M.Sc. (Biochemistry) / Pseudomonas, as originally defined by Migula (1894), was a genus encompassing an assembly of rod-shaped, Gram-negative, aerobic gammaproteobacteria, possessing one or more polar flagella for motility. This vague description resulted in the misclassification of polarly flagellated proteobacteria that had not been completely characterized. Re-evaluation of the taxonomy of Pseudomonads was set into motion by advancements in molecular biology. Following this revision, Pseudomonads were clustered based on the homogeneity in their 16S rRNA sequence (Anzai et al., 2000). Research on Pseudomonas species has gained momentum owing to their significant prospects in biotechnological processes. Their exploitation for use as cleaning, biocontrol and bioremediation agents dates back to the 1980s (Sebastian and Kolattukudy, 1988; Weller, 1988)...

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Lipids are stored in a cell for providing energy. The main advantages of storing lipids over carbohydrates like glycogen is that, lipids yield more energy after oxidation because they represent the highly reduced form of carbon, needs less space and water for storage. Conservation of chemical energy in the form of biologically inert form is by storing molecules like triacylglycerol (TAG) and Steryl esters (SE). Triacylglycerol is the major storage form of energy in all eukaryotic cells. During the periods of nutritional excess and nutritional stress, all organisms like bacteria, yeast, animals, and plants can able to do the critical function of synthesizing the triacylglycerol. TAG is an energy store and a repository of essential and non-essential fatty acids and precursors for phospholipid biosynthesis. TAG synthesis mainly takes place in endoplasmic reticulum in mammals and in plants, it takes place in plastid and mitochondria. Triacylglycerol synthesis discovered by Kennedy starts with glycerol 3- phosphate. Glycerol 3-phosphate gets acylate to form lysophosphatic acid (LPA), which in turn acylate to form phosphatic acid (PA) and the reactions are catalyzed by glycerol 3-phosphate acyltransferase (GPAT) and LPA acyltransferase (LPAT) respectively. PA undergoes phosphorylation by PA phosphatase enzyme to give diacylglycerol (DAG). Further acylation of DAG gives rise to TAG and the reaction is catalyzed by diacylglycerol acyltransferase (DGAT). There are several DGAT classes were identified they are DGAT1, DGAT2, PDAT and bifunctional TAG/wax ester synthase. However all the enzymes involved in Kennedy TAG biosynthetic pathway as well as the enzymes of all different DGAT classes are membrane bound enzymes. Through our studies an another DGAT class that is soluble and cytosolic DGAT was first identified in peanut and also in yeast, Rhodotorula glutinis in which a soluble cytosolic complex itself has been identified. The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in R. glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of alternate TAG biosynthetic pathway in oleaginous yeasts. A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX2GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

La cuticule, une matrice lipidique extracellulaire constituée de cires et d’un squelette de cutine, est la barrière de défense la plus externe des plantes face à leur environnement. Elle intervient dans de nombreuses propriétés agronomiques comme la conservation post récolte, les propriétés mécaniques ou bien l’aspect du fruit, dont la brillance. Afin d’isoler des mutants de cuticule, le criblage d’une collection de mutants EMS de tomate a été entrepris, en se basant sur la brillance des fruits, conduisant à la sélection de 24 mutants. Chez ceux-ci, des analyses biochimiques ont montré de fortes variations de charge et de composition de la cuticule, notamment chez les mutants de cutine. La caractérisation de 4 mutants remarquables a été entreprise afin d’identifier les mutations responsables des phénotypes de brillance. Le mutant le plus affecté, présentant une charge en cutine réduite de 85% par rapport au type sauvage, a révélé une mutation du gène SlGDSL2 codant pour une acylhydrolase à motif GDSL, responsable de la polymérisation de la cutine. Afin d’étudier la formation et la régulation de la cutine, la suite du travail a consisté à obtenir et à caractériser des simples et des doubles mutants affectés dans la synthèse des monomères de cutine, le transport apoplastique et la polymérisation de la cutine. / The cuticle, an extracellular lipid matrix consisting of waxes and of a cutin skeleton is the outermost plants protection barrier against their environment. The cuticle is involved in many agronomic traits such as post-harvest storage, biomechanical or fruit appearance properties like surface brightness. In order to isolate cuticle mutants, the screening of an EMS tomato mutants collection has been undertaken, based on fruit brightness, leading to the selection of 24 mutants. Biochemical analyzes have shown wide variations in cuticle loads and compositions, especially in cutin mutants. The characterization of 4 remarkable mutants was undertaken to identify the mutations responsible for brightness phenotypes. The most affected mutant shows a cutin load reduced by 85% compared to the wild type, and is due to a mutation in the SlGDSL2 gene, encoding a GDSL-motive acylhydrolase enzyme, responsible for the cutin polymerization. In order to further study the cutin formation and regulation, the next work was to obtain and characterize single and double mutants affected in cutin monomer synthesis, apoplastic transport and cutin polymerization.

Feed intake assessment is a valuable tool for herd management decisions. The use of markers, either internal or external, is currently the most used technique for estimating feed intake in production animals. Three experiments were conducted with beef and dairy cattle. In dairy cows fed a corn silage based diet with 45% concentrate, the average fecal recovery (FR) of TiO2 was higher than FR of Cr2O3 and both FR were more than unity. The FR was unaffected by the fecal sampling procedure but, estimates for dry-matter digestibility (DMD) and fecal output (FO) were affected. The Cr2O3 + indigestible acid detergent fiber (iADF) marker pair produced accurate dry-matter intake (DMI) estimates. In steers fed diets with different forage sources FR of TiO2 was close to 1 and this marker produced adequate FO estimates. The FR was affected by the forage source that composed the diet and none of the marker pairs produced accurate DMI estimates. In young Nelore bulls fed corn silage or Tifton-85 hay based diets with varying F:C ratios, the average FR of internal markers acetyl bromide lignin (ABL), indigestible neutral detergent fiber (iNDF), iADF and cutin was different from 100%. Markers Cr2O3 and TiO2 had FR lower and higher than 100%, respectively. The combination of internal and external markers to estimate DMI produced satisfactory and accurate results, particularly Cr2O3 based pairs with iNDF and iADF. Internal markers ABL and cutin deserve more attention. The GRAB sampling procedures yielded accurate DMD, FO and DMI estimates, which were as good as or better than BULK sampling procedures. Regardless of the fecal sampling method and the composition of the diet, it is necessary to establish the FR of the markers to be used in each experiment, in order to obtain correct estimates. None of the markers had complete FR consistent throughout the 3 experiments. Cutin and ABL had lower than unity FR on all diets and failed to produce accurate DMD estimates. When either one was paired with Cr2O3 or TiO2, DMI estimates were also not accurate. Of the indigestible fibers, iNDF and iADF, the later was more accurate in estimating DMI when paired with Cr2O3. Consistently throughout the experiments, FR was lower than and higher than unity for internal and external markers, respectively. Corrected FO estimates were accurate for Cr2O3 on various diets under GRAB sampling. The same was not true for TiO2, which even after FR correction was applied failed to produce accurate FO estimates consistently. The method of dosing the external markers is extremely important and greatly affects and determines results. Whichever the method, it must allow the animals to display normal feeding behavior and not affect performance. The GRAB sampling procedures can replace TFC (once FR is established) which may open new possibilities for pasture based or collectively housed animals. / A determinação do consumo de alimentos é uma valiosa ferramenta na hora da tomada de decisões de manejo do rebanho. O uso de marcadores, sejam internos ou externos, é atualmente a técnica mais utilizada para estimar o consumo em animais de produção. Três experimentos foram conduzidos. Em vacas de leite alimentadas com silagem de milho com 45% de concentrado, a recuperação fecal (RF) média de TiO2 foi maior do que a RF de Cr2O3 e ambas foram maiores do que a unidade. A RF não foi afetada pelo método de coleta de fezes, mas, as estimativas de digestibilidade da matéria seca e produção fecal (PF) foram. A dupla de marcadores Cr2O3+i fibra detergente ácido indigestível (iADF) produziu estimativas de consumo de matéria seca (CMS) acuradas. Em novilhos alimentados com diferentes fontes de forragem, a RF do TiO2 foi próxima a 1 e este marcador produziu estimativas de PF adequadas. A RF foi afetada pela fonte de forragem que compunha a dieta e nenhum dos marcadores produziu estimativas de CMS acuradas. Em tourinhos Nelore alimentados com dietas compostas de silagem de milho ou feno de Tifton-85 e diferentes relações de volumoso:concentrado, a RF média dos marcadores internos lignina brometo de acetila (LBA), fibra detergente ácido indigestível (iFDN), (iFDA) e cutina foi diferente de 100%. Os marcadores Cr2O3 e TiO2 tiveram RF menor e maior que 100%, respectivamente. A combinação de marcadores internos e externos para estimar o CMS produziu resultados acurados e satisfatórios, principalmente as duplas Cr2O3 e iFDN ou iFDA. Os marcadores internos LBA e cutina merecem mais estudos. O método de coleta GRAB produziu estimativas de DMS, PF e CMS acuradas, as quais foram iguais ou melhores que as produzidas pelo método de coleta BULK. Independentemente do método de coleta de fezes e da composição da dieta, é necessário estabelecer a RF real dos marcadores que serão usados em experimento para obter estimativas corretas. Nenhum dos marcadores teve RF completa e consistente nos 3 experimentos. A cutina e LBA tiveram RF menor do que a unidade em todas as dietas e falharam em produzir estimativas acuradas de DMS. Quando qualquer dos dois foi pareado com Cr2O3 ou TiO2 as estimativas de CMS também não foram acuradas. Das fibras indigestíveis, iFDN e iFDA, a segunda foi mais acurada em estimar o CMS quando pareada com Cr2O3. As estimativas de PF corrigida foram acuradas para Cr2O3 em diferentes dietas sob metodologia de coleta GRAB. O mesmo não foi observado para o TiO2, que produziu estimativas enviesadas inclusive após a correção. O método de dosagem dos marcadores externos é extremadamente importante já que afeta e determina em grande medida os resultados. Qualquer que seja o método, este deve permitir ao animal comportar-se e alimentar-se normalmente sem comprometer seu desempenho. Os métodos de coleta GRAB podem substituir a coleta total de fezes (CTF) (após determinação da RF) o que poderá abrir novas possibilidades para estudos com animais em pastagem ou alojados em baias coletivas.

Com o objetivo de comparar as estimativas dos coeficientes de digestibilidade dos nutrientes de dietas para eqüinos compostas por alfafa em cubos, alfafa em cubos com óleo de soja, alfafa em ramas e alfafa em ramas com óleo de soja, através do método de coleta total de fezes com o uso dos indicadores cutina, óxido crômico e lignina em detergente ácido, foram utilizados 4 eqüinos machos, sem raça definida, com idade aproximada de 10 meses e peso médio de 197Kg (170 kg a 216 kg). O experimento teve duração de 11 dias, sendo 8 de adaptação e 3 de coleta. O delineamento experimental utilizado foi o quadrado latino 4X4. A ponderação dos coeficientes de digestibilidade da matéria orgânica (CDMO) pelos indicadores foi efetuada através de um modelo o qual considerava a diferença entre CDMO pelo indicador e pela coleta total de fezes, chamado de viés. Para a avaliação dos indicadores utilizados, foram medidas a acurácia, a precisão e a robustez. A acurácia e a precisão foram determinadas pela comparação dos dados preditos com os observados. A robustez foi determinada pela comparação dos valores preditos menos os observados (vieses) com outros fatores estudados (EE na dieta, ED na dieta e peso vivo). Neste estudo, a cutina não se mostrou adequada como indicador interno, pois superestimou a digestibilidade aparente da matéria orgânica( DAMO), apresentou a menor acurácia e menor precisão. O óxido crômico apresentou baixa recuperação fecal e subestimou a DAMO, embora tenha sido o mais preciso. A LDA foi o indicador que obteve a melhor recuperação fecal e foi o mais acurado, portanto, o indicador mais adequado, todos em relação à coleta total de fezes na estimativa da digestibilidade aparente da matéria orgânica de dietas para eqüinos. / This research aimed to compare organic matter digestibility coefficient (OMDC) of diets composed of alfalfa cubes, alfalfa cubes with soybean oil, alfalfa hay and alfalfa hay with soybean oil by total feces collection and use the internal markers cutin and acid detergent lignin (ADL) and the external marker chromic oxide. Four male weanling horses (approximately 10-months old, 197.25 kg BW average) were used in a 4 x4 Latin Square design. Evaluation of OMDC estimated by markers was performed by a model considering the bias, i.e., the difference between OMDC estimated by marker and by total feces collection. It was evaluated accuracy, precision and robustness of those three markers. The cutin overestimated the OMDC and showed to be the less accurate and precise. The chromic oxide underestimated the OMDC and its recovery was low, but it was the most precise. The ADL recovery did not differ from 100% and it was the most accurate, therefore the most appropriated marker for this kind of diet.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 157-170).

Our research involves the isolation and identification of the possible chemical compounds in black-eyed Susans that may be responsible for the olfactory attraction of the crab spider Misumenoides formosipes to the inflorescences of these plants. In olfactometric bioassays, 80% of 30 male spiders moved towards olfactory-only cues from R. hirta inflorescences over a water control (P = 0.0014). The bulk extract was separated using flash column chromatography (silica column) with a series of solvents. Spiders in olfactometer bioassays showed a significant preference for the fractions collected using 100% dichloromethane over the solvent-only control (P=0.039). The 100% dichloromethane pooled fractions were separated using solid phase extraction (SPE). Three compounds were isolated and identified using TLC, infrared and NMR spectroscopy. Two compounds were identified as contaminants, di(2-ethylhexyl) phthalate and erucamide, found in the flash column chromatography apparatus and SPE apparatus, respectively. A long-chain crystalline hydrocarbon wax was extracted from R. hirta inflorescences. Research shows that several insects use the lipids of the wax layer, specifically various long-chain alkanes and alcohols, as cues in host plant selection or as kairomones, chemical cues used in communication from one organism to another [3]. It also shows that the waxes can act as absorbents or release agents for biologically active material. Thus, the long-chain hydrocarbon wax interacting with the volatile components could play a major role in attracting the male crab spiders to the R. hirta inflorescences / Introduction and background -- Olfactory bioassay studies of M. formosipes -- Chromatographic separation of components in the 100% dichloromethane fractions -- Identification of the possible attractants in the 100% dichloromethane fractions using spectroscopic methods. / Department of Chemistry

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Com o objetivo de comparar as estimativas dos coeficientes de digestibilidade dos nutrientes de dietas para eqüinos compostas por alfafa em cubos, alfafa em cubos com óleo de soja, alfafa em ramas e alfafa em ramas com óleo de soja, através do método de coleta total de fezes com o uso dos indicadores cutina, óxido crômico e lignina em detergente ácido, foram utilizados 4 eqüinos machos, sem raça definida, com idade aproximada de 10 meses e peso médio de 197Kg (170 kg a 216 kg). O experimento teve duração de 11 dias, sendo 8 de adaptação e 3 de coleta. O delineamento experimental utilizado foi o quadrado latino 4X4. A ponderação dos coeficientes de digestibilidade da matéria orgânica (CDMO) pelos indicadores foi efetuada através de um modelo o qual considerava a diferença entre CDMO pelo indicador e pela coleta total de fezes, chamado de viés. Para a avaliação dos indicadores utilizados, foram medidas a acurácia, a precisão e a robustez. A acurácia e a precisão foram determinadas pela comparação dos dados preditos com os observados. A robustez foi determinada pela comparação dos valores preditos menos os observados (vieses) com outros fatores estudados (EE na dieta, ED na dieta e peso vivo). Neste estudo, a cutina não se mostrou adequada como indicador interno, pois superestimou a digestibilidade aparente da matéria orgânica( DAMO), apresentou a menor acurácia e menor precisão. O óxido crômico apresentou baixa recuperação fecal e subestimou a DAMO, embora tenha sido o mais preciso. A LDA foi o indicador que obteve a melhor recuperação fecal e foi o mais acurado, portanto, o indicador mais adequado, todos em relação à coleta total de fezes na estimativa da digestibilidade aparente da matéria orgânica de dietas para eqüinos. / This research aimed to compare organic matter digestibility coefficient (OMDC) of diets composed of alfalfa cubes, alfalfa cubes with soybean oil, alfalfa hay and alfalfa hay with soybean oil by total feces collection and use the internal markers cutin and acid detergent lignin (ADL) and the external marker chromic oxide. Four male weanling horses (approximately 10-months old, 197.25 kg BW average) were used in a 4 x4 Latin Square design. Evaluation of OMDC estimated by markers was performed by a model considering the bias, i.e., the difference between OMDC estimated by marker and by total feces collection. It was evaluated accuracy, precision and robustness of those three markers. The cutin overestimated the OMDC and showed to be the less accurate and precise. The chromic oxide underestimated the OMDC and its recovery was low, but it was the most precise. The ADL recovery did not differ from 100% and it was the most accurate, therefore the most appropriated marker for this kind of diet.