Master of Science / Department of Grain Science and Industry / David L. Wetzel / Isogenic waxy wheat lines differ from their non waxy (normal) parents in functionality, end use, and chemical (i.e. amylopectin/amylose, lipid) contents. Other investigations of waxy and parent wheats involved the carbohydrate and protein fractions. The goal of this work is to apply chemical images to discriminate between the waxy and parent wheats and define the contribution of contrasting lipid profiles. Recent waxy topics include current interest in plant breeding activity to develop new lines that incorporate desirable traits with advantageous success in baking and milling, and the differences needed in milling techniques for waxy versus normal wheats that may be associated with lipids. From our empirical preliminary success in sorting parent wheat kernels from waxy wheat full null specimens by nearIR chemical imaging it was anticipated that using fundamental vibrational spectra in the mid infrared would provide the chemical basis of discrimination. FT-IR microspectroscopic in situ probing and imaging of kernel frozen sections was applied to genetically pure, well documented isogenic breeding lines. With the use of high spatial resolution, elucidation of fundamental vibrations of mid IR provides chemical manifestation of the genetic expression that differentiates waxy wheats from their parent wheats. Comparison between numerous contiguous pixels, typically 3,000 for each type, establishes a consensus and a mean spectrum with characteristic bands for waxy and parent. Extractions with solvents of differing polarity were employed to aid in lipid extraction in situ and kernel extracted endosperm. Differences between kernel sections of waxy and parent are observed using FT-IR microspectroscopic imaging. However, revealing lipid class contribution to the molecular bands required infrared analysis after selective extraction. Triple mass spectrometry of lipid molecule ions was used for compositional analyses to enhance lipid class profile distinction. A normal and waxy advanced breeding line wheat were also analyzed via the same methods. It was noted that digalactoslydyglyercides are the most abundant lipids in all samples, however the relative lipid profiles of normal wheat versus waxy wheat differ as well as tetraploid versus hexaploid. It is observed that in the endosperm of all parent wheat versus waxy wheat specimens analyzed, all waxy wheat specimens contained higher lipid content. Methods were also applied to partial waxy isogenic cultivars to determine detection limits that correspond to the degree of waxy genetic expression.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / The preparation of mesoporous/macroporous silica materials and polarity gradient thin film are introduced in this thesis. These porous silica materials and gradient materials have the potential applications as stationary phases for chemical separations, as materials for combinatorial catalysis and as absorbent/adsorbent layers for use in chemical or biological sensors. Single molecule spectroscopy is used to probe the chemical interaction between single dye molecule and porous silica matrix. Bulk fluorescence spectroscopy is used to investigate the properties of gradient film. In Chapter one, the applications of single molecule spectroscopic methods to sol-gel silica materials are reviewed, which covers a subset of the recent literature in this area and provided salient examples of the new information that can be obtained by single molecule studies. In Chapter two, both the sample preparation and experiment setup are covered. In Chapter three, the preparation of mesoporous silica film is presented. Single molecule spectroscopy is used to probe the mass transport and molecule-matrix interactions in mesoporous thin-film systems. Three different dyes of varying size, charge, and hydrophilicity are used. Silica films with/without surfactant or containing different kind surfactant are studied. The results provide new information on mass transport through the films, evidence of reversible surface adsorption, and quantitative information on variations in these phenomena with film hydration. In Chapter four, a new model describing how to explore the actual dye concentration in single molecule experiment with considering the molecule orientation is presented, which is verified to be correct by both experimental and simulated data. In Chapter five, the growth process of Methylsilsesquioxane (MSQ) particle is studied by single molecule spectroscopy, in which, the MSQ particle is treated as “native” dye molecule. In Chapter six, silica films incorporating polarity gradients are produced by using “infusion-withdrawal dip-coating” method. The gradient film is characterized by bulk fluorescence spectroscopy, water contact angle and FTIR. In Chapter seven, a brief conclusion is drawn and future directions are presented.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / As the field of separation science evolves so do the techniques, tools and capabilities of the discipline. The introduction of microfluidics stemmed from a desire to perform traditional analyses faster and on a much smaller scale. The small device sizes exploited in microfluidics permits the investigation of very small volumes of very dilute samples yielding information inaccessible by traditional macroscale techniques. All of the chapters presented in this dissertation illustrate attempts to supplement current microscale techniques with new tools, techniques and analysis schemes for looking at biologically relevant analyses. In chapter two I present the development and characterization of an amphiphilic polymer that has potential as a material for the fabrication of microfluidic devices. This material is composed of a poly(dimethylsiloxane)-poly(ethylene oxide) block copolymer and is dramatically more hydrophilic than the other polymeric materials currently used for the fabrication of microfluidic templates, mainly poly(dimethylsiloxane). Biomolecules such as proteins are notoriously hydrophobic and will tend to adsorb to other hydrophobic surfaces thus the use of a hydrophilic material may serve to reduce or eliminate this problem. The amphiphilic material is of a suitable durability for micromolding and molded channel architectures can be sealed between two layers of the material by simple conformal contact permitting the execution of high speed electrophoretic separations. Chapter three contains initial results obtained while investigating the fluorescent labeling and electrophoretic separation of ecdysteroids. Ecdysteroids are hormones found in insects that are responsible for controlling the process of molting. Here we attempted to analyze these molecules by employing a reactive fluorescent probe, BODIPY FL® hydrazide, that would target the α,β-unsaturated ketone group on the steroid, permitting its analysis by capillary electrophoresis with laser induced fluorescence detection. While optimistic initial results were obtained with the labeling and analysis of similar functional groups on model compounds such as progesterone, labeling of the ecdysteroid molecules was never achieved to a degree that would permit reliable analysis. In chapter four I report the development and use of a microimmunoaffinity column for the analysis of insect serine protease inhibitors, or serpins. These proteins play a very important role in the regulation of insect immune responses and their activity may play an integral role in the effective transmission of the malaria parasite by the mosquito Anopheles gambiae. A microimmunoaffinity column was constructed from magnets, poly(dimethylsiloxane), fused silica capillary and Protein A coated magnetic microspheres. In these initial studies, purified antibodies to serpin protein, as well as purified serpin protein, were used to prepare and investigate the ability to isolate, preconcentrate, and elute serpin proteins for subsequent analysis. By implementing this miniaturized system which incorporates very small fluid volumes we hoped to extend this technique to the analysis of very small samples, and eventually to the analysis of individual small insects. Our work indicates that it is possible to isolate, elute, and detect serpin protein on a traditional western blot membrane. Chapter five presents the development of a novel polymer blend for the fabrication of paper-based microfluidic devices and use of these devices in the performance of diagnostically relevant clinical assays. We took the concept of paper-based microfluidic devices and improved upon the current photoactive polymers used for their fabrication by developing a polymer blend using an acryloxy modified siloxane polymer as well as a commercially available photoactive adhesive, Norland Optical Adhesive 74. This blended polymer resulted in a dramatic reduction in fabrication time as well as improved resolution permitting the reliable patterning of small feature sizes. We also report for the first time a demonstration of these devices performing a two-step spatially separated online chemical derivatization facilitating the analysis of urinary ketones. These devices are predominantly used for the analysis of urine, and their application was extended to the quantitation of nitrite in saliva for the purposes of hemodialysis monitoring. While varied in application, all of the data presented in this dissertation exploits the power of miniaturization to improve current methods of analysis and to extend macroscale techniques to trace biological analytes.

L’étude qui suit porte sur l’évaluation du risque écotoxicologique du cérium, l’élément le plus exploité de la famille des lanthanides. La présence grandissante de ce métal dans notre quotidien rend possible son relargage dans l’environnement. Il est donc primordial de comprendre l’impact qu’il aura sur les organismes vivant dans un système aquatique. Une approche centrée sur le modèle du ligand biotique a été utilisée pour évaluer adéquatement l’interaction entre le cérium et un ligand biotique à la surface de l’algue unicellulaire Chlamydomonas reinhardtii. Pour mener une étude sur le risque écotoxicologique d’un élément métallique il faut, avant tout, comprendre la spéciation (répartition sous ses différentes formes chimiques) de l’élément en question. Les premières sections du mémoire vont donc traiter des expériences qui ont été menées pour évaluer la spéciation du cérium dans les conditions expérimentales d’exposition à C. reinhardtii. Il sera question de faire la distinction entre la forme particulaire du métal et sa forme dissoute, de caractériser ces changements par spectroscopie ainsi que d’évaluer le pouvoir complexant de la matière organique naturelle. Les résultats montrent une importante déplétion du métal dissout en solution à pH neutre et basique et une forte interaction avec la matière organique naturelle, peu importe le pH de la solution. Ensuite, les expériences de bioaccumulation seront expliquéesen comparant l’effet du pH, de la présence d’un ion compétiteur et de la présence de matière organique naturelle sur les paramètres d’internalisation du cérium. Les résultats indiquent qu’à pH acide, le comportement du cérium est plus prévisible qu’à pH neutre. Néanmoins, en tenant compte de la complexité des milieux naturels, l’interaction du métal avec les molécules complexantes va diminuer son risque d’interaction avec un organisme vivant. / The following study is on the ecotoxicological risk evaluation of cerium, the most widely exploited element of the lanthanide family. The increasing presence of this element in our everyday lives renders possible its release in the environment. It is therefore of primary importance to understand the impact this metal will have on living organisms in aquatic environments. An approach centered on the biotic ligand model was used here to evaluate the interaction between cerium and a biotic ligand at the surface of the unicellular algae Chlamydomonas reinhardtii. To study the ecotoxicological risk of a metallic element one must understand the speciation (partitioning between its multiple chemical species) of the element in question. The first chapters of this thesis will discuss the experiments performed to evaluate cerium speciation under exposure conditions C. reinhardtii. The issue will be to distinguish between the particulate and dissolved species of the metal, to characterise these changes by spectroscopy, as well as to evaluate the complex formation capacity of the metal with natural organic matter. Results indicate an important depletion of dissolved metal in neutral and alkaline solutions as well as a strong interaction with natural organic matter, regardless of solution pH. Bioaccumulation experiments will then be explained and will compare the effects of pH, the presence of competing ions and the presence of natural organic matter on cerium uptake. Results show that cerium’s behaviour is more predictable under acidic pH conditions. Nonetheless, considering the complexity of the natural environment, the interaction of the metal with natural ligands will most likely reduce the risk of cerium’s interaction with living organisms.

Plusieurs tests médicaux, comme celui du dépistage du cancer du sein, se basent sur l’observation de section tissulaire sous un microscope. Ces tests se basent sur l’interprétation d’un spécialiste et les résultats peuvent varier d’un expert à un autre dû la subjectivité des observations. L’utilisation d’une technique analytique offrant une quantification et une identification de cibles moléculaires dans une section tissulaire permettrait aux experts de produire des diagnostics plus objectifs et diminuerait possiblement le nombre de faux diagnostics. Les travaux présentés dans ce mémoire portent sur le développement d’une technique SPRi-MALDI-IMS permettant l’imagerie en deux dimensions de protéines contenues dans une section tissulaire. La MALDI-IMS est la technique de choix pour l’imagerie de biomolécules dans les sections tissulaires. Par contre, elle ne parvient pas à elle seule à quantifier de façon absolue le matériel adsorbé à la surface. Donc, le couplage de la MALDI-IMS avec la SPRi permet la quantification absolue de protéines en deux dimensions et crée une technique répondant aux besoins des experts médicaux. Pour ce faire, nous avons étudié, l’effet de la chimie de surface sur la nature et la quantité de matériel adsorbé à la surface du capteur. De plus, la cinétique de transfert des protéines du tissu vers le capteur a dû être optimisée afin de produire des empreintes correspondant au tissu d’origine, afin d’atteindre la gamme dynamique des instruments SPRi et MALDI-IMS. La technique résultante de ces optimisations permet d’obtenir les premières images quantitatives et qualitatives de protéines en deux dimensions d’une seule section tissulaire. / Several medical tests, such as breast cancer screening, are based on the observation of tissue section under a microscope. These tests rely on the interpretation of a specialist and the results can vary due to subjectivity of these analyses. Using an analytical technique able to quantify and identify molecular targets in a tissue section would enable experts to produce more objective diagnostic and possibly reduce the number of misdiagnosis. The work presented in this thesis focuses on the development of a SPRi-MALDI-IMS technique for imaging proteins in a tissue section. MALDI-IMS is the contemporary technique of choice for biomolecule imaging in tissue sections. However, absolute quantification of material absorbed to the surface cannot be performed using MALDI-IMS. The absolute quantification of proteins in two dimensions was successfully achieved by coupling of the MALDI-IMS with SPRi. Accordingly, we investigated by studying the nature of the surface chemistry and the amount of material adsorbed on the sensor’s surface. In addition, the kinetics of the protein transfer had to be optimized to produce imprints corresponding to the transferred tissue and to reach the dynamic ranges of both SPRi and MALDI-IMS instruments. The resulting technique led to quantitative and qualitative images of proteins in two dimensions of a single tissue section. It is envisioned that SPRi-MALDI-IMS can eventually meet the needs of medical experts for pathological screening.

Les éléments de terres rares (REEs) sont de plus en plus utilisés dans une multitude d’applications, notamment la fabrication d’aimants, de batteries rechargeables et les écrans de téléviseurs. Ils sont pour la plupart des métaux trivalents peu solubles dans les eaux naturelles. Comme pour les métaux divalents, le risque écologique des REEs est très probablement étroitement lié à leurs spéciations chimiques. Cependant, le comportement du samarium (Sm) dans les matrices environnementales est très peu connu et il n'existe actuellement aucune technique pour évaluer sa spéciation chimique. Dans cette optique, la technique d'échange d'ions (IET) sur la résine Dowex a été optimisée pour mesurer le samarium libre en solution. Les temps d'équilibre ont d'abord été déterminés pour des solutions tamponnées de samarium (Sm 6,7x10-8 M ; MES 1,0 mM M ; pH 6,0) en présence du nitrate de sodium (de 0,01M à 0,5 M). Pour ces diverses forces ioniques, l’équilibre thermodynamique n’est atteint que pour NaNO3 0,5M. Un autre mode d’utilisation de la résine (mode dynamique) a donc été développé pour tenir compte des conditions environnementales et évaluer efficacement le samarium libre. Les impacts des ligands organiques tels le NTA, l’EDTA, le citrate, l’acide malique et l’acide fulvique Suwannee River Standard I (SRFA) ont été étudiés par l’IET en mode dynamique. Une grande corrélation a été trouvée plus entre les taux d’accumulation de samarium sur la résine d’échange pour différents rapports NTA : Sm, EDTA : Sm, SRFA : Sm et le samarium libre. Par contre, aucune corrélation significative n’a été observée pour les ligands citrate et acide malique compte tenu des complexes qu’ils forment avec le samarium et qui s’adsorbent aussi sur la résine Dowex. Les concentrations Sm3+ mesurées par la technique IET ont été fortement corrélées avec celles prédites par le modèle WHAM 7.0 en utilisant la constante de stabilité obtenue par titration de SRFA par extinction de la fluorescence. Par ailleurs, la formation de colloïdes de samarium en fonction du pH influe grandement sur la détermination du samarium libre et doit être prise en compte dans la spéciation du samarium. L'IET assisté par des techniques auxiliaires comme le dosage par extinction de la fluorescence et le SP-ICPMS pourrait être une technique utile pour évaluer les concentrations de Sm biodisponible dans les eaux naturelles. / Samarium is present in several high technology products such as magnets, lasers, etc., however little is known about to what extent it can pollute environmental matrices. For divalent metals such as Cd, Cu and Ni, toxicity has often been closely correlated with the free ion concentration. Unfortunately, there are currently no techniques available for evaluating the speciation of samarium in environmental matrices. In this study, an economical and easy to use ion exchange technique (IET) using a resin has been studied for use in measuring free samarium. In order to optimize the IET for free samarium determinations, equilibrium times were first determined for pH buffered solutions of samarium (6.7x10 8 M Sm, 1.0 mM MES, pH 6.0) in the presence of different concentrations of sodium nitrate (NaNO3: 0.01 M to 0.5 M). Equilibrium was only attained for the highest concentration of NaNO3. Therefore, another technique using dynamic measurements on the Dowex resin was developed. Thus, the impacts of different organic ligands such as EDTA, citrate, malic acid and Suwannee River fulvic acid Standard I (SRFA) have been investigated using IET in dynamic mode. The calibration of the technique using different concentrations of NTA, EDTA, citrate, malic acid and fulvic acid (SRFA) allowed the determination of accumulation rates. Strong correlations were found between accumulation rates and the free samarium when using EDTA and NTA to control free samarium. The correlation was very low in case of citrate and malic acid suggesting that their complexes with samarium were adsorbed on the resin. Finally, fluorescence quenching titrations (FQT) and SP-ICPMS were used to determine samarium speciation. The free samarium determined by IET is strongly correlated to that calculated by WHAM 7.0 using the stability constant from FQT and that from Sonke (2006). However, the free Sm resulted from IEC-SP-ICPMS technique has been overestimated once compared to that found using VMinteQ or WHAM 7.0 models. The Sm particulates determined by SP-ICPMS have demonstrated that pH has a great influence on Sm exchange on resin and has to be considered while measuring free samarium. Thus, the IET should be associated to other techniques such as fluorescence quenching titration and SP-ICPMS for evaluating the bioavailable Sm concentrations in natural waters.

To distinguish among malaria parasites producing human infections, PCR-based amplification of the polymorphic Block 2 region of merozoite surface protein-1 (MSP-1) was used to estimate the incidence and clearance of Plasmodium falciparum infection in areas of Mali with seasonal transmission. Molecular estimates were compared prospectively with slide-based estimates in a cohort of 80 children from 6 months to 9 years of age. The molecular method detects 1.45 to 6 fold more incidence and clearance events than microscopy. However, both methods demonstrate similar variation with peaks at the end of the rainy season and troughs at the end of the dry season. The principal molecular marker used in this study allowed us to detect novel hybrid (MAD20/RO33) sequences in Block 2 of MSP-1 from recombination across allotypes between MAD20 parasites (containing 9 nucleotide repeats) and RO33 parasites (with no repeats). Feeding experiments with F1 Anopheles gambiae demonstrated that formation of hybrid parasites occurs during the meiotic reduction division in anopheline mosquito vector, which converts the diploid zygote to an oocyst midgut. The clinical significance of parasites with hybrid sequences is that they are associated with severe disease. Parasites with hybrid (MAD20/RO33) sequences in Block 2 of MSP-1 were found in 58% (14 of 24) children with cerebral malaria, versus 8% (6 of 73) children with asymptomatic infections, chi2 = 20.86, p < 0.001 / acase@tulane.edu

The compounds 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra-(2-N, N-dimethyldithiocarbamoylethoxy) calix (4) arene, 25,26,27,28-tetra-(2-N, N-dimethyldithiocarbamoylethoxy)calix (4) arene, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra-(2-mercaptoethoxy)calix (4) arene, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra-(2-(methylthio)ethoxy)calix lbrack 4) arene, 25,26,27,28-tetra-(2-(methylthio)ethoxy)calix (4) arene, 25,26,27,28-tetra-(2-(2-pyridylthio)ethoxy)calix (4) arene tetra-N-oxide and 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra-(2-(2-thiophenecarboxy)ethoxy)calix (4) arene have been synthesized. They have been characterized by proton NMR spectroscopy, carbon-13 NMR spectroscopy, infrared spectroscopy and elemental analysis. In addition, the crystal structures of 25,26,27,28-tetra-(2-hydroxyethoxy)calix (4) arene, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra-(2-bromoethoxy)calix (4) arene, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra-(2-N,N-dimethyldithiocarbamoylethoxy) calix (4) arene, 25,26,27,28-tetra-(2-N,N-dimethyldithiocarbamoylethoxy)calix (4) arene and 25,26,27,28-tetra-(2-(methylthio)ethoxy)calix (4) arene have been determined by single crystal X-ray diffraction. These sulfur derivatized calixarene derivatives have been found to be selective extractants for heavy and precious metal ions such as Hg(II), Ag(I), Pd(II), Au(III) into chloroform from water. The potential of these ligands as extraction agents in environmental cleaning operations and industrial separations are discussed / acase@tulane.edu

Cyclic polyesters are of interest, as cyclic topology imparts unique physical properties when compared to linear analogs. The production of high purity cyclic poly(epsilon-caprolactone) (PCL) is achieved by copper catalyzed 'clicking' of &agr;-azido-&ohgr;-alkyne functionalized linear PCL through a cycloaddition reaction resulting in no change of mass of the polymer. Through development of improved methods of calibration, end group mass calculation, and metastable byproduct characterization, the structure of the linear precursor and cyclic products could be defined by MALDI-TOF MS, as well as GPC and NMR. For the first time, MALDI methods were developed to show that the hydrolytic degradation of cyclic PCL is retarded when compared to its linear counterpart, while simultaneous monitoring of the degradation by GPC showed an initial increase in solution volume for the cyclic PCL, offering potentially intriguing material and biomedical applications. Cyclic architecture caused no appreciable change in thermal degradation temperature. Tm and Tc are also consistently higher for cyclic PCL. The gas-phase ion volume, as observed by drift time in ion mobility spectrometry-mass spectrometry (IMS-MS) is smaller for cyclic PCL than linear PCL, and thus IMS-MS is a powerful technique for confirming change in architecture where there is no m/z difference. To this end, a library of various polyester architectures (linear, cyclic, star, and dendrimer) were examined via IMS-MS to correlate architecture to drift time, and IMS-MS is shown to be a powerful tool to elucidate architecture of polymers with respect to molecular weight / acase@tulane.edu

The sediments of Bayou Trepagnier, at the western border of the La Branche wetlands, were contaminated with heavy metals in industrial waste water discharged before the Clean Water Act. Our study of the Pb, Cr, Cu, and Zn content of sediments and associated pore waters at 27 sites along the bayou began in June of 1994. At the beginning of this study, cooling water and storm water runoff from the Shell Norco Manufacturing Complex entered the bayou at its headwaters. This effluent was diverted to the Mississippi River in late February of 1995 as required by the EPA. The effluent, which is warm and slightly brackish, counted for only a small fraction of the water feeding the bayou with the major portions being rainwater runoff from the neighboring swamp along with tidal and wind-driven inputs from Lake Pontchartrain. A profile of the conditions of the bayou in 1994, along with updated monthly analyses for metals dissolved in bottom water samples before and since the diversion, will be presented. It appears that the change in water chemistry due to the removal or the plant discharge has not caused a significant release of contaminates currently sequestered in the sediments / acase@tulane.edu