Doctor of Philosophy, The Ohio State University, 2017, Horticulture and Crop Science

Many plant crops of temperate origins, particularly those that overwinter in the field, are sensitive to cold temperatures. Following exposure to low temperatures, cold-sensitive crops experience disruptions to physiological process and cellular architectures, ultimately resulting in chilling and/or freeze injuries. In order to survive harsh winter, cold-tolerant plants have improved their freezing tolerance (FT) by undergoing a complex series of developmental processes as known as “cold acclimation”, following a period of exposure to low but non-freezing temperatures. As part of the acclimation responses, plants accumulated sugars in a tissue-specific fashion. These sugars have been proposed to play a role in mediating plant FT by serving as cryoprotectants, stabilizing the plasma membrane and/or depressing the cytosolic freezing point. To increase our understanding of the role of sugar metabolism and accumulation during cold stress responses, we have taken a metabolomics and molecular biology-based approach. To monitor the cold-induced changes in sugar levels during the cold acclimation process, an optimized capillary zone electrophoresis (CZE) method was developed and was used to separate and quantify mono-, di-, and oligosaccharides isolated from plant tissues. The optimized CZE method provides an average limit-of-detection (LOD) of 1.5 ng/µL for individual carbohydrates, with comparable or superior to the average LOD for these sugars using gas chromatography-mass spectrometry. This optimized CZE method was used to metabolically profile cold-induced shifts in sugar accumulation in two plant species (rubber-producing dandelions and grapevines), as well as to investigate the effect of ABA treatment on sugar accumulation during the development of FT. In addition to mono-, di-, and tri-saccharides, in studies using dandelions, we also monitored the accumulation of inulin. Inulin is a fructose polymer found in the storage organs of rubber-producing dandelions such as (open full item for complete abstract)

Committee: Joshua Blakeslee (Advisor); Katrina Cornish (Advisor); Imed Dami (Committee Member); Feng Qu (Committee Member) Subjects: Agriculture; Horticulture; Plant Biology; Plant Sciences

PHD, Kent State University, 2014, College of Arts and Sciences / Department of Chemistry

The interaction between nucleic acids and small molecule ligands is continuously generating significant interest due to their widespread biological and bioanalytical applications. We investigated the mechanical property of the binding between aptamers and small molecules. Using an ATP binding aptamer as an example, we observed that the mechanical stability of the aptamers that are bound with ATP is higher than those without a ligand. Therefore, a force-based sensor can be developed to detect small molecules using aptamers as a platform. We determined the dissociation constant, Kd, for aptamer-ligand interactions at the single-molecule level by applying a Hess-like cycle. Our experiments allow the Kd determination from only one ligand concentration which was further validated by our capillary electrophoresis (CE) method. By using only one ligand concentration, such a method not only saves time and material, but also is less susceptible to reduced reproducibility due to run-to-run fluctuations. G-quadruplex forming sequences which are wide spread in the genome particularly in telomeres and promoter regions have been shown to be therapeutic targets. G-quadruplex structures have been extensively studied using mostly conventional methods. However, the mechanical stability, thermodynamics, kinetics properties of these interactions at the single-molecule level, remains to be fully understood. While the formation of these G-quadruplex structures is highly dynamic, they are mechanically stabilized upon ligand binding which may affect their biological functions. Small molecules which bind to nucleic acid structures may interfere with vital cellular processes such as transcription and protein translation during cell division by acting as energy barriers or mechanical blockage. Therefore, understanding stability of nucleic structures from a mechanical stability stand point is critical to fully explore their therapeutic potentials. We have investigated the human te (open full item for complete abstract)

Committee: Hanbin Mao PhD (Advisor); Songping Huang PhD (Committee Member); Michael Tubergen PhD (Committee Member); Gail Fraizer PhD (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Biophysics; Molecular Biology

Doctor of Philosophy (PhD), Wright State University, 2012, Biomedical Sciences PhD

The pharmacokinetic difference between drug enantiomers is the impetus for developing analytical techniques to assess enantiomeric purity. Capillary electrophoresis (CE) is an analytical technique that is used for characterizing drug-protein binding. The pitfall to using CE for drug-protein binding studies is protein chiral selectors tend to adsorb onto capillary walls and cause changes in electroosmotic flow that lead to decreased enantioselection and migration time irreproducibility between consecutive injections. The experimental parameters for minimizing the adverse effects of protein adsorption are not clear from the literature. Rinsing protocols to improve enantioselection and migration time repeatability were developed using the tryptophan-bovine serum albumin system as a model. The enantioselection of bovine serum albumin (BSA) could be improved by: 1) increasing separation voltage; 2) using sample buffer ionic strength at least 3 orders of magnitude less than the separation buffer; 3) limiting the equilibration time with separation buffer; and 4) allowing for protein diffusion. Rinsing the capillary with sodium hydroxide, followed by water improved migration time repeatability RSD from 24.7% to 1.8% (n = 4). Drug-protein binding is contingent upon the three dimensional structure of the binding site, and the presence of other competing drug molecules. Drug-drug displacement is difficult to predict and the effects of protein glycation on binding of drugs is not well defined. To highlight the use of CE for addressing questions of biochemical interest, CE was applied to characterize drug-drug displacement and the effects of protein glycation on the enantioselection of drugs by BSA. The tryptophan-bovine serum albumin, 5-fluoro-tryptophan-bovine serum albumin, and 5-hydroxy-tryptophan-bovine serum albumin systems were used as models. A CE method for studying competitive binding was established using ibuprofen as the displacer molecule. Accurate calculatio (open full item for complete abstract)

Committee: Roger Gilpin PhD (Advisor); Gerald Alter PhD (Committee Member); Kenneth Turnbull PhD (Committee Member); Daniel Ketcha PhD (Committee Member); Richard Sherwood PhD (Committee Member) Subjects: Analytical Chemistry; Chemistry; Pharmaceuticals

PhD, University of Cincinnati, 2001, Arts and Sciences : Chemistry

This dissertation comprises of two parts, each discussing different aspects of cyclodextrin chemistry. The research has been performed in an effort to further investigate inclusion properties of cyclodextrins and demonstrate their practical utilization in analytical chemistry. In the first part of the dissertation, cyclodextrin complexes of α- and β-cyclodextrin with nonionic surfactants containing linear alkyl and oligooxyethylene residues are the subject of fundamental research. Several techniques were employed for the determination of the complex stoichiometry and calculation of the stability constants. While UV-Vis spectrophotometry and NMR provided qualitative description of the structure of inclusion complexes, other techniques, such as titration microcalorimetry and capillary electrophoresis, yielded more quantitative data. The second and the third chapter will describe inclusion complexes between C 12 E 6 , and α- and β-CD, respectively. The effect of benzoate on the stability constants of these inclusion complexes was investigated and evidence of ternary complexes will be presented. The fourth chapter is primarily focused on the investigation of the interactions of a homologous series of nonionic surfactants C x E y containing a linear hydrocarbon, in an effort to establish some structural parameters that could affect the strength and the stoichiometry the interaction with both, α- and β-CD. In the second part of this dissertation, application of sulfated β-cyclodextrin to capillary free flow electrophoretic (CFFE) chiral separation is discussed. In recent years, there has been an effort to develop alternative preparative scale chiral separations. Cyclodextrins have proven to be an effective chiral selector, providing a medium for successful chiral separations. Fundamental principles of preparative scale electrophoretic separations will be presented. The concentration distribution of sulfated β-CD, determined in-situ within the CFFE separation chamber, will (open full item for complete abstract)

Committee: Apryll Stalcup (Advisor) Subjects: Chemistry, Analytical

PhD, University of Cincinnati, 2001, Arts and Sciences : Chemistry

The present dissertation discusses the application of two popular and important separation techniques for the separation of chiral compounds and resolution of complex mixtures found in biological samples. Chapter 1 focuses on the general advances and principles of Capillary Electrophoresis, some of the experimental variables that need to be considered for its successful application, and the description of the different modes or methods commonly employed. Different capillary electrophoretic applications are encompassed in Chapters 2, 3 and 4. Electrophoretic enantioseparations of ten different chiral compounds, including acidic, neutral and basic, using cyclodextrins as chiral selectors is the subject of Chapter 2. Three different functionalized β-cyclodextrins, two sulfated β-cyclodextrins and a phosphated β-cyclodextrin, are compared. Effects of different experimental parameters such as degree of substitution of the cyclodextrin, pH of the background electrolyte as well as the concentration of the functionalized β-cyclodextrin were evaluated with respect to enantioseparation. Currently, phenolic mixtures found in a variety of extracts are the topic of several studies. Polyphenols antioxidant properties have been associated with health benefits in humans. Chapter 3 and 4 describes the application of several electrolyte solutions in capillary electrophoresis for the resolution of such phenolic mixtures into their individual components. In both chapters, the mechanism as well as reproducibility of separation is emphasized. Chapter 3 includes the application of tetraalkylammonium tetrafluoroborate salts, while chapter 4 illustrates the screening and potential application of different 1-alkyl-3-methylimidazolium based ionic liquids for the separation of polyphenols found in grape seed extracts. Selenium speciation analyses in Brassica juncea plant tissues using High Performance Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometry is described in Chapt (open full item for complete abstract)

Committee: Apryll Stalcup (Advisor) Subjects:

PhD, University of Cincinnati, 2001, Arts and Sciences : Chemistry

A comparison of capillary electrophoresis (CE) migration times using standard on-column UV detection and inductively coupled plasma - mass spectrometry (ICP-MS) is presented for two different CE separations. The first is a separation of five arsenic compounds, four anionic species and one neutral, using reverse polarity in a chromate buffer with an electroosmotic flow modifier. The second CE separation employs normal polarity for the separation of eleven lanthanide cations using a HIBA buffer with an indirect UV detection reagent. Migration times observed for CE-ICP-MS electropherograms are comparable to migration times acquired with UV detection, and may be adjusted by changes in the make-up liquid level position. The precision of peak areas (<2%) and migration times (<4%) obtained using CE-ICP-MS is comparable to that obtained using online UV absorbance detection. CE-ICP-MS limits of detection are in the low ng ml-1 range for the analytes studied. Then, CE is coupled on-line to a double-focusing sector field inductively coupled plasma-mass spectrometer (DF-ICP-MS) for the analysis of mixtures of lanthanide elements in aqueous samples with natural isotope abundances and in a sample taken from an irradiated tantalum target particle reactor containing artificial nuclide abundances. Detection limits for the most abundant isotopes of lanthanides were 0.1ppb to 5 ppb, an improvement of as much as one order of magnitude compared to a quadrupole ICP-MS system. Abundances for the most abundant isotopes of lanthanides were found to be within 0.1 - 2% of table values for natural samples while isotopes present in smaller amounts were within 3 - 5% of table values. CE-ICP-MS is also used for the chiral speciation of DL-selenomethionine using Marfey's reagent for chiral derivatization and applied to selenized yeast samples extracted with Proteinase K. Finally, two anion exchange chromatography systems were interfaced to ICP-MS for determination of bromate in ozonated drinking w (open full item for complete abstract)

Committee: Joseph Caruso (Advisor) Subjects: Chemistry, Analytical

PhD, University of Cincinnati, 2007, Arts and Sciences : Chemistry

Elemental speciation is the investigation of the chemical form ofmetal and non- metal containing species in environmental and biological systems for the determination of species specific essentiality and toxicity. Speciation analysis is performed by combining modern separation techniques with state-of-the-art element specific mass spectrometry. Separation techniques used in this work include: capillary electrophoresis (CE), high performance liquid chromatography (HPLC) and gas chromatography (GC). Inductively coupled plasma mass spectrometry (ICPMS) is the instrument of choice for ultra-trace elemental speciation analyses due to the excellent sensitivity and selectivity specific to this mass spectrometer. Modern innovations in analytical instrumentation specific for elemental speciation have provided researchers with resources for the development of new hyphenated techniques and analytical methods. The specific goal of this dissertation is to describe modern advancements specific to elemental speciation. In the first section a novel interface coupling CE and hydride generation with ICPMS detection for arsenic speciation is described. The novel concentric tube interface design allowed for the separation, hydride generation, and detection of four arsenic species in less than 10 minutes. The majority of this dissertation focuses on method development for the analysis of organophosphorus chemical warfare agent (CWA) degradation products. Recent increases in worldwide terrorist activity as well as the threat of chemical weapon attacks have led to the demand for rapid and reliable analytical techniques for CWA analysis. Methods utilizing both HPLC and GC separation techniques couple with 31P element specific detection with ICPMS for the analysis of organophosphorus chemical warfare agent degradation products are described. These works are the first to utilize 31P detection with ICPMS for the analysis of chemical warfare agent degradation products.

Committee: Dr. Joseph A. Caruso (Committee Chair); Dr. William R. Heineman (Committee Member); Dr. James Mack (Committee Member) Subjects: Chemistry

PhD, University of Cincinnati, 2008, Arts and Sciences : Chemistry

The work presented in this dissertation aims to advance the application of using cyclodextrins to encapsulate flavoring compounds for enhanced retention and protection from environmental surroundings. Specifically, results of investigations are presented on the study of beta-cyclodextrin (β-CD) inclusion of 2-furfurylthiol (FFT) in the presence and absence of different fatty acids (FAs). In Chapter 1, an overview is given on CD chemistry: its discovery, structural characteristics, inclusion complexation, and uses in industrial and chemical applications. The concept of ternary CD complexes is introduced, in which auxiliary compounds are used in conjunction with CD to achieve desirable traits not realized by simple binary inclusion complexes. The natural occurrence of some sulfur containing aroma and flavor compounds is discussed, specifically the importance of furanthiols in aroma and flavor chemistry. Chapter 1 concludes with an overview of the focused project of this dissertation, and its relation to past work in this field. Chapter 2 describes studies on FFT complexation with β-CD in solution. Nuclear magnetic resonance (NMR) spectroscopy and its application to study CD inclusion complexes are detailed. Results of these experiments provide evidence for cavity inclusion of FFT with β-CD, and a structure of the complex is proposed. A quantitative binding study is also discussed. Chapter 3 details binding studies between FAs and β-CD, using capillary electrophoresis and isothermal titration calorimetry. Results obtained from these techniques can be used concurrently to quantitatively characterize these binary systems. Chapter 4 carries the focus toward the study of solid encapsulation products of FFT with β-CD and β-CD/FAs in combination, with the goal of preparing a solid FFT formulation with ideal retention and environmental protection capabilities. Results are presented for FFT loading and retention in different solid materials that were prepared. The complex (open full item for complete abstract)

Committee: Apryll Stalcup PhD (Committee Chair); Patrick Limbach PhD (Committee Member); James Mack PhD (Committee Member) Subjects: Analytical Chemistry; Chemistry

PhD, University of Cincinnati, 2006, Arts and Sciences : Chemistry

This thesis is dedicated to two separate topics. First topic is related to investigations of the optical properties of spectroelectochemical sensor. The modeling methodology of spectroelectrochemical sensor is described and applied to the Ru(bpy)32+ and Nafion film system. Ru(bpy)32+ sorption is carried out experimentally in a standard sensor configuration. Theoretical absorbance values are obtained for this system based on independently determined optical constants of Nafion film after Ru(bpy)32+ sorption. The developed methodology is applied for studies of thin film ATR spectra. Various thicknesses, pre-soaked Nafion films are studied. Spectral scans over the range of wavelength and angle of incidence reveal significant distortions for films with thickness above 200 nm. Spectra exhibit additional absorbance maxima, shifted considerably away from the true k maxima. The effect is observed if k is on the order of 0.01. Ru(bpy)32+ sorption was studied in situ and by explicit finite difference method. Significant spectral distortions are observed due to leaky waveguiding mode distortions during sorption. The second part of this thesis explores a laser induced fluorescence multi-lane detection system. Plastic materials suitable for optical detection were evaluated over the visible spectrum (403, 488, 532 and 633 nm excitations). Commercially available cyclic olefin copolymers (COC), polycarbonate (PC) and poly(methylmethacrylate) (PMMA) materials and home-made chips are investigated. COC and PMMA and represent the best materials for chip fabrications and exhibit autofluorescence only a few (3 to 5) times higher than Borofloat glass. An optical multi-lane detection system was demonstrated. A linearly expanded beam (~6 mm) was focused across 14 microfluidic channels. As a result, a fluorescent analyte could be detected simultaneously in all channels. Separation of fluorescein and fluorescein isothiocyanate was demonstrated on a glass multi-lane chip.

Committee: Dr. Carl Seliskar (Advisor) Subjects: Chemistry, Analytical

PhD, University of Cincinnati, 2006, Arts and Sciences : Chemistry

Microchip capillary electrophoresis (CE) is the miniaturized form of traditional CE, a powerful electrical separation technique performed in a capillary with micron diameters. The advantages of microchip CE over traditional CE include shorter analysis time and less consumption of chemicals and reagent. The objective of the microchip program in our group is to develop microchip CE instrumentation and methodologies for high throughput screening of pharmaceuticals. As an important step, my work was focused on the development of methodologies for binding studies and trace protein assays in single-lane glass microchips. A pair of thrombin and thrombin-binding aptamer was chosen as a model system to demonstrate the feasibility of microchip CE in binding studies. The combination of a short detection length (1.0 cm) and a high electric field (670 V/cm) succeeded in the detection of the aptamer-thrombin complex and reduction of thrombin adsorption. The limit of detection (LOD) reached 5.0 nM of thrombin. To determine the equilibrium dissociation constant of aptamer-thrombin, a simple hydrodynamic injection method in microchips was developed. This hydrodynamic injection significantly reduces sample bias although the dispensing bias still exists. Using the hydrodynamic injection method, the dissociation constant of aptamer-thrombin was determined to be 23 nM which is consistent with reported results. Moreover, an accurate and fast frontal analysis method was developed for the determination of dissociation constant with a model pair of aptamer and immunoglobulin E (IgE). The capability of microchip CE coupled with a laser-induced fluorescence (LIF) detection system in the detection of trace protein was demonstrated using a 66-mer photoaptamer to detect 165-mer vascular endothelial growth factor (VEGF165). A LOD of 1.0 nM was achieved for the detection of VEGF165 using the fluorescently-labeled photoaptamer. To enhance detection sensitivity of analyte in microchip CE, a field-am (open full item for complete abstract)

Committee: Dr. William Heineman (Advisor) Subjects: Chemistry, Analytical

Doctor of Philosophy, University of Toledo, 2008, Chemistry

Cholinergic neurons play a vital role in cognition and memory. Changes in the cholinergic system are associated with severe neurodegenerative disorders such as Alzheimer's disease (AD) and dementia. Hence, it is important to develop analytical strategies that can elucidate mechanisms of such selective cholinergic degradation and also be able to monitor the subtle changes that occur. Biomolecules that can selectively interact with disease specific biomarkers have potential applications in the development of biosensors. These biocatalysts, when integrated with conductive supports produce transducing signals leading to selective detection of the biomarkers. A key to such effective bioelectronic transduction is integration of various biointerfaces and their efficient electronic communication with the underlying conducting support.The primary focus of the work has been the separation and quantification of cholinergic metabolites using capillary electrophoresis (CE) coupled with electrochemical detection (EC). CE allowed efficient separation of nL sample volumes while enzyme modified microelectrodes (MEs) enabled selective amperometric detection of choline (Ch) and acetylcholine (ACh). This method was used to study the rate of Ch uptake by the High Affinity Choline Uptake transporter protein (CHT) in mouse synaptosomes. The Michaelis-Menten constant of CHT was determined to be 0.79 μM. Further, a cholinomimetic bis-catechol hexamethonium analogue (DTH) was examined for its ability to selectively inhibit Ch uptake by CHT. The IC50 value of DTH was determined to be 76 μM. KI of this inhibitor determined by Dixon plots was calculated as 73 μM. DTH inhibited CHT via a mixed inhibition mode. The results obtained were in logical conclusion with established studies regarding structural aspects and affinity of CHT. Furthermore, ME techniques were employed to develop two amperometric enzyme microsensor systems as detectors for CE for monitoring low micromolar concentrations of Ch (open full item for complete abstract)

Committee: Jon Kirchhoff (Committee Chair) Subjects: Analytical Chemistry

Doctor of Philosophy (PhD), Ohio University, 2004, Chemistry (Arts and Sciences)

Capillary electrophoresis (CE) is a versatile tool for the isolation, separation, and detection of date-rape and club drugs. Advanced applications of electrophoretic techniques allow for the separation of small, similarly charged compounds by the utilization of mobile phase additives and chromatographic stationary phases. Two novel screening procedures were developed using mobile phase additives. The first used sodium dodecyl sulfate (SDS) to detect gamma-hydroxybutyric acid (GHB), gamma-butyrolactone, as well as eight classical and low-dose benzodiazepines. Although SDS previously had been used for the separation of drugs, this was the first simultaneous separation of benzodiazepines and GHB. The second method used the additive hydroxypropyl-beta-cyclodextrin to detect 1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), and three piperazine analogs. An additional aspect of this project was to develop new systems for the analysis of the above drugs using capillary electrochromatography (CEC). A stationary phase in the capillary permitted the analysis of neutral drugs without resorting to pseudostationary phases. The buffers used in CEC were comparable to those used in high performance liquid chromatography (HPLC), yet were more compatible with mass spectrometric detection. While LC-like stationary phases are still among the most popular utilized for CEC, monolithic polymer materials have the potential to provide an environment more suited to the generation of electroosmotic flow, in combination with partitioning, necessary for optimal analysis. Pore size and chromatographic evaluations of various poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases were examined for their utility in small molecule separations. Miniaturization of the techniques described above can result in devices that are small, portable, and disposable. The microfluidic analysis of four nitrated benzodiazepines was accomplished on a commercial device using (open full item for complete abstract)

Committee: Bruce McCord (Advisor) Subjects:

Doctor of Philosophy, Miami University, 2004, Chemistry and Biochemistry

This dissertation presents investigations into the use of single-bounce attenuated total internal reflection (ATR) infrared (IR) microspectroscopy as a detector for analytes in solution. The detection of analytes in nonmoving (static) solutions, flow injection, and separation systems are presented. Detection limits of succinylcholine chloride in water are shown to be as low as 21 ppm. A small sampling volume (180 fL) is important in reducing band broadening, and improving mass detection limits in low flow environments. Flow injection measurements demonstrate that the flow rate, injection volume, capillary diameter, and internal reflectance element (IRE) capillary separation distances are important. Improved analyte selectivity over traditional spectroscopic techniques is evaluated using high-performance liquid chromatography, capillary electrophoresis (CE), and polymer-mediated CE. Background electrolyte concentration has been found to play a large role in separation resolution and peak characteristics, which in turn affects infrared spectral quality. The choice of detector, and the preconcentration of analytes in solution using a reverse phase coating applied to a zirconia sol-gel, can have a large effect on detection limit. Future directions that reduce the detection limit, set-up time, and the creation of an infrared integrated cell are outlined. Finally, undergraduate analytical and physical chemistry labs are presented using modular spectrometers to construct UV/Vis, fluorescence, Raman, and atomic emission instruments.

Committee: Andre' Sommer (Advisor) Subjects: Chemistry, Analytical

Doctor of Philosophy, Miami University, 2003, Chemistry and Biochemistry

The first two projects describe new applications of flow injection capillary electrophoresis (CE). The first study focuses on the determination of lactate or oxalate using injected lactate oxidase (LO) and peroxidase with UV detection. Two reactions, catalyzed by (LO) and peroxidase, are initiated by a single injection of the enzymes and the substrate 2,2'-azino-bis(3-ethylene-thiazoline-6-sulfonic acid) (ABTS) into the capillary previously filled with the sample (lactate or lactate-oxalate mixture) and the running buffer containing NADH. The oxidized ABTS product upon reaction with NADH is converted to NAD+, which is detected at 266 nm with a sample throughput of 7 min including wash steps. Linearity for lactate is established from 0.0025 – 1mM and serum samples are analyzed with an average recovery of 101%. This method can also be applied to the determination of oxalate as an inhibitor of LO. The second topic describes the determination of the concentration of cardiolipin using CE with on-line N-nonylacridine orange (NAO) dye interaction and spectrophotometric detection at 497 nm. Other phospholipids do not interfere and a detection limit of 0.05 iM was found. In a blind study, actual mitochondrial cell membrane samples in the µL range, taken from cells before or after UV light exposure, are analyzed using the CE method. The last two topics describe preconcentration of pharmaceuticals and phospholipids with nano-electrospray ionization mass spectrometry (nano-ESI MS). An analytical method is developed for the quantitative determination of dicyclomine in serum with cyclopentolate as the internal standard by off-line nano-ESI MS with reversed phase mini-solid phase extraction (mini-SPE). Different length (0.5cm, 1cm) reversed phase (C4 and C18) mini-SPE cartridges are prepared by packing gel-loader pipette tips for optimization of the preconcentration effect. A factor of 100 is possible using the 1 cm C18 or C4 cartridge. Spiked serum samples are quantitatively dete (open full item for complete abstract)

Committee: Neil Danielson (Advisor) Subjects: Chemistry, Analytical

MS, Kent State University, 2010, College of Arts and Sciences / Department of Chemistry

Phosphatidyl inositides (PIs) are important regulators of cell signaling. Phosphoinositide 3-kinase (PI3-K)-activated signaling plays a key role in the development of cancer. Therefore various anticancer treatments target this pathway. The aim of this work was to develop an optimized method to separate fluorescent PIPs quickly and efficiently at room temperature using CE-LIF. The reason for the development of a method capable of separating the PIPs at room temperature was that, it could be used for single cell studies. In this regard, the effect of various cations on the separation of the PIs and their seven PIP derivatives was investigated. The effect of pH, temperature, voltage, as well as other buffer mixtures were also studied. Based on this developed method, the activity of PI3-K and PTEN enzymes in NIH 3T3 cells and MDA-MB 231 cells was also studied. The bioactivity of PIPs in these cells was investigated to ascertain whether preliminary CE studies would prove that they were able to be converted inside the cells into other phosphorylated derivatives. Lipid kinases and phosphatases play active roles in cell signaling. These have serious implications in many disease states. For example, accumulation of PIP3 leads to metastatic cancers. Therefore, inhibition studies were also performed to find out whether PI3-K inhibitors were able to block the conversion of PIP2 to PIP3. This is of particular interest in cell-based assays and research involving cancer drug development.

Committee: Simon M Mwongela PhD (Advisor); Songping Huang PhD (Committee Member); Bansidhar Datta PhD (Committee Member) Subjects: Biochemistry