Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-11, Section: B, page: 6759. Adviser: Chad M. Rienstra. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2008. / Title from home page (viewed Oct. 8, 2009). Source: Dissertation Abstracts International, Volume: 70-02, Section: B, page: 0994. Adviser: Martin F. Jarrold.

Thesis (Ph.D.)--Indiana University, Dept. of Chemistry, 2009. / Title from PDF t.p. (viewed on Jul 22, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7531. Adviser: James P. Reilly.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3482. Adviser: Neil L. Kelleher. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6578. Adviser: David E. Clemmer. Title from dissertation home page (viewed Oct. 11, 2006).

<p> Conformational heterogeneity and protein dynamics play important roles in molecular recognition, but are experimentally difficult to characterize with sufficient temporal and spatial resolution. Infrared (IR) spectroscopy can probe protein dynamics on sub-ps timescales; and further, the small size of vibrational chromophores combined with site-selective incorporation of spectrally isolated IR probes provides high spatial resolution. Herein, we site-specifically introduce nitrile and carbon-deuterium bonds at distinct sites in the Src-homology 3 (SH3) domain from yeast protein Sho1 and its proline-rich peptide binding partner from Pbs2 to examine the underlying mechanisms of molecular recognition via IR spectroscopy. Further, we present efforts at developing instrumentation aimed at improving characterization of weakly absorbing vibrational probes in strongly absorbing solvent.</p><p> Nitrile probes were introduced at six distinct sites in the SH3 domain via amber codon suppression. Variation between the observed absorbance bands indicates site specific differences in conformational heterogeneity imposed by protein domain. Residue-specific changes upon peptide binding are observed at incorporated nitrile moieties, but are more dramatically observed for deuterated vibrational probes incorporated within the peptide binding partner. Deuterated amino acids were incorporated at highly conserved proline residues within the peptide ligand. Upon binding, absorbance bands are observed which indicate population of multiple conformational states in the bound complex. Only single resonances were observed by characterization of the same labeled bonds by NMR, suggesting rapid interconversion on the NMR timescale. Results suggest that the SH3 domain recognizes its peptide binding partner with at least elements of an induced-fit mechanism.</p><p> Characterization of the vibrational probes used above can be challenging due to the path length limitation imposed by the presence of strongly absorbing solvent water. This places an upper bound on the achievable signal strength which can obscure small (&micro;OD) absorbance bands. To confront this limitation we have constructed an absorbance spectrometer with a quantum cascade laser (QCL) source. The instrument allows characterization of samples of increased path length with similar signal-to-noise ratios as in FT IR measurements. Achievable signal-to-noise ratios are limited by QCL source noise; we present several approaches, one electronic and one interferometric, aimed at limiting the deleterious effect of QCL fluctuations.</p><p>

<p> Electrokinetic chromatography is a variation of capillary electrophoresis that allows for the separation of nonionic analytes by selective interaction with an ionic pseudostationary phase dissolved in the background electrolyte. The utility of electrokinetic chromatography to characterize pseudostationary phases and pseudostationary phase&ndash;solute interactions has been recognized since its introduction. The objective of this dissertation was to use electrokinetic chromatography and copolymer stabilized lipid bilayer nanodiscs as a pseudostationary phase to characterize small molecule-lipid bilayer interactions. </p><p> Styrene-maleic acid copolymers were used to stabilize cylindrical sections of lipid bilayer in solution, forming nanodiscs. The nanodiscs are formed based on strong hydrophobic interactions between the styrene moiety, on the copolymer, and the alkyl tails of the lipids. Using the nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was characterized. Linear solvation energy relationship analysis was employed to characterize the changes in solvent environment of the nanodiscs of varied copolymer to lipid ratio, copolymer chemistry and molecular weight, and lipid composition. Increases in the lipid to copolymer ratio resulted in smaller, more cohesive nanodiscs with greater electrophoretic mobility. Nanodisc structures with copolymers of different chemistry and molecular weight were compared and showed changes in solvent characteristics and selectivity. Seven phospholipid and sphingomyelin nanodiscs of different lipid composition were characterized. Changes in lipid head group structure had a significant effect on bilayer?solute interactions. In most cases, changes in alkyl tail structure had no discernible effect on solvation environment. </p><p> The nanodisc pseudostationary phase was also used to study sphingomyelin stereochemistry. Various studies have produced conflicting results regarding whether interactions with lipid bilayers are or can be stereoselective. Using sphingomyelin nanodiscs stereoselective interactions between a pair of atropisomers, R-(+)/(S)-(&ndash;) 1,1'-Bi-2-naphthol, were demonstrated. </p><p> Finally the dissociation constants between sphingomyelin nanodiscs and solvochromatic analytes were measured and then validated using steady state fluorescence. Using nanodisc affinity capillary electrophoresis, dissociation constants were derived on the same order of magnitude as the dissociation constants derived using the fluorescent technique. Future directions of this project will be to study peptide and protein interactions with lipid bilayers of interest.</p><p>

<p> N-nitrosodimethylamine is a carcinogenic disinfection byproduct which can be reformed after wastewater treatment if appropriate conditions are present. In this study various NDMA formation precursors over a range of conditions have been studied to attempt to quantify this NDMA reformation potential. Efforts were made in this study to demonstrate the effect of time, pH, and multiple different precursor chemicals on the reformation process. Dimethylamine (DMA) has been demonstrated to be a precursor chemical, and was chosen as a standard for initial experiments. The concentration of this precursor was kept constant at 100 ppt (parts per trillion) and solutions at three different pH&rsquo;s were tested over a 24-hour time period to see if any significant NDMA formation occurred. The impacts of various treated wastewater constituent chemicals including H₂O₂, monochloramine, dimethylhydrazine, etc. were also performed in this study. Significant difficulties occurred for the analysis of these samples, with large background interferences occurring in the method of analysis. Based on the data obtained, it appears that the formation potential of NDMA with DMA as a precursor was found to be the most efficient compound at basic pH (pH 9) and very basic (pH 13). This is consistent with the last step in wastewater remediation, where quicklime is added to the water to regulate the pH and for protection of distribution system. In addition, preliminary experiments were conducted using Ranitidine as a NDMA reformation precursor.</p><p>

<p>The work in this dissertation presents microfluidic methods developed for the study of biological dynamics. The requirements for the methods development was to create approaches with the ability to perform dynamic cell stimulation, measurement, and sample preparation. The methods presented herein were initially developed for the study of pancreatic islet biology but are expected to be translatable to other applications. In another study, a method to interface transmission electron microscopy (TEM) with microfluidics methods was developed. </p><p> The primary biological topic of interest investigated was the mechanisms of inter-islet synchronization. To test this, a microfluidic device fabricated from poly(dimethylsiloxane) (PDMS) was used to culture and stimulate pancreatic islets. Intracellular calcium ([Ca²⁺]i) imaging was performed with a fluorescent indicator, Fura-2-acetoxymethyl ester (Fura-2 AM). Under constant glucose (11 mM), islets demonstrated asynchronous and heterogeneous [Ca²⁺]i oscillations that drifted in period. However, when exposed to a glucose wave (11+/&ndash; 1 mM, 5 min period) islets were entrained to a common and consistent [Ca²⁺]i oscillation mode. The effect of islet entrainment on cellular function was investigated by measuring gene expression levels with microarray profiling. Calcium-dependent genes were found to be differentially expressed. Furthermore, it was speculated that islet entrained produced a beneficial effect on cell function and upkeep. </p><p> While [Ca²⁺]i imaging is an acceptable proxy measurement for insulin, it is not a viable reporter for other islet peptides and direct measurement is desired. Electrophoretic affinity assays can be performed on a microfluidic device in a serial manner to measure peptide release from an on-chip cell culture in near real-time. Successful analysis of electrophoretic affinity assays depends strongly on the preservation of the affinity complex during separations. Elevated separation temperatures due to Joule heating promotes complex dissociation leading to a reduction in sensitivity. To address this limitation, a method to cool a glass microfluidic chip for performing an affinity assay for insulin was achieved by a Peltier cooler localized over the separation channel. The Peltier cooler allowed for rapid stabilization of temperatures, with 21&nbsp;&deg;C the lowest temperature that was possible to use without producing detrimental thermal gradients throughout the device. Kinetic capillary electrophoresis analysis was utilized as a diagnostic of the affinity assay and indicated that optimal conditions were at the highest attainable separation voltage, 6&nbsp;kV, and the lowest separation temperature, 21&nbsp;&deg;C, leading to 3.4% dissociation of the complex peak during the separation. These optimum conditions were used to generate a calibration curve and produced 1&nbsp;nM limits of detection (LOD), representing a 10-fold improvement over non-thermostated conditions. </p><p> To date, most approaches for measurement of rapid changes in insulin levels rely on separations, making the assays difficult to translate to non-specialist laboratories. To enable rapid measurements of secretion dynamics from a single islet in a manner that will be more suitable for transfer to non-specialized laboratories, a microfluidic online fluorescence anisotropy immunoassay was developed. A single islet was housed inside a microfluidic chamber and stimulated with varying glucose levels from a gravity-based perfusion system. The total effluent of the islet chamber containing the islet secretions was mixed with gravity-driven solutions of insulin antibody and cyanine-5 (Cy5) labeled insulin. After mixing was complete, a linearly polarized 635 nm laser was used to excite the immunoassay mixture and the emission was split into parallel and perpendicular components for determination of anisotropy. Key factors for reproducible anisotropy measurements, including temperature homogeneity and flow rate stability were optimized, which resulted in a 4 nM LOD for insulin with &lt; 1% RSD of anisotropy values. The capability of this system for measuring insulin secretion from single islets was shown by stimulating an islet with varying glucose levels. As the entire analysis is performed optically, this system should be readily transferable to other laboratories. </p><p> To increase the number of analytes that can be simultaneously monitored by a fluorescence anisotropy immunoassay, frequency encoding was introduced. As a demonstration of the method, simultaneous competitive immunoassays for insulin and glucagon were performed by measuring the ratio of bound and free Cy5-insulin and fluorescein isothiocyanate (FITC)-glucagon in the presence of their respective antibodies. A vertically polarized 635 nm laser was pulsed at 73 Hz and used to excite Cy5-insulin, while a vertically polarized 488 nm laser pulsed at 137 Hz excited FITC-glucagon. The total emission was split into parallel and perpendicular polarizations and collected onto separate photomultiplier tubes. The signals from each channel were demodulated using a fast Fourier transform, resolving the contributions from each fluorophore. Anisotropy calculations were carried out using the magnitude of the peaks in the frequency domain. The method produced the expected shape of the calibration curves with LOD of 0.6 and 5 nM for insulin and glucagon, respectively. (Abstract shortened by ProQuest.) </p><p>

<p> Single cell metabolomics provides new insights into understanding cellular heterogeneity of small molecules, and individual cell response to environmental perturbations. With high sensitivity and specificity, mass spectrometry (MS) has become an important tool for analyzing metabolites, lipids, and peptides in individual cells. Facing significant challenges, single cell and subcellular analysis by MS requires technical advances to answer fundamental biological questions, for example the phenotypic variations of genetically identical cells. The work presented in this dissertation describes my efforts to develop and apply capillary microsampling MS with ion mobility separation (IMS) for the analysis of single cells and subcellular compartments. </p><p> Chapter 1 introduces MS based analytical techniques for single cell and subcellular analysis. Recent advances of sampling and ionization methods for MS analysis of volume-limited samples are reviewed with emphasis on ambient ionization techniques, cell micromanipulation methods, and rapid gas phase separations. </p><p> In Chapter 2, the application of capillary microsampling electrospray ionization (ESI)-IMS-MS for metabolic and lipidomic analysis of single <i> Arabidopsis thaliana</i> epidermal cells is presented. Distinct metabolite compositions and metabolic pathways are identified among basal and pavement cells, and trichomes. These three specialized epidermal cells serve different functions in the plant leaf, and our single cell MS data reveals the corresponding metabolic pathways. </p><p> In Chapter 3, it describes the utilization of capillary microsampling ESI-IMS-MS for the analysis of metabolites and lipids in single human hepatocellular carcinoma cells. Cellular physiological states and their heterogeneity in response to xenobiotics treatment, and lipid turnover rates are explored. Here, IMS helps to enhance molecular coverage, facilitate metabolite and lipid identification, resolve isobaric ions, and minimize background interference. Comparing cells affected by metabolic modulators to unaffected counterparts reveals dramatic reduction in the availability of energy in the former. </p><p> In Chapter 4, the combination of fluorescence microscopy with capillary microsampling ESI-IMS-MS for selective analysis of identified cell subpopulations at a single cell level is demonstrated. Molecular differences and heterogeneity corresponding to cells in distinct mitotic stages are explored. Pairwise correlations between relative metabolite levels among individual mitotic cells are also studied. </p><p> In Chapter 5, the subcellular distributions of neuropeptides in individual identified neurons are explored by capillary microsampling ESI-IMS-MS. Distinct peptide distributions between the cytoplasm and nucleus are revealed. Mass spectra provide direct evidence for high abundance of these peptides in the nucleus despite the scarcity of immunostaining results supporting their presence there. A new neuropeptide is discovered and sequenced by MS in a single cell. </p><p> In Chapter 6, the current state of single cell and subcellular metabolomics is discussed. Major challenges include the low-throughput of current sampling techniques, low molecular coverage of metabolites, lipids and peptides, and external perturbations introduced by the sampling and ionization processes. In addition to exploring new solutions to these challenges, future advances will lead to the development of systems biology at the single cell level, to nano- and micro-fabricated tools to study perturbations in a lab-in-a-cell framework, and to coupling with optical manipulations and microfluidic techniques to investigate subcellular heterogeneity.</p><p>