Thesis (Ph.D)--Murdoch University, 2007. / Thesis submitted to the Faculty of Sustainability, Environmental and Life Sciences. Includes bibliographical references (leaves [134]-158).

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph. D.)--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. Title from title screen of research.pdf file (viewed on June 17, 2009) Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: X-ray structure of PAF acetylhydrolase. Includes bibliographical references (p. 161-175). Also available online through Digital Dissertations.

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2006. / Donald Doyle, Committee Member ; Harish Radhakrishna, Committee Member ; Alfred Merrill, Committee Member ; Marion Sewer, Committee Chair Includes bibliographical references.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2007. / Title from second page of PDF document. Includes bibliographical references.

Entre as estratégias que os protozoários do gênero Leishmania apresentam para o escape da resposta imune do hospedeiro vertebrado, há a ocorrência de um tipo de morte celular programada, conhecida como apoptose. Quando em contato com o macrófago, a Leishmania é fagocitada de forma silenciosa, evitando a resposta inflamatória do hospedeiro vertebrado. Alguns autores defendem que a Leishmania mimetiza a apoptose, expondo entre outras moléculas um fosfolipídio que sinalizaria para o macrófago que está em apoptose, e esse mecanismo é denominado na literatura como mimetismo apoptótico. O objetivo desta tese foi elucidar como ocorre esse escape com o enfoque nos fosfolipídios presentes e expostos em parasitas mutantes de L. (L.) amazonensis com características fenotípicas distintas, utilizando diferentes estratégias: transfecção com cosmídeos contendo frações do genoma de L. (L.) amazonensis; identificação e clonagem do gene pi4k contido no cosmídeo em vetor de expressão em Leishmania; seleção de parasitas resistentes a miltefosina, mantidos ou não sob pressão do antibiótico, seleção de parasitas na 28a passagem em cultura; seleção de parasitas purificados de macrófagos de linhagem RAW. A caracterização desses mutantes foi realizada em relação à ligação de anexina V-FITC, infectividade em macrófagos da linhagem RAW, tomada de fosfolipídios fluorescentes (NBD), IC50 de células tratadas com os antibióticos duramicina, miltefosina e anfotericina B. De acordo com os ensaios de ligação à anexina V-FITC, identificamos que os mutantes pi4k-pSNBR e os mutantes resistentes à miltefosina apresentaram maior ligação à anexina V-FITC. O gene que codifica a fosfatidilinositol (PI) 4-kinase, fez com que os parasitas que continham tanto o cosmídeo como o superexpressor pi4k, apresentassem menor infectividade em relação ao controle selvagem. O mesmo ocorreu para os parasitas resistentes à miltefosina. Em contrapartida, os parasitas derivados desses resistentes, mas mantidos sem pressão do antibiótico, recuperaram os valores de infectividade comparáveis ao grupo controle. Interessante é que os parasitas resistentes à miltefosina, mantidos ou não sob pressão, assim como o parasita superexpressor do gene pi4k apresentaram maior ligação à anexina V-FITC em relação ao controle selvagem, indicando que a ligação à anexina V-FITC não se correlaciona com infectividade. Parasitas resistentes à miltefosina, mantidos ou não sob pressão apresentaram maior sensibilidade à duramicina, e quando tratados com anfotericina B, esses parasitas apresentaram maior resistência. Uma outra abordagem analisada nessa tese foi elucidar qual o fosfolipídio é reconhecido pelo macrófago durante a infecção por L. (L.) amazonensis. Como resultado, observamos que os lipossomas contendo PC levam à diminuição dose-dependente da infecção, o que não foi visto em PS ou PC:PE. Esse resultado sugere a importância de PC para o estabelecimento da infectividade. / Among the strategies that Leishmania protozoans present to escape the immune response of the vertebrate host, there is a type of programmed cell death, known as apoptosis. When in contact with the macrophage, Leishmania is phagocytized in a silent manner, avoiding the inflammatory response of the vertebrate host. Some authors argue that Leishmania mimics apoptosis, exposing among other molecules a phospholipid that would signal to the macrophage that is in apoptosis, and this mechanism is denominated in the literature as apoptotic mimicry. The objective of this thesis was to elucidate how this escape occurs with the focus on the phospholipids present and exposed in mutant parasites of L. (L.) amazonensis, with distinct phenotypic characteristics, using different strategies, such as selection of parasites showing higher attachment to annexin V-FITC. After transfection with cosmids containing the genome of L. (L.) amazonensis; identification and cloning of the pi4k gene contained in the cosmid in Leishmania expression vector; selection of parasites resistant to miltefosine, whether or not under antibiotic pressure, selection of parasites at the 28th passage in culture; selection of purified strains of RAW lineage macrophages. The characterization of these mutants was performed in relation to the annexin V-FITC binding, infectivity in RAW lineage macrophages, fluorescent phospholipid (NBD) uptake, IC50 of cells treated with the antibiotics duramycin, miltefosine and amphotericin B. According to the binding assays to Annexin V-FITC, we have identified that pi4k-pSNBR mutants and miltefosin-resistant mutants showed higher attachment to annexin V-FITC. The gene coding for phosphatidylinositol (PI) 4-kinase caused the parasites containing both the cosmid and the pi4k superexpressor to have less infectivity than the wild-type control. The same occurred for parasites resistant to miltefosine. In contrast, the parasites derived from these resistant, but kept without antibiotic pressure, recovered infectivity values, comparable to the control group. Interestingly, miltefosine-resistant parasites, whether or not under pressure, as well as the overexpressing parasite of the pi4k gene showed greater attachment to annexin V-FITC over wild-type control, indicating that attachment to annexin V-FITC does not correlate with infectivity. Parasites resistant to miltefosine, whether or not under pressure, showed greater sensitivity to duramycin, and when treated with amphotericin B, these parasites showed greater resistance. Another approach analyzed in this thesis was to elucidate the phospholipid recognized by the macrophage during infection by L. (L.) amazonensis. As a result, we observed that PC-containing liposomes lead to dose-dependent decrease of infection, which has not been seen in PS or PC: PE. This result suggests the importance of PC for the establishment of infectivity.

Orientadores: Lireny Aparecida Guaraldo Gonçalves, Luiz Antonio Viotto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T21:14:27Z (GMT). No. of bitstreams: 1 Soares_MarinaldadaSilva_D.pdf: 1635248 bytes, checksum: f576efc88ef55b05d1817c84d8468ee7 (MD5) Previous issue date: 2004 / Resumo: Tradicionalmente o óleo de soja bruto é degomado por adição de água ou solução ácida. A tecnologia de membranas aplicada ao processamento de óleos vegetais tem se tornado importante, pois utiliza baixas temperaturas, não necessita de tratamento de águas residuais, retém compostos indesejáveis, além de preservar compostos minoritários importantes no óleo. Os objetivos deste trabalho foram otimizar as condições de ultrafiltração de miscela de óleo de soja bruto como alternativa à degomagem clássica, avaliar as características físico-químicas e sensoriais do óleo degomado e desodorizado em escala piloto e obter lecitina de soja através da concentração do retentado. A ultrafiltração da miscela foi realizada à 40 ºC, em unidade piloto NETZSCH utilizando duas membranas cerâmicas em alumina, pré-condicionadas, com diâmetro de poro de 0,01mm, de 19 e 37 canais. O efeito da pressão transmembrana e do teor de fósforo na alimentação foram avaliados com relação ao fluxo de permeado e retenção de fosfolipídios. A faixa de pressão transmembrana utilizada nos tratamentos foi de 0,6 a 2,0 bar, sob velocidade tangencial de 3,5 m/s. Foi estabelecido um planejamento experimental fatorial 22 completo, com 3 pontos centrais e 4 pontos axiais, para cada membrana. O teor de fósforo na alimentação não exerceu efeito considerável no percentual de retenção, entretanto teve efeito negativo sobre o fluxo de permeado nas duas membranas. Valores maiores de pressão transmembrana favoreceram o aumento de fluxo e de retenção para membrana de 19 canais. Entretanto, para membrana de 37 canais, apenas a retenção foi favorecida pelo aumento da pressão, sendo que um grande aumento da pressão (acima de 1,5 bar) para esta membrana, teria efeito negativo sobre o seu fluxo. Os premeados obtidos nos tratamentos que apresentaram melhor retenção de fosfolipídios (>98%) para as duas membranas, ou seja, valores de fósforo abaixo do nível máximo exigido pela indústria de 10 mg.kg-1, foram desodorizados em unidade piloto de desodorização com vaso de inox encamisado de 3 litros, sob vácuo de 12 mmHg, a 230ºC, por 90 minutos, utilizando nitrogênio como gás de arraste. Após determinações físico químicas que asseguraram a qualidade, os produtos obtidos foram levados à analise sensorial de aceitação ao nível de consumidor e comparados com um óleo de soja refinado comercial. Os óleos desodorizados obtidos não apresentaram diferença significativa (p £ 0,05) entre si e comparados ao óleo de soja refinado comercial para os atributos aroma e sabor. A lecitina obtida a partir da ultrafiltração dos retentados estava de acordo com os padrões do Food Chemical Codex, com 53 % de insolúveis em acetona / Abstract: Crude soybean oil is traditionally degummed by water addition or phosphoric acid. The membrane technology applied to vegetable oils processing has become important because it allows to use temperatures, reduces waste water treatment, retains undesirable products, besides preserving important minor compounds in the oil. The objectives of this work were to optimize the conditions of crude soybean oil micelle ultra filtration as an alternative to the traditional degumming, evaluate the physical-chemical and sensorial characteristics of the degummed and deodorized oil in pilot plant scale and to obtain soy lecithin through retentate concentration. The ultrafiltration was accomplished at 40o C, in a NETZSCH pilot unit utilizing two pre-conditioned ceramic in alumina membranes with 0.01 mm pore diameter of 19 and 37 channels. The feed transmembrane pressure effect and the phosphorous content were evaluated regarding to the permeate flux and retention of phospholipids. The transmembrane pressure range used in the treatments was from 0.6 to 2.0 bar under tangential velocity of 3.5m/s. A complete 22 experimental design was established with 3 central points and 4 axial points for each membrane. The phosphorous content has not carried on considerable effect in the retention, nevertheless it had a negative effect over the permeate flux in the two membranes. Greater transmembrane pressure values favor the flux and retention increase in the 19 channels membrane. However in 37 channels membranes only the retention was favored by the increasing pressure while, a great pressure increase (above 1.5 bar), presented a negative effect on the its flux. The treatments that showed better phospholipids retention (>98%) for the two membranes, phosphorous values below the maximum level of 10 mg/kg required by the industry were deodorized in a deodorization pilot unity with stainless steel jacketed reactor with 3 liters capacity, under 12 mmHg vacuum, at 230 ºC, for 1.5 hours, using nitrogen as carrier gas. After physical-chemical determinations that assured the quality, the obtained products were analyzed regarding to the sensorial acceptance at the consumer level and compared with commercial soybean refined oil. The statistical analysis have not shown significative difference (p £ 0.05) among the two desodorized oils and commercial soybean neutrative-bleached-desodorized oil for the aroma and flavor atributes. The obtained lecithin was in accordance with the standards of Food Chemical Codex, with 53% of acetone insoluble / Doutorado / Doutor em Tecnologia de Alimentos

Many factors affect membrane composition in ectotherms, including allometry, temperature, toxins such as PCB-153, and osmotic stress. This thesis seeks to describe the relationship between membrane composition, size, and phylogeny in twelve species of cypriniform fish; to describe interactions between the homeoviscous responses to temperature and to PCB-153 in goldfish and rainbow trout; and to describe the membrane response to hypoosmotic stress in goldfish. Commonalities in these patterns provide insight into shared mechanisms of phospholipid modulation. In particular, such similarities indicate whether the membrane pacemaker theory of metabolism, which connects allometric relationships between body size, membrane phospholipids, and metabolic rate, can serve as a general framework for understanding membrane composition. Chapter 2 investigates how cypriniform membrane unsaturation decreases with mass through different fatty acid substitutions than in endotherms, but these fatty acids are in turn shown to be due to the species’ relatedness to one another rather than to purely physiological causes. In Chapter 3, PCB-153 is shown to increase cholesterol in liver and brain, while high temperature primarily reduces phospholipid unsaturation. In Chapter 4, these patterns are further explored in trout. As in goldfish, cholesterol modulation is the primary response to PCB-153, whereas temperature primarily reduces phospholipid unsaturation. Trout show more pervasive fatty acid changes than goldfish in all tissues except the liver, which does not respond to PCB exposure, suggesting that PCB-153 pushes trout’s homeoviscous response to a limit that similarly-exposed goldfish do not face. Chapter 5 shows that goldfish intestines decrease membrane saturation; kidneys decrease membrane cholesterol; gills decrease neither; and muscles decrease both in response to long-term exposure to hypoosmotic conditions. The intestine and kidney are both involved in recovering ions from body fluids, but gills suppress ion loss and muscle concentrates ions from the bloodstream. Temperature, osmotic stress, PCB-153, and increasing body size are all addressed via a similar set of membrane responses in fish, which fits with the membrane pacemaker theory’s predictions regarding membrane composition, metabolic rate, and size.

Lipidomics is a relatively new field under the heading of systems biology. Due to its infancy, the field suffers from significant ‘growing pains’, one of which is the lack of bioinformatic analytic resources that other “-omics” fields enjoy. Here, I describe the creation and validation of the glycerophospholipid identification program VaLID. Using an in silico approach, we generated a comprehensive database containing all of the glycerophospholipids within multiple sub-classes: those containing chains of 0 to 30 carbons with up to 6 unsaturations and various linkages. Using Java, I created a web- based computer interface with a search engine and a visualization tool to access this database. In comparing results to current programs, I found that VaLID consistently contained more identity predictions than did the current gold standard LipidMAPS. Results from several tests with real datasets confirm that VaLID is more than capable as a phospholipid identification tool for use in lipidomics.