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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

High Mass Accuracy Coupled to Spatially-Directed Proteomics for Improved Protein Identifications in Imaging Mass Spectrometry Experiments

Rizzo, David Geoffrey 08 June 2016 (has links)
<p>Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the study of protein distributions in biological tissue specimens. This has traditionally been performed using time-of-flight (TOF) systems due to their large practical mass range, high dynamic range, and high throughput of the TOF analyzer. While many proteins are detected with this technology, the unambiguous identification of these analytes remains challenging. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. This project incorporates high mass resolving power and high mass accuracy Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) into protein IMS experiments. With sample preparation and instrument methodologies tailored for improved protein signal detection, proteins up to 17 kDa were detected with resolving powers of 75,000 and mass accuracies less than 5 ppm. Additionally, in situ digestions were utilized to investigate proteins above current observable mass ranges of the FTICR. Incubation times for enzymatic digestions were compared using IMS approaches to evaluate maximum peptide signal while minimizing delocalization.</p> <p>In order to improve confidence in protein and peptide identifications, identification strategies that preserve some form of spatial information were employed. Initially, liquid microextractions were used to selectively interrogate regions of tissue after in situ digestion. Localization of extracted peptides provided an additional level if information to correlate back to the IMS data. To further decrease the extraction diameter, hydrogel technologies for spatially-localized protein digestion/extraction were modified. Parameters such as percent polyacrylamide used in hydrogel construction as well as concentration of trypsin with which the hydrogel is loaded were investigated to maximize the number of protein identifications from LC-MS/MS analysis of hydrogel extracts. Increased polyacrylamide concentrations led to more rigid polymers, which were more amenable to perforating hydrogels using small punch biopsies. Hydrogels were fabricated with diameters as small as 260 µm, while still providing over 600 protein identifications. These improved methods to the hydrogel process allow researchers to target smaller biological features for robust spatially-localized proteomic analyses. Integrating high mass accuracy instrumentation with regio-specific proteomics experiments allows for confident identifications of proteins, providing insight into underlying biology of heterogeneous samples.</p>

Tip-Enhanced Laser Ablation Sample Transfer for Mass Spectrometry

Seneviratne, Chinthaka Aravinda 03 May 2016 (has links)
In this research, atomic force microscope tip-enhanced laser ablation mass spectrometry (AFM TELA-MS), an ambient sub-micrometer scale sampling method for offline MS was developed. AFM TELA was used to transfer molecules from thin films to a suspended silver wire for off-line mass spectrometry using laser desorption ionization (LDI) and matrix-assisted laser desorption ionization (MALDI). An AFM with a 30 nm radius gold-coated silicon tip was used to image the sample and to hold the tip 15 nm from the surface for material removal using a pulsed Nd:YAG laser, which provides output at wavelengths of 532 nm in the visible, 1064 nm in the near IR, or the 355 nm UV wavelength. The laser is mildly focused onto the AFM tip and the fluence is set just below the far-field ablation threshold to irradiate the AFM tip for material removal with a smaller spot size than a laser focused with a conventional lens system. The AFM is used to image ablation craters and place the tip at the area being analyzed. For small molecules, approximately 100 fg of material was ablated from each of the 1 µm ablation spots and transferred with approximately 3% efficiency. AFM-TELA of large biomolecules was also demonstrated at 3% efficiency and a mass range up to 600 Da. AFM-TELA studies with different laser parameters indicated that the tip-enhanced material ejection depends on laser wavelength, polarization, fluence, and number of laser shots used for material ejection, but not on the absorption of the sample itself. The utility of AFM-TELA was applied to sampling of rat brain tissue. The ability of producing sub-micrometer scale craters, capture on a suspended silver wire and detection of lipids were demonstrated using off-line MALDI MS.

Environmental influences on arctic halogen chemistry| Investigation of melt onset and snowpack properties

Burd, Justine Amanda 04 May 2016 (has links)
<p> Reactive halogen radicals (e.g. Br, Cl and their oxide forms) dominate tropospheric oxidation mechanisms during Arctic springtime (Feb. &ndash; Apr.) by depleting ozone and changing the fate of pollutants. During ozone depletion events, reactive bromine radicals rapidly oxidize mercury which gets subsequently deposited, becoming more bioavailable. During Arctic springtime, a heterogeneous surface reaction (referred to as BrO recycling) between hypobromous acid (HOBr) and bromide (Br-) rapidly increases the abundance of reactive bromine episodically up to 40 pptv peaks. However, as spring transitions to summer (May - June), elevated reactive bromine levels suddenly decrease. There are two key requirements to maintain BrO recycling including surface area and sea salt (i.e. bromide) abundance. This study investigated environmental factors that impact BrO recycling during late spring (May-June) in the Arctic, including temperature, snowpack depth and rain/snow precipitation events. Near horizon BrO was measured using Multi-AXis Differential Optical Absorption Spectroscopy (MAX-DOAS) at Barrow, AK and above frozen Arctic sea ice. The late spring &ldquo;end&rdquo; to elevated reactive bromine (referred as the Seasonal End Date, SED) was objectively determined at all sites (N=12). Air temperature-derived melt onset dates were determined for all sites (<i>N</i>=12) and occurred within two days of the SED (<i>RMS</i> = 1.8 days, <i>R<sup>2</sup></i> = 0.989). Through these studies, we determined BrO recycling is hindered by melt onset of snowpack, ending the reactive bromine season. </p>

Visible Light and Electron Transfer Chemistry for O-Glycosylation and Remote Hydroxylation

Spell, Mark Louis 05 May 2016 (has links)
This dissertation focuses on the synthesis of dauer pheromone analogs for biological evaluation, the development of visible light promoted O-glycosylation methods and the development of an Ir(ppy)3 catalyzed C-H hydroxylation method. Chapter 1 describes the synthesis of dauer pheromone analogs. C.elegans is a small nematode that enters a dauer stage when encountering unfavorable environmental conditions. The dauer stage is initiated by the nematodes chemosensation of the dauer pheromone caused by the down regulation biochemical pathways TGF-â and IGF-1. These biochemical pathways are also seen in Homo Sapiens and play a role in a number of biological processes. Understanding how these pathways work in C.elegans can give us a better understanding of how they function in humans. In order to see the effect structure has on binding to the GCPRs that initiate dauer formation a series of dauer pheromone analogs differing in chain length and degree of unsaturation were synthesized. These analogous were then tested to see there dauer inducing activity to give a structure to activity relationship. Chapter 2 centers upon the development of an alpha selective glycosylation of alcohols with selenoglycosides using visible light. Selenoglycosides are highly stable glycosyl donors. There stability is useful in oligosaccharide synthesis. This stability, however, requires harsh reaction conditions to activate them. The development of a mild and easily performed O-glycosylation method using selenoglycosides is described here. Chapter 3 focuses on the development of a metal free, visible light promoted O-glycosylation method using thioglycosides. Thioglycosides like selenoglycosides are commonly used in oligosaccharide synthesis. The harsh reaction conditions used to activate them has been a long standing issue in the carbohydrate community. The development of a mild, catalyst free O-glycosylation method using thioglycosides is described here. The method uses 4-methoxyphenyl-3-butenylthioglucoside donors in the presence of easily handled and bench stable Umemotos reagent to provide good yields of disaccharides, it is selective for the â-anomer when using the glycosyl donor with an acetate group at the 2- position, and the method is orthogonal. Chapter 4 details the development of a C-H hydroxylation method using fac-Ir(ppy)3 catalyst. 1-6 and 1-7-hydrogen atom transfers from unactivated aliphatics were performed with the aid of a Tzo directing group. The resulting radical then underwent redox chemistry followed by attack of water to give hydroxylated products in moderate yield.

Electrochemical Instrumentation for Real-Time Monitoring of Biological Model Systems

Cognata, Andrew David 11 April 2016 (has links)
New technologies for model biological systems aid in drug development and toxicology by providing an in vitro environment that closely mimics in vivo conditions but require new instrumentation to meet needs in monitoring and analysis. Techniques adapted from the existing Multianalyte Microphysiometer, an instrument used for the quantitative analysis of cell metabolism, were applied in the optimization of a screen-printed platinum electrode platform capable of monitoring levels of glucose, lactate, oxygen, and solution acidification in complex mixtures such as cell medium for integration into biological model systems. Various modifications including enzyme and metal films provide sensitivity and selectivity to the electrodes. Improvements were made in the function of the screen-printed electrode platform and preliminary steps in the integration of the electrodes with a hollow-fiber bioreactor intended to simulate the environment of the blood-brain barrier were performed. Challenges in the interfacing of biological model systems with fundamentally unsterile instruments are identified and addressed.

Enhanced separations strategies for complex sample characterization using ion mobility-mass spectrometry

Montenegro Burke, Jose Rafael 21 April 2016 (has links)
Ion mobility-mass spectrometry (IM-MS) is an analytical technique used to separate gas-phase ions based on their structural properties such as size and shape as well as their mass, in the IM and MS dimensions, respectively. Furthermore, this analytical technique has become a widely used tool for a wide variety of fields. Its great sensitivity and selectivity provides scientists numerous advantages for the analysis of complex samples of numerous analyte types (biomolecules, synthetic polymers, metals, etc.) spanning a broad dynamic range. It is well known, that MS can be greatly improved when a separation is performed prior to ionization, more commonly liquid chromatography (LC). However, in the pursuit of faster separations and increased analyte coverage, both gas- and condensed phase techniques such as ion mobility (IM), supercritical fluid chromatography (SFC) and size exclusion chromatography (SEC) have been implemented. Their orthogonality was explored in the metabolome characterization of a wide variety of biological and synthetic samples.

Molecularly Imprinted Polymers for Enantiomer Separations and Biomolecular Sensors

Hebert, Britney Lyn 28 July 2016 (has links)
Molecularly Imprinted Polymers (MIPs) encompass a wide range of applications by changing the different components, e.g. the template, crosslinker or functional monomers. Of interest among these different applications are separations and sensors. Separations by MIPs traditionally use a chiral pure template but in some cases that chiral pure template may not be available for imprinting. Using chiral (N-α-bismethacyloyl-L-alanine) and achiral (N,O-bisacrylamide ethanolamine) crosslinkers we investigated imprinting of scalemic and racemic template mixtures of Boc-tyrosine enantiomers. The achiral and chiral crosslinkers yielded similar results for the partial separation of enantiomers by scalemic imprinted polymers because separation and recognition are not dependent on diastereomeric interactions here. The racemic imprinted polymers, however, required the chiral crosslinker for chiral differentiation. Surprisingly, variable D or L bias was observed in the L-NALA racemic imprints with equal probability over multiple replicates of polymer synthesis. The binding of the template to the polymer was evaluated in both batch rebinding and chromatographic modes, and the results will be discussed in detail. Another important area of MIPs is their applicability in sensor devices, especially for biological targets. A proven method of development of a sensor by molecular imprinting is by incorporating MIPs in a stimuli-responsive hydrogel. An imprinted hydrogel was developed to detect a DNA mir21 mimic using complementary aptamers in both a capillary hydrogel format and thin film hydrogel diffraction grating. The hydrogels imprinted for the DNA mir21 target were responsive to the re-introduction of the target sequence and selective among similar nucleotide sequences. It was also shown that an imprinted system was required; the pre-polymer complex of both the aptamers with the DNA mir21 mimic was necessary to achieve maximum stimuli response detected by shrinking of the hydrogel sensor.

Synthetic Efforts Towards the Synthesis of Prostaglandin PGF2a

Pollard, Amy Marie 29 July 2016 (has links)
This dissertation describes strategies for synthesizing prostaglandin PGF2α. Our synthetic design creates the stereochemistry needed for the core and side chains of the target prostaglandin PGF2 alpha and PGF2 alpha synthase selective analogues while incorporating iodocyclization desymmetrization of acyclic dienes. A model system for 4-(iodomethyl)-6-methyl-1,3-dioxan-2-one was developed and synthesized for our target compound 4-(2-(benzyloxy)ethyl)-6-(iodomethyl)-5-vinyl-1,3-dioxan-2-one. Both compounds were successfully synthesized providing useful stereocenters for completing the synthesis of prostaglandin PGF2 alpha. Efforts toward total stereochemical control of PGF2α include the partial syntheses of bis-diethylanimedimethylsilane and of (4S,5S)-2-((1E,3E)-penta-1,3-dien-1-yl)-4,5-diphenyl-1,3-ditosyl-1,3,2-diazaborolidine.

Spectroscopic Study of the Conformational Changes Involved in the Activation of CaMKII

Rodriguez Mena, Francisco Guillermo 22 July 2016 (has links)
The CaMKII enzyme is involved in diverse biological processes including control of heart rate and synaptic plasticity. CaMKII is activated by binding of calmodulin (CaM) and ATP. Its intricate structure and dynamics are thought to underlie its mode of self-regulation. The enzyme consists of catalytic, regulatory (auto-inhibitory), and association domains. Unregulated CaMKII activity has been implicated in cardiac pathologies. As such, a better understanding of the activation mechanism of the enzyme could contribute to the development of CaMKII-specific inhibitors. The hypothesis tested was that binding of ATP to CaMKII alters the flexibility of the regulatory domain and induces conformational rearrangements in the catalytic domain of the kinase. Toward this end, studies were made on monomeric constructs lacking the association domain. Spin labels were placed at specific positions in the regulatory and catalytic domains for distance measurements by Double Electron-Electron Resonance (DEER) in basal, +ATP, and +CaM conditions. Additionally, the effect of ATP on the CaM-binding region was studied on a per-residue basis by NMR. The findings demonstrate 1) a re-structuring of the regulatory domain upon CaM binding, 2) a change in regulatory domain dynamics due to ATP binding, consistent with increased exposure of the CaM-binding segment to the solvent, and 3) shorter distances within the catalytic domain after addition of ATP, in concordance with a closed catalytic domain conformation.

Design and Implementation of QCM Virtual Sensing Schemes for Analyses of Volatile Organic Compounds

Speller, Nicholas Colby 07 July 2016 (has links)
Sensor arrays have evolved as powerful approaches for providing detection and discrimination of volatile organic compounds (VOCs) as required across numerous analytical applications. Such systems typically comprise a number of cross reactive sensor elements, which generate analyte specific response patterns upon exposure to VOCs, and are known as multisensor arrays. When evaluated using statistical methods, these response patterns facilitate classification of VOCs. As an alternative, a single dynamically operated sensor could also be used to generate analyte specific response patterns. This approach is known as a virtual sensor array (VSA) and can exhibit significant advantages when compared to MSAs. Some advantages include lower power consumption, sensor drift, material cost, and experimental preparatory time. Furthermore, several dynamically operated sensors could be used in tandem (using the MSA and VSA scheme in a complementary fashion) to fabricate virtual multisensor arrays (V-MSAs). Such systems would exhibit greater data density than either the MSA or VSA, and are promising for samples that are particularly challenging to discriminate. Among the various systems utilized for VOC discrimination, sorption based systems hold considerable promise because they are simple and inexpensive yet highly effective. This dissertation is focused on the development of array sensing schemes using ionic liquids (ILs), a group of uniform materials based on organic salts (GUMBOS), and binary blends of either IL or GUMBOS with polymer as recognition elements and the quartz crystal microbalance (QCM) as the transducer. Towards this end, ILs, which are defined as organic salts with melting points below 100 °C, and group of uniform materials based on organic salts (GUMBOS) which extend the melting range of ILs to 250 °C to encompass similar solid phase salts, were used to design the first examples of QCM based VSAs, and V-MSAs, for pure VOC and complex mixture analyses. Furthermore binary blends of organic salts and polymer were used to fabricate the first VSA with the capability to identify and approximate molecular weight of pure VOCs. By and large, the studies presented here demonstrate the excellent potential of these materials and techniques for advancement of vapor phase measurement science.

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