecurity and bioenergy
Creator: Reese, Kristen Leigh
Date: 2020
Collection: Electronic Theses & Dissertations
Description: All organisms emit volatile organic compounds (VOCs) and profiling of volatiles (volatilomics) is finding diverse applications. Some VOCs are consistently present throughout the lifecycles of organisms, while other VOCs are biomarkers, quantifiable indicators of changes in physiological state or reflective of environmental stresses. This dissertation describes research into volatile biomarkers of different microorganisms in the context of biosecurity and bioenergy. Untargeted analyses of...
Show moreAll organisms emit volatile organic compounds (VOCs) and profiling of volatiles (volatilomics) is finding diverse applications. Some VOCs are consistently present throughout the lifecycles of organisms, while other VOCs are biomarkers, quantifiable indicators of changes in physiological state or reflective of environmental stresses. This dissertation describes research into volatile biomarkers of different microorganisms in the context of biosecurity and bioenergy. Untargeted analyses of microbial biomarkers were accomplished using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In the context of biosecurity, pathogenic bacteria can be used as the basis for a bio-terrorism attack. There is a need for deeper understanding of the chemical signatures of organisms, in particular when they infect individuals, and a need for methods for detecting these pathogens in the context of infections of humans. Current research has performed metabolite profiling of VOCs emitted in culture by surrogates for potential bacterial bioterrorism agents, Bacillus anthracis Sterne and Francisella tularensis novicida in conjunction with measurements of VOCs released by their fully virulent counterparts, F. tularensis SCHU S4 and B. anthracis Ames, both on the CDC category A bioterrorism and disease agent list. Methyl ketones, alcohols, esters, carboxylic acids, and nitrogen- and sulfur-containing compounds were attributed to the bacteria. The two genera showed distinct VOC profiles whereas the taxa within each genus showed subtler differences in VOC profiles. Growth phase influenced absolute and relative VOC abundances, indicating the potential for markers to discriminate growth phases. This in vitro determination of VOC profiles laid groundwork for non-invasive probing of bacterial metabolism.Towards bioenergy efforts, microalgae present a renewable alternative to producing biofuels. However, biofuels are more costly per gallon compared to non-renewable fossil fuels due to production and harvesting costs. Therefore, research driving increases in biomass production are of interest, specifically (1) better early-warning tools to anticipate and/or diagnose the presence of predators and (2) understanding algae-bacteria interactions, as they are challenging to manage and may help or harm algal productivity. Research towards part (1) aimed to better define the physiological state of algae ponds. A biofuel-relevant alga, Microchloropsis salina, was infected with a predator, the rotifer Brachionus plicatilis. SPME-GC-MS aided discovery of seven putative culture crash biomarkers, including carotenoid degradation products trans-β-ionone and β-cyclocitral, over several timepoints during active crashing of algal ponds that were not observed in healthy controls. These biomarkers offer potential as diagnostic tools to signal the need for crash mitigation strategies, as signals were detected before observed losses in algal cell density. Research towards part (2) aimed to detect and identify VOC biomarkers related to the micro-scale interactions of a model system of alga P. tricornutum and bacterium Marinobacter spp. 3-2. The presence of Marinobacter spp. 3-2, either in the form of live bacterial cells or sterile exudates, caused modest inhibition in growth rates of P. tricornutum. Substantial differences in VOC biomarker profiles were observed between 1) co-cultures of both organisms, 2) P. tricornutum exposed to Marinobacter spp. 3-2 exudates, and 3) Marinobacter spp. 3-2 exposed to P. tricornutum exudates, all relative to the VOC biomarker profiles of corresponding monocultures. Increasing the knowledge base of algae-bacterial interactions will enable a deeper understanding of the basic science of microorganism signaling.
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Title: Effects of external factors on the fluidity of supported lipid bilayers and cell membranes
Creator: Hossain, Masroor
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Biological plasma membranes are essential for a cell’s proper function. They play key roles in cell-communication, structural support, and are involved in key processes, such as inducing anesthesia. They are composed of hundreds of distinct molecules with the majority being phospholipids, followed by proteins and carbohydrates. We take advantage of this well-crafted system by creating artificial bilayer models, whose parameters are well-controlled. Ultimately, the goal of this research is to...
Show moreBiological plasma membranes are essential for a cell’s proper function. They play key roles in cell-communication, structural support, and are involved in key processes, such as inducing anesthesia. They are composed of hundreds of distinct molecules with the majority being phospholipids, followed by proteins and carbohydrates. We take advantage of this well-crafted system by creating artificial bilayer models, whose parameters are well-controlled. Ultimately, the goal of this research is to understand the complex interplay of molecules in a model bilayer through translational diffusion constants and connect the results to live plasma membranes. The model bilayer composition was simplified by focusing on major lipids, namely cholesterol, 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), sphingomyelin, and ceramide on mica substrates. Various applications of artificial membranes exist, and we choose to focus on the area of anesthesiology. Though anesthesia use continues to be vital, from minor to major surgical processes, molecular mechanisms are not clear and lack of widespread consensus on theories cause debate among researchers. The main contenders are anesthetics acting directly on proteins or indirectly by dissolving in lipids and affecting transmembrane protein functions. Drawing from previous work in the Blanchard research group, we apply ethanol and n-butanol, on our models to understand interactions of general anesthetics with the bilayer. With perylene or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) as fluorescent probes (sensitive to phospholipid headgroup or tail mobility, respectively), the fluorescent recovery after photobleaching (FRAP) technique results in fluorescence recovery curves of the supported lipid bilayers, from which translational diffusion constants are extracted. The values are related directly to fluidity and, as predicted, application of the short-chain alcohols led to higher diffusion coefficients overall. However, high alcohol concentrations resulted in lower diffusion coefficients potentially due to bilayer interdigitation. On both the macroscopic and microscopic scale, ceramide rigidified the system. As a whole, further investigations pointed to the heterogeneous morphology of the models. Size-dependent FRAP measurements led to an important observation that anomalous diffusion is occurring. Ultimately, results from the models need to be connected with those on live cell membranes and we had this experience with a collaborative project with the Busik laboratory in the physiology department at Michigan State University. Their group works on various projects, one of which involves treatment options for retinal-based degradation in diabetic subjects. Increased cholesterol generally decreases membrane fluidity, and related to our research goals, the drug N, N-dimethyl-3β-hydroxy-cholenamide (DMHCA), a selective LXR agonist, rejuvenated circulating angiogenic cell (CAC) membrane fluidity which has potential in vascular repair of people afflicted with the diabetic retinopathy complication.
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Title: The integration of computational methods and nonlinear multiphoton multimodal microscopy imaging for the analysis of unstained human and animal tissues
Creator: Murashova, Gabrielle Alyse
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Nonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption ...
Show moreNonlinear multiphoton multimodal microscopy (NMMM) used in biological imaging is a technique that explores the combinatorial use of different multiphoton signals, or modalities, to achieve contrast in stained and unstained biological tissues. NMMM is a nonlinear laser-matter interaction (LMI), which utilizes multiple photons at once (multiphoton processes, MP). The statistical probability of multiple photons arriving at a focal point at the same time is dependent on the two-photon absorption (TPA) cross-section of the molecule being studied and is incredibly difficult to satisfy using typical incoherent light, say from a light bulb. Therefore, the stimulated emission of coherent photons by pulsed lasers are used for NMMM applications in biomedical imaging and diagnostics.In this dissertation, I hypothesized that due to the near-IR wavelength of the Ytterbium(Yb)-fiber laser (1070 nm), the four MP-two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF) and third harmonic generation (THG), generated by focusing this ultrafast laser, will provide contrast to unstained tissues sufficient for augmenting current histological staining methods used in disease diagnostics. Additionally, I hypothesized that these NMMM images (NMMMIs) can benefit from computational methods to accurately separate their overlapping endogenous MP signals, as well as train a neural network for image classification to detect neoplastic, inflammatory, and healthy regions in the human oral mucosa. Chapter II of this dissertation explores the use of NMMM to study the effects of storage on donated red blood cells (RBCs) using non-invasive 2PEF and THG without breaching the blood storage bag. Unlike the lack of RBC fluorescence previously reported, we show that with two-photon (2P) excitation from an 800 nm source, and three-photon (3P) excitation from a 1060 nm source, there was sufficient fluorescent signal from hemoglobin as well as other endogenous fluorophores. Chapter III employs NMMM to establish the endogenous MP signals present in healthy excised and unstained mouse and Cynomolgus monkey retinas using 2PEF, 3PEF, SHG, and THG. We show the first epi-direction detected cross-section and depth-resolved images of unstained isolated retinas obtained using NMMM with an ultrafast fiber laser centered at 1070 nm and a 303038 fs pulse. Two spectrally and temporally distinct regions were shown; one from the nerve fiber layer (NFL) to the inner receptor layer (IRL), and one from the retinal pigmented epithelium (RPE) and choroid. Chapter IV focuses on the use of minimal NMMM signals from a 1070 nm Yb-fiber laser to match and augment H&E-like contrast in human oral squamous cell carcinoma (OSCC) biopsies. In addition to performing depth-resolved (DR) imaging directly from the paraffin block and matching H&E-like contrast, we showed how the combination of characteristic inflammatory 2PEF signals undetectable in H&E stained tissues and SHG signals from stromal collagen can be used to analytical distinguish healthy, mild and severe inflammatory, and neoplastic regions and determine neoplastic margins in a three-dimensional (3D) manner. Chapter V focuses on the use of computational methods to solve an inverse problem of the overlapping endogenous fluorescent and harmonic signals within mouse retinas. The least-squares fitting algorithm was most effective at accurately assigning photons from the NMMMIs to their source. This work, unlike commercial software, permits using custom signal source reference spectra from endogenous molecules, not from fluorescent tags and stains. Finally, Chapter VI explores the use of the OSCC images to train a neural network image classifier to achieve the overall goal of classifying the NMMMIs into three categories-healthy, inflammatory, and neoplastic. This work determined that even with a small dataset (< 215 images), the features present in NMMMIs in combination with tiling, transfer learning can train an image classifier to classify healthy, inflammatory, and neoplastic OSCC regions with 70% accuracy.My research successfully shows the potential of using NMMM in tandem with computational methods to augment current diagnostic protocols used by the health care system with the potential to improve patient outcomes as well as decrease pathology departmental costs. These results should facilitate the continued study and development of NMMM so that in the future, NMMM can be used for clinical applications.
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Title: Chromophore rotational dynamics as a probe of local organization in bulk and interfacial systems
Creator: Mize, Hannah
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "The use of chromophore rotational motion to study local organization is well established. Under certain conditions, the same measurements can be used to evaluate the effect(s) of thermal energy transfer from the chromophore to the bath. Such information is of central importance to gaining a fundamental understanding of the intermolecular interactions that are ultimately responsible for bulk system properties, and to the creation of biomimetic mono- and bilayer structures. This thesis...
Show more"The use of chromophore rotational motion to study local organization is well established. Under certain conditions, the same measurements can be used to evaluate the effect(s) of thermal energy transfer from the chromophore to the bath. Such information is of central importance to gaining a fundamental understanding of the intermolecular interactions that are ultimately responsible for bulk system properties, and to the creation of biomimetic mono- and bilayer structures. This thesis includes studies that address each of these areas. The first study, focusing on understanding the molecular scale consequences of thermal energy dissipation in bulk systems, reports on the rotational diffusion dynamics of tetracene in a series of n-alkane solvents, where differences in those dynamics were measured for excitation to the S1 and S2 states. These data demonstrated that for excitation to the S2 state, fast nonradiative relaxation to the S1 state produces transient heating of the solvent surrounding the chromophore, with the details depending on the identity of the solvent bath. The second study aims at understanding the influence of the aqueous overlayer in contact with a planar bilayer on molecular scale organization and phase separation. These data reveal organization-dependent changes in the bilayer that depend on both the pH and the ionic strength of the aqueous overlayer. These findings are of direct relevance to the use of such films in the construction of biomimetic sensors, for example, where organization is expected to influence the ability of the bilayer to host biomolecules in their active forms and to mediate permeability."--Page ii.
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Title: Leveraging capillary zone electrophoresis-mass spectrometry for multi-level proteomics
Creator: Shen, Xiaojing
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Mass spectrometry (MS) coupled with online liquid-phase separation is the major tool for large-scale bottom-up proteomics (peptide-centric), top-down proteomics (proteoform-centric), and native proteomics (protein complex-centric). While liquid chromatography (LC)-MS is the dominant method for proteomics at different levels, capillary zone electrophoresis (CZE)-MS has emerged as a valuable and complementary technique, which provides high-capacity separation and highly sensitive detection of...
Show moreMass spectrometry (MS) coupled with online liquid-phase separation is the major tool for large-scale bottom-up proteomics (peptide-centric), top-down proteomics (proteoform-centric), and native proteomics (protein complex-centric). While liquid chromatography (LC)-MS is the dominant method for proteomics at different levels, capillary zone electrophoresis (CZE)-MS has emerged as a valuable and complementary technique, which provides high-capacity separation and highly sensitive detection of peptides, proteoforms and even protein complexes under native conditions. This work focuses on developing novel CZE-MS/MS methods for multi-level proteomics (bottom-up, top-down, and native).In Chapter 2, a high-throughput bottom-up proteomics workflow was developed by coupling immobilized trypsin-based speedy protein digestion with fast CZE-MS/MS. Immobilized trypsin produced almost the same digestion performance as free trypsin for complex proteomes with about 50-times higher speed (15 min vs. 12 h). Integration of immobilized trypsin (IM)-based rapid protein cleavage and fast CZE-MS/MS enables the identification of thousands of proteins from the mouse brain proteome in only 3 h, which is significantly faster than the typical LC-MS-based bottom-up proteomics workflow (3 h vs. >12 h). The high-throughput workflow was expected to be useful for bottom-up proteomics of human clinical samples (e.g., serum and urine).Chapter 3 presents the first example of CZE-MS/MS with activated ion-electron capture dissociation (AI-ECD) on a high-end quadrupole-time-of-flight (Q-TOF) mass spectrometer for top-down proteomics, enabling high-resolution separation, highly sensitive detection, and extensive gas-phase backbone cleavages of proteoforms. The CZE-AI-ECD method will be useful to the top-down proteomics community for the comprehensive characterization of proteoforms in complex proteomes. Chapter 4 and 5 focus on the development of novel CZE-MS methods for native proteomics, delineating proteins and protein complexes under native conditions. In Chapter 4, a native CZE-MS/MS platform with an Orbitrap mass spectrometer was established for native proteomics of a complex proteome (E. coli), leading to the identification of 23 protein complexes in discovery mode. The work represents the first example of native proteomics via coupling online liquid-phase separation to native MS and MS/MS. The characterization of large protein complexes (up to 200 kDa) was also achieved with a new CZE-MS system on a high-end Q-TOF mass spectrometer.In Chapter 5, a novel native capillary isoelectric focusing (cIEF)-assisted CZE-MS method is presented for the characterization of monoclonal antibodies (mAbs) with large sample loading capacity and high separation resolution. Using the method, the potential separations of different conformations of the SigmaMAb and the detection of its various glyco-proteoforms and homodimer were documented. The method separated the NISTmAb into three peaks with a microliter sample loading volume, corresponding to its different proteoforms. In addition, eight glyco-proteoforms of the NISTmAb and its homodimer were detected. The results demonstrate the potential of the native cIEF-assisted CZE-MS method for advancing the characterization of large proteins (i.e., mAbs) and protein complexes under native conditions.
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Title: Theoretical and experimental studies of multistep electrochemical biosensors
Creator: Rafat, Neda
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Electrochemical biosensors are analytical devices that detect analytes by transforming a biochemical reaction into a quantitative, electrical signal. This class of biosensors has proven valuable in research, quality control, food safety, medical diagnosis, and monitoring of therapeutic efficacy. Electrochemical biosensors integrate the specificity of biological recognition molecules (e.g., antibodies) with the advantages of electrochemical detection techniques (reproducible, quantitative...
Show moreElectrochemical biosensors are analytical devices that detect analytes by transforming a biochemical reaction into a quantitative, electrical signal. This class of biosensors has proven valuable in research, quality control, food safety, medical diagnosis, and monitoring of therapeutic efficacy. Electrochemical biosensors integrate the specificity of biological recognition molecules (e.g., antibodies) with the advantages of electrochemical detection techniques (reproducible, quantitative electrical output) to provide sensitive and specific analytical devices. Miniaturized amperometric biosensors that use redox enzymes to generate an electric current in response to the voltage applied at a working electrode have been successfully commercialized.Mechanistic mathematical models that describe the multiple mass-transfer and chemical-reaction steps that give rise to the electrical output are needed to help design, optimize, and validate electrochemical biosensors for medical and environmental applications.In this work, experimental and theoretical studies of two types of multistep electrochemical biosensors were performed. An electrochemical immunosensor (EI) was fabricated on screen-printed electrodes (SPEs) for detection of a model protein (mouse IgG) by integrating principles of an enzyme-labled immunosorbent assay (ELISA) using horseradish peroxidase (HRP) as the labeling enzyme and an electrochemical transducer. Experimental conditions such as substrates concentration, pH, and applied voltage were optimized using a fractional factorial design. A mathematical model was developed to simulate the EI's steady-state signal by solving the non-linear ordinary differential equations including enzyme kinetics and diffusion-based mass transfer rates for all the reactants. A new concept, current-control coefficient, was introduced to measure the extend to each reaction step limited the current density. The model allows the rate limiting step to be identified and experimental conditions that optimize detection sensitivity to be determined.In addition, experimental and theoretical studies of an inhibition-based bi-enzyme electrochemical biosensor (IBE) for a model inhibitor of acetylcholinesterase (AChE), phenylmethylsulfonyl fluoride (PMSF), were conducted. The IBE was fabricated by co-immobilization of AChE and tyrosinase (Tyr) on the gold working electrode of a SPE. The inclusion of a hydrolase enzyme (AChE) and an oxidase enzyme (Tyr) provided an amplification system which improved the biosensor's sensitivity significantly. A comprehensive mathematical model was developed to simulate the time-dependent electrochemical signal in the IBE. The unsteady-state model was developed by solving a system of non-linear partial differential equations including enzymatic reactions, inhibition kinetics of AChE by an inhibitor (PMSF), and diffusion-based mass transfer steps. The model successfully simulated the IBE's response to the substrate (phenylacetate) and the inhibitor. Using the model along with the current-control coefficient and sensitivity parameters, the effect of the governing factors on the IBE's performance was studied. The model allowed to optimize the governing factors to achieve optimum sensitivity for detection of the inhibitor and design the biosensor to achieve specific performance criteria.
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Title: Molecular Dynamics Controlling the Robustness of Cu(II)-Complexed Thin Films and the Excited-State Behavior of Photoactive Reagents
Creator: Capistran, Briana Ashley
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Understanding the molecular interactions that control chemical processes is essential for developing novel advancements in any chemical industry, especially for the fields of surface modifications and photoactive chemical reagents. Monolayers and thin films are essential to a wide range of surface chemistry and materials science applications, such as chemical separations, heterogeneous catalysis, and tribology. However, adlayers currently utilized have fixed properties upon deposition onto...
Show moreUnderstanding the molecular interactions that control chemical processes is essential for developing novel advancements in any chemical industry, especially for the fields of surface modifications and photoactive chemical reagents. Monolayers and thin films are essential to a wide range of surface chemistry and materials science applications, such as chemical separations, heterogeneous catalysis, and tribology. However, adlayers currently utilized have fixed properties upon deposition onto substrates. Alternatively, photoactive reagents, specifically super-photobases, are pivotal for controlling the temporal and spatial extents of photochemical reactions used in industries such as precision chemistry and high-speed chemical sensing. Nonetheless, few known super-photobases currently exist, and little is understood about the molecular dynamics that give rise to such reactive properties upon photoexcitation. As such, this two-part work focused on the characterization of metal ion-complexed films as candidates for the creation of films with reversible properties, as well as the investigation of the excited-state behavior and spectral dynamics of super-photobase precursor molecules.The first part of this work focused on the study of the molecular properties that lead to the robustness of Cu2+-complexed amphiphilic Langmuir-Blodgett films. Incorporation of Cu2+ ions into the film system resulted in the unique formation of a highly ordered, rigid film system resistant to collapse. Film orientation and mobility were determined to be controlled by Cu2+-amphiphile interactions and a shift in dominating intermolecular forces as subphase pH increased. The unique presence of a rigid film at high surface pressures and pH conditions was attributed to the extrusion of Cu2+-amphiphile moieties into the aqueous subphase rather than traditional amphiphile buckling, which occurs at low pH conditions. In the second part of this work, the excited-state behavior of FR0, a substituted fluorene precursor to the recently developed super-photobase FR0-SB, was studied to determine the state-dependent solvent-solute interactions that give rise to the unique reactivity of the photobase molecule upon photoexcitation (ΔpKb ~ 15). Using time-resolved fluorescence spectroscopy and quantum chemical calculations, the electronic structure and relaxation dynamics of FR0 were examined in protic and aprotic solvents. Hydrogen-bonding interactions in protic solvents, particularly those in the S2 excited state, were determined to mediate relaxation dynamics. Solvent hydroxyl functionality concentration was also determined to play a role in such dynamics. Examination of the behaviors of structurally modified versions of FR0 demonstrated that the spectral dynamics observed for FR0 are structurally invariant, but that the nature of hydrogen bonding changes depending on the placement and type of modification. Overall, this work has led to a fundamental characterization of unique classes of surface modifications and photoactivated chemical reagents. First, the dynamics leading to unusually robust thin films were determined, the understanding of which can lead to the creation of selective interfaces using such films. Second, the intermolecular interactions lending themselves to unique spectral properties of super-photobase precursor molecules were identified, which can serve as a vantage point for the future synthesis and development of additional photobase compounds. In both aspects of this work, such advancements contribute to the furthering of each field by providing important fundamental frameworks for future applications in surface chemistry and photoactive reaction chemistry.
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Title: Method development for capillary electrophoresis mass spectrometry (CE-MS)-based proteomics and application to uncovering proteome dynamics of zebrafish embryos during early embryogenesis
Creator: Chen, Daoyang
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Reversed-phase liquid chromatography (RPLC) coupling with tandem MS (MS/MS) is often the method of choice in both peptide-centric bottom-up proteomics (BUP) and proteoform-centric top-down proteomics (TDP) studies. In recent years, capillary zone electrophoresis (CZE)-MS has attracted attention as another platform in proteomics due to high separation efficiency, high sensitivity, and complementarity to LC-MS. This work is dedicated to developing novel CE-MS-based methods for large-scale...
Show moreReversed-phase liquid chromatography (RPLC) coupling with tandem MS (MS/MS) is often the method of choice in both peptide-centric bottom-up proteomics (BUP) and proteoform-centric top-down proteomics (TDP) studies. In recent years, capillary zone electrophoresis (CZE)-MS has attracted attention as another platform in proteomics due to high separation efficiency, high sensitivity, and complementarity to LC-MS. This work is dedicated to developing novel CE-MS-based methods for large-scale proteomics and applies them to study the proteome dynamics of zebrafish embryos during early embryogenesis.In Chapter 2, a sample stacking method, dynamic pH junction, was systematically investigated and employed to improve CZE’s sample loading capacity for large-scale BUP. The results of the optimized system represent the highest loading capacity, the highest peak capacity, and the widest separation window of CZE for peptide separation to date. The automated CZE-MS system opened the door to using CZE-MS for large-scale BUP. In Chapter 3, for the first time, a strong cation exchange (SCX)-RPLC-CZE-MS/MS platform was established for deep BUP and phosphoproteomics. The platform approached comparable performance to the modern 2D-LC-MS/MS for deep proteomic sequencing evident by identifying 8200 protein groups and 65,000 unique peptides from a mouse brain proteome digest, 11,555 phosphopeptides from the HCT116 cell line. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein, peptide, and phosphopeptide IDs. In Chapter 4, a quantitative BUP study was performed on zebrafish embryos across four developmental stages during the maternal-to-zygotic transition (MZT) via coupling isobaric tag for relative and absolute quantitation (iTRAQ) chemistry with both RPLC-MS/MS and CZE-MS/MS. Expression kinetics of nearly 5000 proteins including over 100 transcription factors (TFs) across four early embryonic stages were determined. The protein expression profiles fall into several different clusters and accurately reflect the important events during early embryogenesis. Further studies of the expression profiles of TFs revealed that the differentially expressed TFs during the MZT show wave-like expression patterns. Top-down proteomics (TDP) aims to directly characterize proteoforms in cells. CZE-MS/MS has been demonstrated as a useful tool for TDP. In Chapter 5, for the first time, we evaluated various semiempirical models for predicting proteoforms’ electrophoretic mobility using large-scale TDP data sets from earlier CZE–MS/MS studies. Linear correlations were achieved between the experimental and predicted μef of E. coli proteoforms and histone proteoforms (R2 = 0.98), demonstrating that the μef of proteoforms in CZE-MS can be predicted accurately, which could be useful for validating the confidence of proteoform IDs from a database search. In Chapter 6, we concluded the results of this dissertation and provided our expectations for future studies.
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Title: Surface energy characterization and competitive surface equilibria using contact angle
Creator: Stubbs, Barrack Perez
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Surface energy measurements are usually difficult to assess when not considering a solid-air or liquid-air interface. Contact angles was used as a method of obtaining the interfacial energy of a silanol-water surface using carbon tetrachloride as a probing liquid. The theory for this work operated under the assumption that the pKa of a silanol surface could be measured by obtaining a titration like curve as a function of pH. A decrease in contact angle of about 7° was observed at a pH (pKa)...
Show moreSurface energy measurements are usually difficult to assess when not considering a solid-air or liquid-air interface. Contact angles was used as a method of obtaining the interfacial energy of a silanol-water surface using carbon tetrachloride as a probing liquid. The theory for this work operated under the assumption that the pKa of a silanol surface could be measured by obtaining a titration like curve as a function of pH. A decrease in contact angle of about 7° was observed at a pH (pKa) of 3.5. This corresponds to an energy difference of 2 mN/m. -- Abstract.
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Title: Fundamental studies of competitive equilibria at silica surfaces
Creator: Stubbs, Barrack Perez
Date: 2019
Collection: Electronic Theses & Dissertations
Description: "The fundamental interactions such as physisorption at solid-liquid interfaces are not fully characterized. The physical properties and chemical reactivity at the silica surface is one that is subject to much investigation. A variety of analytical techniques, including solid state nuclear magnetic resonance (SS-NMR ) and high-performance liquid chromatography (HPLC) were utilized to help characterize the surface of silica. These techniques were combined in order to understand the fundamental...
Show more"The fundamental interactions such as physisorption at solid-liquid interfaces are not fully characterized. The physical properties and chemical reactivity at the silica surface is one that is subject to much investigation. A variety of analytical techniques, including solid state nuclear magnetic resonance (SS-NMR ) and high-performance liquid chromatography (HPLC) were utilized to help characterize the surface of silica. These techniques were combined in order to understand the fundamental mechanisms of surface interactions such as adsorption mechanisms and chemical reactivity. The pH, ionic strength, and mobile phase compositions were varied throughout the experiments as a basis for assessing reactivity.The SS-NMR data shows a correlation between pH and labile surface hydrogens. At higher pH values, the transfer of excitation from labile surface hydrogens to surface silicon atoms is diminished. A change in peak ratios that correspond to labile primary silanols and surface bridging siloxanes was observed. The relative concentration of bridging siloxanes is constant whereas the signal from primary silanols is greatly diminished.Aqueous, normal-phase HPLC shows the various interactions of phenol with the silica surface. Multiple peaks were generated from a single analyte when the water/methanol concentration was at least 20%/80%. At 90% and 100% methanol, a single peak is shown for phenol. The ratio and number of peaks depends on conditions such as solvent composition, pH, ionic strength, flow rate, and temperature. We argue that the distribution of surface silanol sites is responsible for the generation of multiple peaks that are seen in the extreme aqueous conditions. These sites, as well as the water in the mobile phase, modify the mass transport term in the Van Deemter equation. The conditions and results obtained in these experiments are explained below with an emphasis on mass transport.Additionally, other analytical techniques such as scanning electron microscopy, thermogravimetric analysis, and inductively coupled plasma were used to help characterize the silica surface. Through these techniques, values for surface silanol concentration ranging from 1.6micromole/m2 to 7.0micromole/m2 were obtained. The physical morphology of the silica surface was also characterized before and after exposure to basic environments."--Pages ii-iii.
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Title: Mass spectrometric strategies for profiling of electrophilic oxylipin metabolites and their glutathione conjugates
Creator: Bao, Jiangyin
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Plants cannot escape from herbivory and other environmental stresses including cold and drought, and have evolved sophisticated defense systems to survive. Improving food production has become one of the most urgent problems facing humankind with 7 billion population and limited land available. Plants also serve as the base of the earth's sustainable fuel supply, and synthesize a diverse suite of natural compounds that help defend them against stress, but also are potential pharmaceutics....
Show morePlants cannot escape from herbivory and other environmental stresses including cold and drought, and have evolved sophisticated defense systems to survive. Improving food production has become one of the most urgent problems facing humankind with 7 billion population and limited land available. Plants also serve as the base of the earth's sustainable fuel supply, and synthesize a diverse suite of natural compounds that help defend them against stress, but also are potential pharmaceutics. Improving our understanding of plant defense systems is a key factor in using plants as natural resources to provide solutions to these problems. To defend themselves, plants synthesize oxidized fatty acids, or oxylipins, to regulate gene expression in response to stress. In response to wounding and certain stresses, many plants synthesize the cyclopentenone oxylipin 12-oxo-phytodienoic acid (OPDA) as a precursor of the master regulatory hormone jasmonic acid (JA). JA is then conjugated to isoleucine in cytoplasm to produce the universal defense gene regulator JA-isoleucine. OPDA has been shown to be an independent but not fully understood metabolite that regulates plant defenses. Reactive electrophiles such as OPDA are subject to conjugation to the tripeptide glutathione. This process is catalyzed by an assortment of glutathione transferase enzymes, and is expected to deactivate the biological functions of electrophiles. However, understanding of the functions of specific GSTs (>50 in model plant Arabidopsis from sequenced genome), particularly in plants, is limited.The research described in this dissertation has aimed to reveal biological functions of GSTs in defense response to mechanical wounding in Arabidopsis. The basic strategy has combined information about GST protein levels with profiling of metabolites in a GST knockout mutant to correlate metabolic phenotypes with genotypes, for deduction of gene functions. 8 GSTs were identified in wild type Arabidopsis leaves, and one of them, AtGSTU5, was shown to be highly accumulated after mechanical wounding. Several Arabidopsis knockout mutants, including AtGSTu5, were grown for wounding experiments and phenotype assessment using non-targeted metabolite profiling. The glutathione conjugate of OPDA was quantified and shown to be accumulated after mechanical wounding in leaves of wild type and other tau-family GST knockout mutants of Arabidopsis, but not in the knockout mutant AtGSTu5. This finding suggests that AtGSTU5 is responsible for in vivo glutathione conjugation of OPDA. A new LC-TOF-MS protocol was developed to explore the range of endogenous glutathione conjugates in extracts of Arabidopsis leaves. A family of novel glutathione conjugates is also discovered and proposed to be derived from OPDA-containing galactolipids. In the identification and quantitation of plant oxylipins, tandem mass spectrometry (MS/MS) data are often the primary source of metabolite structural information, but ion fragmentation pathways are not well understood for negative ions. This dissertation describes a novel fragmentation mechanism in negative mode involving charge-directed hydride migration for OPDA and its lower homolog dinorOPDA. This mechanism has potential to guide structure elucidation of unknown oxylipins. For other cyclopentenone prostaglandins with greater degrees of unsaturation, however, fragmentation behavior differed. Relative amounts of specific product ions distinguished prostaglandins with identical side chains but opposite cyclopentenone ring orientations.
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Title: DEVELOPING ULTRASENSITVE MS-BASED PROTEOMIC PLATFORMS FOR THE CHARACTERIZATION OF MASS-LIMITED SAMPLES
Creator: Yang, Zhichang
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Single cell analysis such as single cell sequencing has shed invaluable light on cellular heterogeneity and understanding molecular mechanisms such as cell differentiation. Modern single cell sequencing techniques have achieved throughput of analyzing thousands of single cells per day with sequencing depth of thousands of genes. Proteins play vital roles in almost all biological processes and have been crucial biomarkers for disease diagnosis and drug development. Unlike DNA and RNA molecules...
Show moreSingle cell analysis such as single cell sequencing has shed invaluable light on cellular heterogeneity and understanding molecular mechanisms such as cell differentiation. Modern single cell sequencing techniques have achieved throughput of analyzing thousands of single cells per day with sequencing depth of thousands of genes. Proteins play vital roles in almost all biological processes and have been crucial biomarkers for disease diagnosis and drug development. Unlike DNA and RNA molecules, protein molecules can’t be amplified, making the large-scale characterization of proteins (proteomics) challenging for mass-limited samples such as single cells. Novel proteomics methodologies with extremely high sensitivity are vital for analysis of mass-limited samples. This work focuses on developing ultrasensitive Mass Spectrometry (MS)-based proteomics platforms to enable large-scale proteome profiling of mass-limited samples. In Chapter 2, we applied nanoRPLC-CZE-MS/MS platform for large scale proteome profiling on 5 μg of a MCF7 cell digest. The digest was fractionated by the nanoRPLC, followed by dynamic pH junction based CZE-MS/MS. The nanoRPLC-CZE-MS/MS produced over 7500 protein IDs and nearly 60000 peptide IDs from the 5-μg MCF7 proteome digest. It reduced the required amount of complex proteome digests for LC-CZE-MS/MS-based deep bottom-up proteomics by two orders of magnitude. In Chapter 3, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500-ng peptides using a Q-Exactive HF mass spectrometer. In addition, we coupled single spot solid phase sample preparation (SP3) method for sample preparation on 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells. In Chapter 4, we developed a Nanoparticle-aided Nanoreactor for Nanoproteomics (Nano3) technique for processing few mammalian cells for bottom-up proteomics. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins followed by nanoRPLC-MS/MS analysis. The Nano3 method identified 40-times higher number of proteins from 2-ng mouse brain protein samples compared to the low volume SP3 method. The Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 1084 ± 287 (N=4) protein IDs from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. In Chapter 5, we developed a universal sample preparation method for denaturing top-down proteomics (dTDP), and the method combined the sodium dodecyl sulfate (SDS)-based protein extraction and the membrane ultrafiltration (MU)-based protein cleanup. The MU method outperformed CMP (chloroform-methanol precipitation) and SP3 methods, resulting in high and reproducible protein recovery from both E. coli cell (59±3%) and human HepG2 cell (86±5%) samples without a significant bias. The assay afforded identification of various post-translational modifications and protein containing transmembrane domains through top-down analysis.
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Title: Manipulating antibody-antigen interactions in microporous membranes for selective antibody and protein purification
Creator: Bennett, Austin Landry
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Monoclonal antibodies are among the most successful and most expensive therapeutic agents. Rapid, inexpensive antibody capture should decrease the cost of both production and detection of these remarkable drugs. Affinity membranes can potentially capture protein more rapidly than traditional bead-based methods without the large pressure drops of columns.This research employed an Fc (fragment, crystallizable) binding peptide (denoted as KK12) and an Fab (fragment, antigen-binding) binding...
Show moreMonoclonal antibodies are among the most successful and most expensive therapeutic agents. Rapid, inexpensive antibody capture should decrease the cost of both production and detection of these remarkable drugs. Affinity membranes can potentially capture protein more rapidly than traditional bead-based methods without the large pressure drops of columns.This research employed an Fc (fragment, crystallizable) binding peptide (denoted as KK12) and an Fab (fragment, antigen-binding) binding peptide (denoted as K19) for rapid antibody capture in membranes. KK12-modified membranes captured 8.40 mg of Herceptin and 9.96 mg of Avastin per mL of membrane. The antibodies eluted in 100 mM Gly (pH 2.7), but the membranes were not reusable. K19-modified membranes capture 16.3 mg of Herceptin per mL of membrane and showed minimal binding of Avastin, as expected because K19 mimics the HER2 Herceptin antigen. Furthermore, K19-modified membranes selectively captured Herceptin from human serum and showed no significant non-specific adsorption. Antibody-containing membranes may also enable rapid, high-throughput immunoprecipitation. Anti-(hemagglutinin A) (HA) antibodies successfully captured HA-tagged regulator G-protein signaling 2 (HA-RGS2) from cell lysate and HA-RGS2 subsequently eluted in 5% formic acid. Non-specific adsorption, however, is a persistent problem that must be addressed prior to downstream analyses of eluted proteins. Anti-(C-peptide) antibodies captured up to 4 pmoles of C-peptide. This is a promising result for future studies that will employ membrane-immobilized anti-(C-peptide) to selectively capture C-peptide from cell secretions to help determine the role of this peptide in diabetic complications.
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Title: Electrically driven ion separations using membranes coated with polyelectrolyte multilayers or conductive films
Creator: Zhu, Yan (Graduate of Michigan State University)
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Ion-exchange membranes are attractive for salt recovery and brackish water desalination because they provide high selectivities between cations and anions. Nevertheless, typical ion-exchange membranes show only modest selectivities among cations or anions, and such selectivities are important in applications including water softening, acid recovery, and salt purification. This dissertation explores coating of membranes with polyelectrolyte multilayers (PEMs) or conductive films to enhance...
Show moreIon-exchange membranes are attractive for salt recovery and brackish water desalination because they provide high selectivities between cations and anions. Nevertheless, typical ion-exchange membranes show only modest selectivities among cations or anions, and such selectivities are important in applications including water softening, acid recovery, and salt purification. This dissertation explores coating of membranes with polyelectrolyte multilayers (PEMs) or conductive films to enhance selectivity in electrically driven ion transport. Adsorption of PEMs on Nafion membranes gives rise to high monovalent/divalent cation selectivities in electrodialysis (ED), but the high cost of Nafion may preclude its use in many ED applications. This work demonstrates that relatively inexpensive Fujifilm cation-exchange membranes modified with protonated poly(allylamine) (PAH)/poly (4-styrenesulfonate) (PSS) films have extremely high K+/Mg2+ cation selectivities >1000 in ED. The high exclusion of Mg2+ suggests a complete and dense PEM, presumably because the smooth Fujifilm surface allows formation of a continuous coating. The PEM formed on the anode side of the membrane is essential for the high selectivity, whereas the cathode-side coating contributes only a small amount to resistance of the membrane system and little selectivity. Current density-voltage curves and transference numbers suggest that water splitting occurs at overlimiting currents. Overall, these highly selective membranes may be attractive for salt purification and salt recovery using ED. Although PEM-modified cation-exchange membranes show high cation-selectivity during ED, the current efficiency is only ~0.5, which implies that unwanted ions carry 50% of current. Adsorption of (PDADMAC/PSS)n films on Nafion membranes leads to high monovalent /divalent cation selectivity in ED, and moreover, the monovalent cation current efficiency is as high as 0.8. (PDADMAC/PSS)3PDADMAC films give the highest current efficiency in both K+/Mg2+ and Li+/Co2+ separations. The high current efficiency presumably results from the high aqueous swelling of (PDADMAC/PSS)n films to increase the monovalent cation permeance. However, (PDADMAC/PSS)n films are not stable in solutions with high salt concentrations, so future work should aim to increase the film stability. The high selectivity of PEMs partly stems from the high surface charge of the thin film. Thus, instead of introducing membrane surface charge through adsorption of polyelectrolytes, this work also aimed to develop conductive membranes to create surface charge using an electrical potential applied between the conducting membrane and a reference electrode in the source phase. The potential-dependent surface charge should alter cation and anion partitioning into the membrane. Electroless deposition of gold followed by electrosynthesis of poly(3,4-ethylenedioxythiophene) gives highly conductive membrane with sheet resistances <100 Ω/. Unfortunately, the ion fluxes do not significantly change with applied potential. Finally, this dissertation investigates the possible mechanism of high salt rejection in nanofiltration through PEM-coated NF270. The surface charge may create regions of nonelectroneutrality in the membrane, and the low concentration of the excluded ion in this region should control the resistance to salt transport.
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