The possibility of separating homologous phenols by liquid-liquid extraction with aqueous sodium hydroxide has been considered. In particular, phenol-o-cresol mixtures have received attention and the system phenol, o-cresol, water, and sodium hydroxide has been defined both with respect to the boundary of the two-phase region and the phase equilibria involved. Some qualitative and quantitative considerations have been given to possible extraction operations. It is concluded that extraction in the quaternary system is likely to be attractive only in the case of particular alkaline phenolic feeds. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

Petroleum, one of the most complex mixtures on Earth, has been extensively studied for decades. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and the ultrahigh resolution it provides, however, has dramatically transformed our understanding of petroleum by providing detailed molecular characterization. Nevertheless, many properties of crude oil, and the molecular characteristics that drive them, remain poorly understood, especially in regards to the role and impact of oxygen in oils. In the work presented here in-depth characterization of oxygen in petroleum is explored, to augment understanding of petroleum behavior due to oxygen. Although current FT-ICR MS instrument easily provides ultrahigh resolution, there is a constant for more as the complexity of petroleum means there is also some molecule just out of the obtainable analytical reach. Spectral segmenting on petroleum, in which narrow mass windows are acquired instead of all masses at once as in conventional broadband mass spectrometry, was performed in order to increase resolution and dynamic range to explore the species typically “missed” in conventional FT-ICR MS analysis. From the performance improvements provided by spectral segmenting, it was observed that molecules with high heteroatom content, including extensive oxygen content, are observed at low abundances, and that they may play a critical role in petroleum behavior despite their low abundances. Because of this the behavior of oxygen in petroleum is further explored. Naphthenic acids (carboxylic acids) in petroleum are known to be corrosive, yet no known correlation between naphthenic acid content and specific petroleum corrosivity has ever been established, other than more acidic crudes are generally more corrosive. In one proposed corrosion mechanism, a ketone is formed with a structure related to the structure of the corrosive acid. A method to detect and characterize these ketones in petroleum is developed and validated with model acids in an oil matrix, and then applied to more complex mixtures of naphthenic acids and acids derived from a vacuum gas oil to provide insight into which acids are most corrosive. Oxygen in petroleum plays an important role when petroleum is exposed to the environment. FT-ICR MS is used with traditional gas chromatography mass spectrometry (GC/MS) techniques to highlight how it is possible to use oxygen, in addition to other properties and heteroatoms, to track the relation of petroleum as it flows from reservoir to an oil seep and ultimately at the sea surface. In addition to highlighting petroleum subterranean connectivity, an initial characterization is obtained from which information about how petroleum transforms as it migrates is provided. Asphaltenes are perhaps the most poorly understood fraction of petroleum, with their molecular structures under intensive debate. Analysis by high resolution GC/MS of pyrolysis products of “classical” asphaltenes from bitumen and of “environmental” asphaltenes from a tarball is performed. It is found that high resolution is necessary for accurate characterization of even “simple” pyrolysis products, and that environmental asphaltenes are enriched in oxygen relative to the classical bitumen asphaltenes. The exact oxygen structures observe also provides insight into the weathering processes that the tarball underwent in its formation. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / January 17, 2018. / Corrosion, FT-ICR MS, GC-MS, Petroleum, Solid Phase Extraction / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; Jinfeng Zhang, University Representative; Christian Bleiholder, Committee Member; Wei Yang, Committee Member; Ryan P. Rodgers, Committee Member.

No description available.

Membrane proteins contribute about 30% of the proteome and about 60% of the drugs in the market target membrane proteins. But only about 1.5% of structures in Protein Data Bank are membrane proteins which reflect the challenges in membrane protein studies. In this dissertation work M2 full-length protein from Influenza A, a drug target to treat Influenza, is studied in lipid bilayer environment. ssNMR, the best technique to date to study membrane proteins at atomic resolution, has been utilized and intermonomer distance measurement is carried out throughout the study. Differently isotopically labeled protein mixtures were utilized for experiments to obtain unambiguous measurements, even though this method significantly reduces the sensitivity. Influence of amantadine, an antiviral drug, towards distinctive residues at different locations of the channel, such as N-terminus channel pore opening, channel interior and C-terminus of the channel has been characterized. M2 is known to be sensitive to its environment and here the influence of factors such as drugs, pH and membrane constituents such as cholesterol is studied. Distance information obtained for M2 full-length is compared with some of the existing M2 structures of truncated constructs and the importance of studying M2 protein as much as its native-like environment is emphasized. Among multiple drug resistant mutations of M2, S31N mutation contribute almost 100% of drug resistant strains observed in flu infected humans. Therefore, studies involving S31N mutation is an essential part of M2 proton channel studies. Hence, multiple experiments has been performed to obtain structural insights of this oligomeric protein and to compare them with that of M2 wild-type protein, in addition to study of its effect to environmental conditions such as pH. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2015. / July 01, 2015. / Amantadine binding, Cholesterol binding of M2 protein, Influenza A M2 protein, Inter-monomer distance measurements, S31N mutant of M2 protein, Solid-state NMR bspectroscopy / Includes bibliographical references. / Timothy A. Cross, Professor Directing Dissertation; Debra A. Fadool, University Representative; William T. Cooper, Committee Member; Alan G. Marshall, Committee Member.

The work in this dissertation presents methods for study of glucose stimulated insulin secretion as well as other hormones involved in glucose homeostasis. A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver sinusoidal glucose waveforms to the islet chamber. Insulin levels in the perfusate were measured using an online competitive electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration points ranging from 2–8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3–6 min. Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations to this forcing period. Exposing groups of 6–10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of oscillatory insulin secretion. A dual detection microscopy system was developed to simultaneously image intracellular messengers and monitor insulin secretion from islets of Langerhans. Glucose stimulated insulin secretion plays a critical role in glucose homeostasis, but the underlying mechanism is still unclear. To develop an automated system to simultaneously study the correlation between intracellular events and insulin secretion, fluorescence imaging and laser induced fluorescence detection for insulin immunoassay were integrated into a single microscopy system. Intracellular calcium ([Ca2+]i) was used as a representative secondary messenger and studied with insulin secretion from islets during exposure to constant and oscillatory glucose levels. Both of [Ca2+]i and insulin were oscillatory during constant glucose levels and entrained to a small amplitude sinusoidal wave of glucose (0.5 – 2 mM). [Ca2+]i and insulin oscillations were temporally but not necessarily quantitatively correlated. Oscillatory glucose waveforms amplified insulin oscillation amplitude without further increasing [Ca2+]i, which could be related to amplifying pathway. The developed system was also applied to study the effect of glucokinase activator 22 on [Ca2+]i and insulin secretion. These results indicated the robustness of the developed method, which can be potentially expanded to include other intracellular messengers to study the mechanism of glucose stimulated insulin secretion. A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of constant glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4−10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis. The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror image aptamers, so-called Spiegelmers, is demonstrated. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the Doctor of Philosophy. / Spring Semester 2016. / April 4, 2016. / affinity assay, capillary electrophoresis, hormone, islet of Langerhans, microfluidics / Includes bibliographical references. / Michael G. Roper, Professor Directing Dissertation; P. Bryant Chase, University Representative; John G. Dorsey, Committee Member; Kenneth L. Knappenberger, Jr., Committee Member.

Histone variants and post-translational modifications (PTMs) are known to play central roles in genome regulation and maintenance. Quantitavely characterization of the variants and the associated PTMs is the first step to understand their biological functions. However, many variants and the PTM combinations are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. In this dissertation, a method of identification of histone H4 proteoforms is developed, revealing the challenge of top-down proteomics. Then a method of proteoform identification and relative quantitation of histones H2A and H2B by FT-ICR MS and MS/MS is developed, yielding quantitative identification of all detected H2A and H2B isobaric and isomeric proteoforms with a label-free approach in HeLa. The method is further extended to 1) normal and HIV-infected U937 cells; 2) the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) stem cells sorted into early G1, late G1, S, and G2/M phases; 3) MDA-MB-231 breast cancer cells synchronized to S and M phases. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological roles of many previously unstudied histone variants and PTMs. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2016. / June 27, 2016. / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; David M. Gilbert, University Representative; Michael G. Roper, Committee Member; Timothy M. Logan, Committee Member.

High resolution mass spectrometry, especially Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) is a widely practiced technique of choice in proteomics and lipidomics due to its high sensitivity, reproducibility and wide dynamic range. FT-ICR MS enables quick assignments of hundreds of peptides and lipids with extreme complexity. Chapter 1 introduces the fundamental of FT ICR phenomena for mass measurement and basic theories of LC-MS based hydrogen deuterium exchange (HDX) technique for high order structure studies. Chapter 1 also introduces the application of mass spectrometry in lipidomics including lipid classification and MS analysis. Chapter 2 described the characterization of the binding interfaces in R2TP complex by hydrogen/deuterium exchange mass spectrometry. The two closely related AAA+ family ATPase Rvb1 and Rvb2 form a tight functional complex with two Hsp90 interactors: Pih1p and Tah1p. The R2TP complex involves in multiple biological processes including apoptosis, PIKK signaling, and RNA polymerase II assembly, and snoRNP biogenesis. The current lack of structural information on R2TP complex prevents a mechanistic understanding of many biological processes. By use of solution-phase HDX MS, we probed the contact surfaces on Pih1p-Tah1p upon Rvb1/2p binding. The present results demonstrate that Pih1p-Tah1p interacts with Rvb1/2p through N-terminal and IDR2 regions of Pih1p. Significantly, HDX also detected a rearrangement of residues 38–60 of Pih1p and 1–44 of Tah1p upon formation of the R2TP complex. Chapter 3 depicts the study of conformations of activated, disease-associated glucokinase variants by a comparative hydrogen/deuterium exchange mass spectrometry. Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Previous biochemical and biophysical studies suggest the existence of two activated variants. HDX results demonstrate that a disordered active site, which is folds upon binding of glucose, is protected from exchange in α helix variant. Additionally, α helix variant displays an increased level of exchange near enzyme’s hinge region. In contrast, β hairpin variant does not show substantial difference in global or local exchange relative to that of wild type GCK. The work elucidates the structural and dynamics origins of GCK’s unique kinetic cooperativity. Chapter 4 investigated the structure of an antibody with ‘Knob-into-hole’ mutations by HDX MS. Bispecific antibodies (BsAbs) have flourished in the biopharmaceutical industry for targeting two distinct antigens simultaneously. ‘Knob-into-hole’ approach is a way to manufacture bispecific antibodies. The applicability and advantage of ‘Knob-into-hole’ engineered bispecific antibody is vast. However, concerns about the conformational change and immunogenicity risks posed by the new approach has have been raised. To better understand the conformations and dynamics impacted by the ‘knob’ and ‘hole’ mutations, HDX MS is used to characterize peptide-level conformational changes of a ‘Knob-into-hole’ engineered antibody. The study shows that there is no significant structural alternation induced by ‘Knob-into-hole’ framework. In Chapter 5, the applicability of resolving HDX-derived isotopic fine structure by ultrahigh resolving power FT ICR mass spectrometry was discussed. In an HDX experiment, labeling protein with deuterium causes the deuterium incorporation, resulting in distributions of various combinations of 13C1H and 12C2H (Δm = 2.9 mDa). The isotopic fine structure typically cannot be used to evaluate deuteration level due to the difficulty of .resolving fine structures for all proteolytic peptides spanning wide mass range from HDX experiments. The introduction of hexapolar cell triples the observed resolving power on 14.5 tesla FT-ICR mass spectrometer, thus we successfully extend the capability of resolving isotopic fine structure to most of identified peptides. Additionally, a new method of analysis of isotopic fine structure-resolved HDX data was proposed to determine degrees of deuterium incorporation. Another research area I have worked on is characterization of polar lipids by LC coupled with FT-ICR mass spectrometry. Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. In Chapter 6, I compared polar lipid compositions for two microalgae, Nannochloropsis oculata and Haematococcus pluvialis, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry ((−) ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. H. pluvialis exhibits more highly polyunsaturated lipids than does N. oculata. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / June 13, 2018. / FT ICR Mass Spectrometry, Hydrogen Deuterium Exchange, Isotopic Fine Structure, Lipidomics, Liquid Chromatography, Protein High Order Structure / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; Hengli Tang, University Representative; John G. Dorsey, Committee Member; Brian G. Miller, Committee Member.

The idea of lines and angles in the two dimensional space is a fairly simple and intuitive idea. However, as we move to N-dimensions, this idea may not be as intuitive. In this project, we are going to take the intuitive ideas in a plane and move to a higher dimensional space. We will introduce some machinery, such as the wedge product and the Hodge star operator, which will assist us to investigate these concepts in N-dimensions. We will then look at what we mean by oriented volume of an object in N-dimensions. This will lead us to interesting generalizations of two well-known ideas: the Pythagorean theorem and the law of cosines

No description available.

A Polish space is a separable topological space that can be metrized by means of a complete metric. A subset A of a Polish space X is analytic if there is a Polish space Z and a continuous function f : Z —> X such that f(Z)= A. After proving that each uncountable Polish space contains a non-Borel analytic subset we conclude that there exists a universally measurable non-Borel set.