Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "December 2008" Includes bibliographical references.

Thesis (Ph. D. in Mathematics)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

Diacylglycerol acyltransferase (DGAT), an integral membrane protein of the endoplasmic reticulum, catalyses the final step in the sn-glycerol-3-phosphate pathway leading to triacylglycerol. Although DGAT has been cloned from a variety of species, including the major oilseed crop of Canada, canola (Brassica napus), little is known about the structure/function of the enzyme. BnDGAT1 is the major isoform of type I DGAT (DGAT-I) in microspore-derived cell suspension cultures of B. napus L. cv Jet Neuf, with the possible existence of a truncated form of BnDGAT1 known as BnDGAT2. In order to gain some insight into the topology of the enzyme, type I DGAT from B. napus was investigated using two approaches: (1) in vitro translation in the presence of microsomes and (2) immunochemical analyses of microsomes isolated from cell suspension cultures, both in combination with proteolytic mapping. Difficulties were encountered with the in vitro translation approach, possibly due to proper incorporation of the polypeptide into microsomal vesicles. Two cytocolic regions were identified in BnDGAT1, and one cytosolic region in putative BnDGAT2, using the immunochemical approach, thus providing some insight into the topology of B. napus DGAT-I. The results here support and nine and eight membrane-spanning topology for BnDGAT1 and BnGAT2, respectively. / xvii, 194 leaves ; 29 cm.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O objetivo desse trabalho foi investigar a produÃÃo de mono (MG) e diglicerÃdeos (DG) a partir da hidrÃlise e/ou glicerÃlise enzimÃtica de um triglicerÃdeo (TG), utilizando-se a tÃcnica de miniemulsÃo. Utilizou-se como substrato o triglicerÃdeo do Ãcido caprÃico, a tricaprilina. Dois tipos de lipases foram utilizadas neste estudo a fim de se estudar a regioseletividade da reaÃÃo de hidrÃlise em miniemulsÃo. A lipase Rhizopus arrhizus (RAL) com regiosseletividade especÃfica sn-1,3 e a lipase Pseudomonas cepacia (PS) devido a sua ausÃncia de regioseletividade. Uma vez que hidrÃlise e glicerÃlise ocorrem na interface Ãleo-Ãgua, diferenÃas na Ãrea interfacial pela variaÃÃo da quantidade de surfactante e a influÃncia da concentraÃÃo das lipases tambÃm foram estudadas. AlÃm disso, a glicerÃlise foi estudada pela adiÃÃo de glicerol na preparaÃÃo da miniemulsÃo com o objetivo de direcionar a formaÃÃo dos produtos para uma maior quantidade de monoglicerÃdeos. Os produtos das reaÃÃes foram caracterizados e quantificados pelas tÃcnicas de H1-RMN e HPLC. Por HPLC obteve-se as quantidades totais de cada componente (MG, DG, Ãcido graxo livre e glicerol) enquanto que por RMN, pode-se calcular as quantidades individuais de cada produto formado (1-MG, 2-MG, 1,2-DG e 1,3-DG). Observando-se as concentraÃÃes dos produtos formados na hidrÃlise catalisada pela lipase PS, pÃde-se concluir que essa lipase, conhecida por sua nÃo-especificidade, catalisou a reaÃÃo preferencialmente na posiÃÃo sn-2, tendo como principais produtos 1,3-DG e 1-MG, com concentraÃÃes mÃximas de 29% e 22%, respectivamente. Por outro lado, a hidrÃlise catalisada pela lipase RAL, teve como principais produtos 1,2-DG e 2- MG, com concentraÃÃes mÃximas de 24% e 18%, respectivamente. Os resultados corroboram com a preferÃncia dessa lipase pela posiÃÃo sn-1,3. Observou-se para a glicerÃlise em miniemulsÃo catalisada pela lipase RAL, que a adiÃÃo de glicerol mudou o perfil de formaÃÃo de 1-MG e 2-MG, alcanÃando um mÃximo de 10-12% e 32-35%, respectivamente, apÃs 4h, comparado com 8 e 22-25% durante hidrÃlise. JÃ para glicerÃlise catalisada pela lipase PS, os resultados mostraram que essa lipase, que apresentou preferÃncia pela posiÃÃo sn-2 na hidrÃlise, passou a ter comportamento similar ao da lipase RAL, sn-1,3 especÃfica. / The aim of this study was to investigate the production of mono (MG) and diglycerides (DG) from the enzymatic hydrolysis and/or glycerolysis of a triglyceride (TG), using the technique of miniemulsion. As substrate, the triglyceride of the caproic acid, the tricaprylin was used. Two types of lipases were used in this study in order to examine the regioselectivity of the hydrolysis reaction in miniemulsion. The Rhizopus arrhizus lipase (RAL) known as specific sn-1,3 and the Pseudomonas cepacia (PS) known as non-specific lipase. Since hydrolysis and glycerolysis occur in the oil-water interface, differences in the interfacial area by varying the amount of surfactant concentration and the influence of the lipase were also studied. Additionally, glycerolysis was studied by adding glycerol to prepare the miniemulsion in order to increase amount of monoglycerides as products. The products were characterized and quantified by the 1H-NMR and HPLC techniques. HPLC afforded the quantification of total amount of each component (MG, DG, free fatty acid and glycerol) whereas by NMR, it was possible to calculate the amounts of each individual isomers (1-MG, 2-MG, 1,2-DG e 1,3-DG). The concentrations of the isomers formed in the lipase PS catalyzed hydrolysis showed that this lipase, known for its non-specificity, catalyzed the reaction preferentially at the sn-2 position, having as main products 1,3-DG and 1-MG, with maximum concentrations of 29% and 22%, respectively. On the other hand, hydrolysis catalyzed by lipase RAL has as main products 1,2-DG and 2-MG with maximum concentrations of 35% and 25% respectively, at 4h. The results corroborate the preference of the lipase for the sn-1,3 position. The initial addition of glycerol to the reaction catalyzed with lipase RAL did not significantly affect the reaction profiles and the formation rates of the diglycerides. However, the introduction of glycerol changes the profile of the formation of 1- and 2-monocaprylin, reaching the concentration maximum of 10-12% and 32-35% after 4h compared to 8% and 22-25% during the hydrolysis reaction, respectively. Lipase PS, an unspecific enzyme (slight preference for the sn-2-position in hydrolysis in miniemulsion) showed in presence of glycerol a behavior similar to RAL, a sn-1,3 specific lipase.

In the last decade, there has been a growing interest in the use of differential sensing for molecular recognition. Inspired by the mammalian olfactory system, differential sensing employs an array of non-selective receptors, which through cross-reactive interactions, create a distinct pattern for each analyte tested. The unique fingerprints obtained for each analyte with differential sensing are studied with statistical analysis techniques, such as principal component analysis and linear discriminant analysis. It was postulated that serum albumin proteins would be applicable to differential sensing schemes due to significant differences in sequence identity between different serum albumin species, and due to the wide range of hydrophobic molecules which are known to bind to these proteins. Consequently, cross-reactive serum albumin arrays were developed, utilizing hydrophobic fluorescent indicators to detect hydrophobic molecules. As such, serum albumin cross-reactive arrays were employed to discriminate subtly different hydrophobic analytes, and mixtures of these analytes, in the form of terpenes and perfumes, plasticizers and plastic explosive mixtures, and glycerides and adipocyte extracts. In this doctoral work, a detailed review of the field of differential sensing, and a thorough study of principal component analysis and linear discriminant analysis in various differential sensing scenarios, are given. These introductory chapters aid in better understanding the methods and techniques applied in later experimental chapters. In chapter 3, serum albumins, a PRODAN indicator, and an additive are shown to discriminate five terpene analytes and terpene doped perfumes. Chapter 4 describes an array with serum albumins, two dansyl fluorophores, and an additive which successfully differentiate the plasticizers found within the plastic explosives C4 and Semtex and simulated C4 and Semtex mixtures. Discrimination of these simulated mixtures was also achieved with this array in the presence of soil contaminants, demonstrating the potential real-world applicability of this sensing ensemble. Finally, chapter 5 details an array consisting of serum albumins, several fluorescent indicators, and a Grubb's olefin metathesis reaction, to differentiate saturated and unsaturated triglycerides, diglycerides, and monoglycerides. Mixtures of glycerides in adipocyte extracts taken from rats with different health states were then successfully discriminated, showing promise for clinical applications in differentiating adipoctyes from pre-diabetic, type 2 diabetic, and non-diabetic individuals. / text