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Microfluidic Methods for the Study of Biological DynamicsMukhitov, Nikita 03 March 2018 (has links)
<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 ([Ca<sup>2+</sup>]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 [Ca<sup>2+</sup>]i oscillations that drifted in period. However, when exposed to a glucose wave (11+/– 1 mM, 5 min period) islets were entrained to a common and consistent [Ca<sup>2+</sup>]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 [Ca<sup>2+</sup>]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 °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 kV, and the lowest separation temperature, 21 °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 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 < 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>
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Emerging Methods for Single Cell MetabolomicsZhang, Linwen 28 April 2018 (has links)
<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>
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Structural Analysis of Bovine Derived HeparinsSt.Ange, Kalib 03 April 2018 (has links)
<p> Bovine heparin is characteristically different than porcine intestinal heparin. These differences include sulfation, molecular weight properties, activity, structure, and shape. Bovine lung heparin has a higher amount of GlcNY6S (where Y can represent Ac or S) while the amount of GlcNY6S is much lower in bovine intestinal heparin. All heparins have high amounts of trisulfated TriS disaccharide but the level of TriS is in lower in bovine intestinal heparin. The amount of NS2S is much higher in bovine intestinal heparin than in bovine lung and porcine intestinal heparins. The average molecular weight of bovine intestinal heparin is similar to porcine intestinal heparin but the molecular weight of bovine lung heparin is much lower. The activities of bovine tissue heparins were comparable to, but lower than, the activity of porcine intestinal heparin. </p><p> The differences in heparins from different sources become much more subtle as they are depolymerized into low molecular weight heparin (LMWH). These differences are often sufficiently small so that they require principle component analysis (PCA) to determine. Differences in the reducing end and the non-reducing end structures in heparin determined by mass spectrometry (MS) as well as differences in the glucosamine and uronic acid residues determined by nuclear magnetic resonance spectroscopy (NMR) were selected for PCA. Using PCA it was possible to link parent heparin starting material to its daughter LMWH. This analysis demonstrated the lower variation between LMW bovine and LMW porcine heparins than between bovine and porcine heparins. This lower variation afforded the LMW bovine heparin similar anti-Xa and anti-IIa activity comparable to commercial heparin. </p><p> The harsh purification process used to prepare heparin leaves the heparin product largely unaffected except for its reducing end tetrasaccharide linkage region. GlyserineAc is present in porcine and bovine heparin but is absent in LMW heparin. We hypothesized that the peracetic acid bleaching adds an O-acetyl group that is selectively lost during β-elimination in the LMWH production process. The tetrasaccharide composition of bovine and porcine heparin is similar as are different batches from the same supplier. This emphasizes how similar processing results in similar heparin regardless of whether bovine or porcine is used. </p><p> Small differences in counterfeit heparin, i.e. blended porcine and bovine heparins were next examined. Porcine heparin and bovine heparin of similar molecular weights to obtain a bovine heparin counterfeit drug of enhanced activity. Such counterfeit blends are undetectable by current methods of analysis. Diffusion ordered spectroscopy (DOSY) method for analysis was developed. DOSY exploits differences in the molecular shape of bovine heparin and porcine heparin and achieved partial separation in the diffusion dimension. Additional spectra for component resolution (SCORE) analysis were used to demonstrate detection and identification of blend mixture.</p><p>
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Further Development of Raman Spectroscopy for Body Fluid Investigation| Forensic Identification, Limit of Detection, and Donor CharacterizationMuro, Claire K. 21 July 2017 (has links)
<p> The challenges to forensic body fluid analysis have placed limitations on the type of information that investigators can acquire and how that information can be collected. In recent years, Raman spectroscopy has proven itself useful for characterizing body fluids. In 2008, a large-scale investigation was undertaken to explore the use of Raman spectroscopy as a means of identifying body fluids. This work resulted in multidimensional Raman spectroscopic signatures for the five main body fluids: semen, peripheral blood, saliva, vaginal fluid, and sweat. These studies were incredibly successful and created the foundation for years of continued research. Accordingly, the studies included in this thesis have been specifically chosen to frame the previous research projects. They include a suite of projects aimed to advance and validate the developed method. </p><p> First, a statistical model was developed to automatically identify and differentiate body fluids based on their Raman spectra. The multidimensional spectroscopic signatures mentioned above are very effective at identification, but they are body fluid-specific. In other words, they individually evaluate whether or not an unknown spectrum is from a particular body fluid, such as blood. Additionally, each signature was built on spectra from a limited number of donors. To improve on this capability, a single classification model was built on the Raman spectra from 60 donors (12 for each body fluid). This model was externally validated with an additional 15 donors in order to objectively assess the model’s performance. All of the external validation donors were correctly identified, illustrating how accurate and robust the model is. </p><p> Second, the limit of detection (LOD) for the classification model was explored as a form of validation. It is vitally important that a method’s limits be established before deploying it into use. The LOD of peripheral blood was investigated. Peripheral blood is unique from other body fluids because its Raman spectrum has been attributed almost entirely to one molecule- hemoglobin. Because hemoglobin is only found in red blood cells (RBCs), the Raman spectrum of peripheral blood essentially results purely from RBCs. Given this, we chose to start with a single RBC, and then increase the volume until identification was successful. We found that we were able to conclusively and confidently identify peripheral blood using a single red blood cell. This limit is 5000X smaller than the amount of blood required for DNA analysis, demonstrating the sensitivity of the developed method. </p><p> Finally, the method was further advanced by incorporating donor characterization into the process. Besides identifying body fluids, the method can now extract “phenotypic” information about the donor. Raman spectroscopy and multivariate data analysis were used to determine the biological sex of saliva donors, and the race of semen donors. These studies will help forensic investigators extract incredibly useful information about a potential suspect or victim, and can be performed directly at a crime scene for instant results. </p><p> Altogether, these studies combine to strengthen the method previously developed by our research group. More importantly, they help to bridge the gap between research and application. Creating a universal method to differentiate and identify body fluids, investigating the method’s LOD, and developing additional techniques to characterize body fluids represents a significant contribution to the field of forensic chemistry. The universal method created within this thesis will be adapted to perform on-site analysis of physical evidence at crime scenes. The methods’ incredible sensitivity has been demonstrated by determining that it can identify peripheral blood based on a single RBC. Finally, by developing models to characterize body fluid donors, investigators will be able to extract useful information about individuals that may have been present at a crime scene. Additional studies are already being conducted to make further improvements, and our method is poised to make a significant contribution to the field of crime scene investigation. </p><p>
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Combining Experimental and In Silico Methods for Comprehensive Compound Dereplication of Natural Products for Mass Spectrometry Based MetabolomicsVaniya, Arpana 01 December 2017 (has links)
<p> Metabolomics is a rapidly growing field in “omics” research where metabolites are analyzed in biological systems. Over the past decade, mass spectrometry (MS) based metabolomics has been used for its superior analytical performance to reveal how these biological systems respond to genetic and environmental changes. MS is both sensitive and selective and is capable for providing comprehensive information for metabolic profiling by combining separation methods such as liquid chromatography (LC-MS) or gas chromatography (GC-MS). However, in untargeted metabolomics identification of small molecules is the bottleneck. In the research described here, I have combined both <i> in silico</i> and experimental methods for compound dereplication of natural products using MS-based metabolomics. </p><p> <b>Chapter 1</b> addresses the advancement of fragmentation and mass spectral trees used for unknown metabolite identification. Tools used for metabolite identification from the past 10 years are discussed, including algorithms, software, mass spectral libraries, and databases that implement fragmentation and mass spectral trees. Due to the inherent complexity of natural products in plants and microbes, unknown compound identification is increasingly difficult and limiting. Resolving this problem requires better computational tools and informative data such as those acquired by multi-stage mass spectrometry (MS<sup>n</sup>). MS<sup>n</sup> yields more fragmentation data and allows for more complex structural elucidation as needed for compounds with positional isomers. The limitation with using tandem mass spectrometry (MS/MS) only is that many ions are shared between positional isomers and full structural information is not available to elucidate an unknown metabolite. Fragmentation and mass spectral trees both describe the fragmentation processes of a metabolite and aid in fragmentation rule generation and substructure identification. The major difference between fragmentation and mass spectral trees is that fragmentation trees use elemental compositions to describe the fragmentation process and mass spectral trees or ion trees use precursor and product ion spectra from MS<sup>n</sup> mass spectral acquisition. As a result, there has been a large increase in efforts to develop MS<sup>n > 2 </sup> data and tools for both structure elucidation and spectral annotations with the use of fragmentation and mass spectral trees in recent years. </p><p> <b>Chapter 2</b> describes research and development of iTree, a MS<sup>n</sup> mass spectral tree library of plant natural products and its aid in compound identification of natural products. In metabolomics, mass spectral library searching is a standard method for compound identification, correctly known as compound dereplication. Mass spectral libraries are either freely or commercially available and can contain both experimental and <i> in silico</i> MS/MS reference spectra. The coverage of MS<sup>n > 2 </sup> reference spectra is much smaller in many of these MS/MS libraries and databases. To date the largest MS<sup>n > 2</sup> libraries are HighChem and mzCloud, which also support mass spectral trees. The chemical coverage of such libraries and databases are very low in comparison to the number of known compounds. iTree was developed to expand the coverage of fragmentation spectra for natural products. iTree contains more than 2,000 natural products and more than 9,000 ion tree spectra annotated with <i>in silico</i> generated substructures from both Mass Frontier 7.0 and CFM-ID. iTree is freely available through MassBank of North America (MoNA), an open-access mass spectral database. As a result of the high number of natural products, and specifically flavonoid aglycones, previously published fragmentation rules were studied and validated. A new rule for flavanonols was proposed as a loss of –CCO to occur specifically for this class. In addition, iTree was used to profile secondary metabolites in the roots and nodules of the host plant <i> Datisca glomerata</i>. More than 100 natural products were identified by combining LC-MS<sup>n</sup>, high resolution LC-MS/MS, and ion tree analysis using iTree. Overall, iTree has shown to provide a method to facilitate metabolite identification for plant natural products. </p><p> Although MS<sup>n > 2</sup> data is more useful for complex structural elucidation, the predominant data used in untargeted metabolomics is MS/MS. For this reason, <i>in silico</i> tools that focus on the interpretation of MS and MS/MS spectra alone must be evaluated. In Chapters 3 through 5, I discuss how the Critical Assessment of Small Molecule Identification (CASMI) has allowed for such an evaluation by presenting unknown challenge data sets to the metabolomics community to evaluate the tools and methods they currently use for unknown compound identification. The results submitted by each user are compared and discussed to provide greater insight into how <i>in silico</i> tools can be further improved to aid in the advancement and accuracy of unknown compound identification methods. </p><p> <b>Chapter 3</b> focuses specifically on the performance of MS-FINDER, a software that uses MS and MS/MS spectra for structural elucidation of unknown compounds, presented in the CASMI 2016 Category 1. (Abstract shortened by ProQuest.) </p><p>
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Kinetic studies of the thermal decomposition of explosives using accelerating rate calorimetryLee, Pauline P January 1986 (has links)
Abstract not available.
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Sequencing synthetic copolymers using electrospray ionization mass spectrometryGiguere, Marie-Soleil January 2003 (has links)
The dissociation of gas-phase synthetic homopolymers poly(butyl acrylate) (PBA), poly(methyl methacrylate) (PMMA) and poly(vinyl acetate) (PVAc) ionized by Li+ or Na+ was investigated by electrospray-ionization tandem mass spectrometry. Based on these results it was possible to use tandem mass spectrometry to sequence three copolymers produced by free radical polymerization (PBA/PVAc, PBA/PMMA and PMMA/PVAc). In the case of PVAc containing copolymers, tandem mass spectrometry yielded partial sequence information including the number of VAc monomers and consecutive BA or MMA monomers while the complete sequence (and mixture of sequences) could be deduced for PBA/PMMA.
The poly(vinyl acetate) ions lose acetic acid molecules from the polymer side chain, and ultimately metal acetate, to form a polyene with a positive charge on the carbon backbone while PBA and PMMA fragment along the polymer backbone. An extensive study was made of the dissociation characteristic of PVAc. Collision-induced dissociation (CID) was performed at different center-of-mass collision energies to study the sequential appearance of the fragment ions. Molecular mechanics/molecular dynamics (MM/MD), Hartree-Fock, semi-empirical and density functional calculations were employed to model the lowest energy dissociation processes.
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Conformational studies of cell division regulator MinE by nuclear magnetic resonance and circular dichroism spectroscopyRamos, Dennis January 2006 (has links)
Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum; namely, MinC, MinD and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation, however, little is actually known about the structure of this functionally critical region. In order to understand how MinE can perform its anti-MinCD function, we have therefore investigated the structural properties of the full-length MinE from N. gonorrhoeae. Results from solution NMR show that, in contrast to previous models, parts of the anti-MinCD domain are stably folded with many functionally important residues forming part of a beta-structure. In addition, this structure may be stabilized by interactions with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The inactive MinE mutant L22D showed even larger evidence of structural perturbations, with significant destabilization of the entire MinE structure. Overall, these results suggest an intimate structural association between the anti-MinCD and topological specificity domains raising the possibility that the functional properties of the two domains could be modulated through this interaction.
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Applications of spectroscopy to the creation and study of nanostructuresHeafey, Eve January 2009 (has links)
With the technological advances of today, comes the increasing need for device miniaturization. This thesis focuses on the construction of nanostructures, approached from both bottom-up (synthesis) and top-down (lithography) methods.
First, a bottom-up method to construct cadmium selenide semiconductor nanoparticles under mild conditions in reverse micelles is described and investigated. A detailed spectroscopic study of nanoparticle growth is provided, whereby the growth was monitored over a period of up to a year. The nanoparticles were removed from the surfactant using ethanol precipitation and were subjected to surface derivatization in order to stabilize them.
Secondly, photoreversible cycloaddition of an anthracene derivative is studied for use in double-exposure lithography. The system was found to be adequately reversible, though possible side photoreactions were investigated. Spectroscopic studies were also conducted and photophysical pathways of the compound were examined. The compound was found to have a cleaving quantum yield of 0.67 +/- 0.04 and was established as an actinometer to assess the efficacy of future candidate compounds.
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Identification of Candidate Exposure Biomarkers for Toxicants in Complex Environmental MatricesOsika, Natalie Ann January 2011 (has links)
The term "biomarker" refers to an indicator employed to monitor the interactions between a xenobiotic and an individual, provide information of an individual's biologic state, or refer to an individual's susceptibility to a xenobiotic, condition or disease. They are currently employed to assess exposure to, or effects from, environmental toxicants. It is important to have biomarkers that can accurately and precisely assess complex mixture exposures, since environmental exposures to single substances rarely occur outside the laboratory setting, and complex exposures are often associated with specific occupational or environmental settings. This project employed microarray technology and subsequent bioinformatic analyses to identify candidate biomarkers of in vitro exposure to coal tar extract. Murine pulmonary epithelial cells were exposed to coal tar extract and gene expression was assessed using microarrays. The results showed that expression of genes involved in steroidogenesis, cytokine-cytokine interaction, angiogenesis, as well as several genes that code for secreted proteins, were altered by coal tar exposure. The entire work constitutes the initial stages of a project that will ultimately identify gene expression and secretome profiles that are specific to coal tar exposure.
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