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Rôle des lipides dans l’infestation, la virulence, et la transformation des promastigotes en amastigotes et implication des acides gras dans la pathogénie de Leishmania / Role of lipids in infestation, virulence, and transformation of promastigotes into amastigotes and involvement of fatty acids in the pathogenesis of LeishmaniaBouazizi, Hana 26 June 2018 (has links)
Les parasites Leishmania sont les agents responsables de la leishmaniose viscérale (LV), mucocutanée (LM) ou cutanée (LC) chez l'homme et de la leishmaniose canine (CanL). Ces parasites sont phagocytés par les macrophages humains et animaux où ils vont se transformer de promastigote en amastigote. Nous avons identifié et analysé les lipides impliqués dans le processus de transformation dans le complexe de Leishmania donovani et infantum. Quatre classes de lipides, les phospholipides, les acides gras libres, les triglycérides et les stérols ont été étudiés. De même, nous avons analysé la composition en acides gras des lipides totaux chez neuf espèces de Leishmania isolées en Tunisie, dont quatre souches de L. infantum, deux souches de L. major et deux souches de L. tropica et une souche de L. infantum de chien. Durant nos premiers travaux, la composition en acides gras des lipides totaux a été analysée et nos résultats montrent une augmentation des acides gras et du cholestérol et une diminution des triglycérides et de l'ergostérol durant la transition entre les promastigotes et les amastigotes. En ce qui concerne les classes de phospholipides, nous avons trouvé une proportion accrue de sphingomyéline et de phosphatidylsérine et une proportion réduite de phosphatidylinositol et de lysophosphatidyléthanolamine. Pour la composition en acides gras, une augmentation significative des acides gras n-7 a été observée chez les amastigotes, quant aux acides gras n-6 totaux, ils ont diminué chez les PL. Plusieurs changements ont également été observés au niveau des TG et des acides gras libres, en particulier, les acides gras n-7 et 20: 4n-6 ont été fortement augmentés, alors que les acides gras n-9 et les précurseurs n-6 ont diminué. Pour la deuxième étude, les résultats trouvés dans nos premiers travaux qui concernent la présence de proportions très élevés de 18 : 2n-6 contre de faible proportion de l’AA (20 :4n-6) ainsi que l’absence ou la très faible proportion de 18 :3n-3 ont été confirmés. De plus, L. major présentait une proportion plus élevée de 14 : 0 (acide myristique), 18: 3n-6 (acide gamma-linoléique) et une plus faible proportion de n-3, y compris 18: 3n-3 (acide alphalinoléique) et 22: 6n-3 (acide docosahexaénoïque) comparé à L. infantum et L. tropica. Après la supplémentation de l'acide oléique (AO), l'acide arachidonique (AA) et l’acide docosahexaénoïque (DHA) sur deux souches de L. infantum utilisées pour infecter les macrophages : un isotype MON-24 responsable de la leishmaniose cutanée et un MON-1 causant la leishmaniose viscérale ; nous avons constatés que les AA et DHA augmentaient la virulence du parasite, tandis que L’AO diminuait leur virulence / Leishmania parasites are the causative agents of visceral (LV), mucocutaneous (LM) or cutaneous (LC) leishmaniasis in humans, canine leishmaniasis in dogs (CanL), and leishmaniases in other mammals .The parasites are phagocytosed by human and animal macrophages where they will transform from promastigote to amastigote. We identified and analyzed the lipids involved in the transformation process in the Leishmania donovani complex. Four classes of lipids, phospholipids (PL), free fatty acids (FA), triglycerides (TG) and sterols were studied.Similarly, we analyzed the fatty acid composition of total lipids in nine Tunisian Leishmania isolates, including five strains of L. infantum (four human and one canine), two strains of L. major and two strains of L. tropica.In our early work, the fatty acid composition of total lipids was analyzed and our results show an increase in fatty acids and cholesterol and a decrease in triglycerides and ergosterol during the transformation of promastigotes into amastigotes. For phospholipid classes, we found an increase in sphingomyelin and phosphatidylserines and a decrease in phosphatidylinositols and lysophosphatidylethanolamines during processing. For the fatty acid composition, a significant increase of n-7 fatty acids was observed in amastigotes, as for n-6 total fatty acids, they decreased in PLs. Several changes have also been observed in TG and free fatty acids, in particular, n-7 and 20: 4n-6 fatty acids have been greatly increased, whereas n-9 fatty acids and n-6 ??precursors have been significantly increased. decreased.The study of the fatty acid composition of the 9 Tunisian strains of Leishmania confirmed, in the first place, our results found in our first works concerning the presencevery high proportions of 18: 2n-6 against low proportion of AA (20: 4n-6) as well as the absence or very low proportion of 18: 3n-3.In addition, the comparison of the fatty acid compositions of the three species studied: L. infantum, L. major and L. tropica showed that L. major had a higher proportion of 14: 0 (myristic acid), 18: 3n- 6 (gamma-linoleic acid) and a lower proportion of n-3, including18: 3n-3 (alpha-linoleic acid) and 22: 6n-3 (docosahexaenoic acid) compared to L. infantum and L. tropica. After supplementation of oleic acid (AO), arachidonic acid (AA) and docosahexaenoic acid (DHA) on two L. infantum strains used to infect macrophages: a MON-24 isotype responsible for cutaneous and a MON-1
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Proposta de um protocolo para a caracterização e análise das propriedades mecânicas de surfactantes exógenos / Proposal of a protocol for the characterization and analysis of the mechanical properties of exogenous surfactantsMuñoz, Diana Maria Martinez 02 October 2013 (has links)
O surfactante pulmonar é uma mistura complexa de fosfolipídios e proteínas, e encontra-se presente na interface ar-líquido dos alvéolos pulmonares. O seu papel principal é reduzir a tensão superficial para manter os alvéolos estáveis. A deficiência ou disfunção do surfactante leva ao colapso alveolar, provocando a falta de oxigenação que ocorre devido ao edema ou a resposta inflamatória nos pulmões. Em recém-nascidos, a imaturidade pulmonar, pela deficiência do surfactante, pode causar a Síndrome de Desconforto Respiratória (SDR). Nos adultos, a Síndrome de Desconforto Respiratório Agudo (SDRA) é a manifestação mais grave da Lesão Pulmonar Aguda (LPA), o tratamento para estas doenças inclui a utilização de surfactantes exógenos. Para entender a funcionalidade do surfactante é necessário caracterizá-lo biofisicamente. A principal característica observada neste estudo foi o espalhamento e recuperação do surfactante na subfase, para a interface ar-líquido. O espalhamento e recuperação foram quantificadas observando o trabalho feito em sucessivos ciclos de compressão e expansão na balança de Wilhelmy. Analisou-se o decaimento do trabalho ao longo dos ciclos até a sua estabilização. Os parâmetros obtidos neste ajuste do decaimento exponencial foram utilizados para a caracterização de dois surfactantes exógenos, o Curosurf® e o Survanta®. As comparações entre eles foram segundo a concentração, as subfases e das velocidades de barreira. O decaimento exponencial do trabalho nos ciclos só ocorreu para concentrações menores de surfactante. Quando em subfase de solução salina ocorreu a melhora na recuperação do surfactante para a interface ar-líquido, em comparação a subfase de água ultrapura. A melhor velocidade de barreira encontrada para otimização da recuperação do surfactante foi 120 mm/min. Foi observado nesse estudo que as propriedades de recuperação do Curosurf® foram melhores em relação ao Survanta®, os parâmetros se mostraram de acordo com os dados clínicos encontrados na literatura, a caracterização da dinâmica do surfactante foi feita de forma diferente de todos os métodos encontrados / Lung surfactant is a complex mixture of phospholipids and proteins, found at the airliquid interface of pulmonary alveoli. The main role is to reduce the surface tension to keep alveoli stable. Surfactant deficiency, or dysfunctional, leads to alveolar collapse, causes a lack of oxygen and it may be due to edema or inflammatory response in the lungs. In newborn babies, pulmonary immaturity, caused by surfactant deficiency, may cause Respiratory Distress Syndrome (RDS). In adults, the Acute Respiratory Distress Syndrome (ARDS) is the gravest manifestation of Acute Lung Injury (ALI), and the treatment includes Mechanical Ventilation (MV) and exogenous surfactants. To understand surfactant functionality, it is necessary to characterize them biophysically. The main characteristic observed in this work was the mobility and recovery of surfactant in the subphase to the air-liquid interface. The mobility and recover were quantified observing the work done in successive cycles of compression and expansion in a Wilhelmy plate tensiometer. The work decay was analyzed over cycles until its stabilization. The parameters obtained for the exponential fitting of decay were used for characterization of two exogenous surfactants, Curosurf® and Survanta®. The comparisons between them were done under concentration, subphases and barrier speeds. The exponential decay of the cycle work only happened for lower concentrations of surfactant. Saline solution subphase improved the surfactant recovery to the air-liquid interface over ultrapure water subphase. A suitable barrier speed founded to optimize surfactant recovery was 120 mm/min. In this study were observed that recovery properties of Curosurf® were better than Survanta®, the parameters agrees with clinical data from the literature, and the dynamic characterization of surfactant was done of different way than founded methods
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Influência da âncora de glicosilfosfatidilinositol na imobilização da fosfatase alcalina de placa óssea imobilizada em sistemas miméticos de membranas celulares: monocamadas de Langmuir e filmes Langmuir-Blodgett de fosfolipídios / Influence of the glycosylphosphatidylinositol anchor in the immobilization of rat osseous plate alkaline phosphatase immobilized in biomimetic systems: phospholipid Langmuir monolayers and Langmuir-Blogett filmsCaseli, Luciano 13 April 2005 (has links)
Nesse trabalho, estudou-se a incorporação da fosfatase alcalina de placa óssea de ratos em dois tipos de sistemas modelo que mimetizassem uma membrana celular: as monocamadas de Langmuir e os filmes Langmuir-Blodgett (LB), ambos formados com fosfolipídios. No intuito de se investigar o papel da âncora de glicosilfosfatidilinositol (GFI), covalentemente ligada ao grupo carboxi-terminal da cadeia polipeptídica, duas formas da enzima foram estudadas: uma com a âncora GFI intacta, solubilizada por ação de um tensoativo não-iônico, e a outra sem a parte hidrofóbica dessa âncora, clivada por ação de uma fosfolipase específica. A primeira forma enzimática foi chamada de FAT (fosfatase alcalina solubilizada por tensoativo), e a segunda de FAC (fosfatase alcalina clivada). Diferenças marcantes na atividade superficial foram observadas entre as duas formas. Comparativamente, a forma FAT adsorve mais rapidamente à interface ar/água que a forma FAC, que mostra um tempo de indução significativo. A incorporação da forma FAT às monocamadas de ácido dimiristoilfosfatídico (DMPA) também ocorre mais rapidamente que a forma FAC. No entanto, o uso de alta força iônica acelerou a adsorção da forma FAC à interface ar/água (com ou sem DMPA). Uma pressão de superfície de exclusão de 20mN/m foi encontrado para a forma FAC, enquanto para a forma FAT, essa pressão representa uma mudança no perfil das curvas pressão x tempo. Isso revelou que, devido à presença da âncora GFI, essa forma enzimática é capaz de incorporar às monocamadas de DMPA, mesmo em altas pressões de superfície. Isotermas superficiais de monocamadas mistas de DMPA e fosfatase alcalina também mostraram diferentes perfis para duas formas enzimáticas estudadas. Enquanto a forma FAT provoca uma alteração na compressibilidade em pressões de até 20mN/m, a forma FAC desloca a curva pressão x área para áreas mais elevadas. Tal fato foi explicado pelo fato da forma FAT incorporar na monocamada preferencialmente com a âncora GFI posicionada entre as cadeias apolares do DMPA, enquanto a forma FAC deva incorporar a cadeia polipeptídica à interface lipídica . Microscopias de fluorescência e no ângulo de Brewster revelaram que a forma FAT provoca agregação espontânea do DMPA na interface ar/água, levando a uma microeterogeneidade, na qual três fases distintas podem ser observadas. A obtenção de espectros de infravermelho na interface ar/água, associada com medidas de atividade catalítica in situ, revelou que a atividade da fosfatase alcalina é modulada pela capacidade de empacotamento interfacial, medida pelo módulo de compressibilidade superficial. Em 20mN/m, há uma reorganização molecular na interface, o que vai restringir a flexibilização da cadeia polipeptídicia, que estará voltada para a interface ar/água. No entanto, ao menos até 30mN/m, nenhuma alteração conformacional foi detectada, como revelada pelos espectros de infravermelho. Filmes LB mistos de DMPA e as duas formas de fosfatase alcalina revelaram que o empacotamento máximo de proteína depende da presença da âncora GFI. Dados usando microgravimetria, atividade enzimática, infravermelho, elipsometria, e microscopia de força atômica mostraram que a adsorção da âncora na interface posiciona o eixo maior do elipsóide, formador da cadeia polipeptídica, paralelamente à matriz lipídica. Na ausência da âncora GFI, o posicionamento da cadeia polipeptídica na interface é aleatório, e para um alto grau de empacotamento, as interações entre os resíduos de aminoácidos intermoleculares favorecerão o posicionamento do eixo maior do elipsóide em uma posição mais perpendicular à interface lipídica / Não consta
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Ensaio de permeabilidade em membrana artificial paralela (PAMPA): investigação das variáveis do ensaio para o estudo da permeabilidade de fármacos. / Parallel artificial membrane permeability assay (PAMPA): investigation of the assay variables in drug permeability studyJuliana Mazza Reis 12 April 2013 (has links)
O acesso à permeabilidade é uma etapa crucial na definição da via de administração de um fármaco, além de ser um dos parâmetros utilizados para a avaliação e seleção de moléculas durante as fases iniciais da pesquisa por novos fármacos. Diversos modelos in vitro têm sido descritos para a realização dos estudos de permeabilidade, mas ainda é evidente a carência de padronização de seus protocolos. O modelo de Permeabilidade em Membrana Artificial Paralela (PAMPA) é rápido e simples, podendo ser facilmente incorporado à rotina de um laboratório. Não obstante, tem apresentado excelente correlação com modelos in vitro quando na avaliação da permeabilidade de fármacos transportados pela via passiva transcelular. O principal objetivo deste trabalho foi investigar a influência das principais variáveis do método de PAMPA e seu impacto na permeabilidade de fármacos. Para tanto, foram selecionados 9 fármacos com características físico-químicas distintas, em função dos seguintes critérios: lipofilicidade (logP); Área de Superfície Polar (PSA); Volume Molecular (VM); pKa; Peso Molecular (PM); Número doadores de ligação de hidrogênio (HBD) e Sistema de Classificação Biofarmacêutica (SCB). Os fármacos selecionados foram listados quanto à sua permeabilidade em jejuno humano (Peff) e em cultura de células Caco-2, além de Fração de Absorção (FA%) em humanos. Para o estudo das variáveis da técnica de PAMPA, a ferramenta de Desenho de Experimentos (DOE) foi utilizada e as seguintes condições de ensaio foram exploradas: gradiente de pH do meio do compartimento doador, presença ou ausência de ácido glicocólico no meio do compartimento receptor, variação do tempo de incubação, presença ou ausência de agitação das placas durante a incubação e variação da composição lipídica da membrana artificial. A combinação fatorial completa das condições de ensaio a serem exploradas resultou em 96 experimentos, os quais foram executados em triplicata. Os resultados obtidos apontaram que as variações do pH do meio do compartimento doador, do tempo de incubação e da composição do meio do compartimento receptor foram as condições do ensaio de PAMPA que mais impactaram a permeabilidade dos fármacos. Os resultados obtidos nos 96 experimentos do DOE foram relacionados, a partir de regressão linear, com os parâmetros fisico-químicos e dados de permeabilidade dos fármacos do estudo. O experimento de DOE que apresentou melhor correlação com os dados de permeabilidade em jejuno humano para os fármacos contemplados neste estudo teve um R2=0,858. As condições de ensaio consideradas para este experimento foram: pH 4,5 no compartimento doador, incubação de 15 horas, ausência de agitação das placas, membrana composta por fosfatidilcolina 10% (p/v) e colesterol 0,5% (p/v) e presença de ácido glicocólico 0,5% (p/v) no compartimento receptor. Dentre as propriedades físico-químicas, o PM, PSA e VM foram propriedades comuns a grupos de fármacos cuja permeabilidade foi favorecida pelas mesmas condições de ensaio de PAMPA. Os maiores valores de permeabilidade em PAMPA foram obtidos para fármacos com Peff entre 1 e 2 *10-4 cm/s, permeabilidade em células Caco-2 entre 20 e 27 *10-6 cm/s, faixa de logP entre 0 e 3 além de PM abaixo de 300 Da e PSA abaixo de 90Å2. / The permeability access is a critical step when defining the route of administration of a drug, besides from being one of the parameters considered for selecting the best candidates during the initial stages of new molecules discovery. For this reason, there is a need to implement high capacity and low cost models, which have a high correlation with in vivo permeability. Several in vitro models have been described for the studies of permeability, but the lack of standardization of protocols is still evident. Parallel Artificial Membrane Permeability Assay (PAMPA) is fast and simple and can be easily incorporated into the routine of a laboratory. Nevertheless, it has shown excellent correlation with in vitro models when evaluating the permeability of drugs that are transported primarily by passive transcellular route. The aim of this project was to investigate the influence of key variables of PAMPA on the permeability of drugs. Therefore, nine drugs with different physicochemical characteristics were selected based on the following criteria: lipophilicity (logP), Polar Surface Area (PSA), Molecular Volume (MV), pKa, Molecular Weight (MW), number of Hydrogen Bond Donors (HBD) and Biopharmaceutical Classification System (BCS). In addition, the drugs were listed for their permeability in human jejunum, Caco-2 and Fraction of Absorption (FA%) in humans. Design of Experiments (DOE) was considered to plan the PAMPA experiments, and the following assay conditions were explored: pH gradient in the donor compartment, glycocholic acid presence or absence in the acceptor compartment, variations in the incubation time, presence or absence of plate agitation and variations in the lipid composition of the artificial membranes. Full factorial combination of the assay variables resulted in 96 experiments, which were performed in triplicate. The results showed that the pH of the donor compartment, the incubation time and acceptor compartment composition were the assay conditions which most impacted the PAMPA results of drugs. The 96 DOE experiments results were then correlated, by linear regression, with both the physic-chemical and permeability data of the selected drugs. The DOE experiment that showed the best correlation with the permeability in human jejunum (R2 = 0.858) for the drugs included in this study had the following test conditions: pH 4.0 in the donor compartment, incubation of 15 hours, without plate agitation, artificial membrane composed of phosphatidylcholine 10% (w/v) and cholesterol 0.5% (w/v) in dodecane and presence of glycocholic acid 0.5% (w/v) in the receptor compartment. Among the studied physicochemical properties, the molecular weight (MW), polar surface area (PSA) and molecular volume (VM) were the best drivers for determining PAMPA most suitable assay conditions. The highest PAMPA values were obtained for drugs with Peff between 1 and 2 *10-4 cm/s and Caco-2 permeability between 20 and 27 *10-6 cm/s, besides from logP between 0 and 3, MW below 300 Da and PSA below 90Å2.
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Complexação de pDNA com nanoemulsões catiônicas : estudos de formulação e toxicidade em células Hep G2Fraga, Michelle January 2007 (has links)
Nanoemulsões catiônicas têm sido recentemente propostas como sistemas carreadores de DNA. O presente trabalho teve por objetivo avaliar a influência de diferentes fosfolipídeos sobre propriedades dos complexos formados entre nanoemulsões e pDNA (pTracerTMCMV2). Em uma primeira etapa, nanoemulsões catiônicas constituídas de triglicerídeos de cadeia média, estearilamina, lecitina de gema de ovo ou fosfolipídeos isolados (DSPC, DOPC, DSPE ou DOPE), glicerol e água foram preparadas através do procedimento de emulsificação espontânea. Independente do tipo de fosfolipídeo empregado, esse procedimento conduziu à obtenção de nanoemulsões catiônicas monodispersas com diâmetro de gotícula e potencial zeta de cerca de 250 nm e +50 mV, respectivamente. A complexação do pDNA com as nanoemulsões catiônicas, avaliada através do retardamento de migração do pDNA em gel de agarose por eletroforese, foi total quando o complexo apresenta uma relação de cargas [+/-] ≥ 1,0. Nestas condições, os complexos formados foram protegidos da degradação pela enzima DNase I. Em uma segunda etapa, a citotoxicidade das nanoemulsões e dos complexos com o pDNA sobre células Hep G2 foi avaliada, através do ensaio de MTT. Os resultados obtidos demonstraram que a adição de quantidades crescentes das nanoemulsões, conduz a uma toxicidade progressiva sobre as células, independente do pH do meio. Dentre as formulações estudadas, aquelas estabilizadas pelos fosfolipídeos DSPC e DSPE, de elevada temperatura de transição de fases, foram marcadamente menos tóxicas, em comparação com as formulações obtidas com lecitina, DOPC e DOPE. Essa mesma tendência foi detectada para os complexos formados com o pDNA. Em uma última etapa, foi realizado um estudo preliminar de transferência gênica em células Hep G2, utilizando a técnica de PCR em tempo real. Dentre as diferentes formulações testadas, a maior quantidade de DNA de GFP detectada parece ser para a formulação obtida com o fosfolipídeo DSPC, ilustrando as potencialidades de uso das nanoemulsões desenvolvidas como reagentes de transfecção de pDNA em células Hep G2. Em conclusão, o conjunto dos resultados obtidos demonstra o efeito dos fosfolipídeos empregados sobre propriedades físico-químicas, complexação, estabilidade, citotoxicidade e transfecção de nanoemulsões catiônicas como sistemas carreadores de pDNA. / Cationic nanoemulsions have been recently considered as potential delivery systems for DNA. The aim of the present work was to evaluate the influence of different phospholipids on the properties of complexes formed between nanoemulsions and pDNA (pTracerTM-CMV2). First, cationic nanoemulsions composed of medium chain triglycerides, stearlyamine, egg lecithin or isolated phospholipids (DSPC, DOPC, DSPE or DOPE), glycerol and water were prepared through spontaneous emulsification process. Independently of the type of phospholipid used this procedure results in monodisperses cationic nanoemulsions with droplet size and zeta potential of about 250 nm and +50 mV, respectively. The complexation of pDNA with cationic nanoemulsions, analyzed by agarose gel retardation assay was total when the complex possesses a charge relation [+/-] ≥ 1.0. In these conditions the complexes were protected from enzymatic degradation by DNase I. After that, the cytotoxicity of the nanoemulsion and the complexes with pDNA in Hep G2 cells was evaluated through MTT assay. The results showed that the addition of increasing amount of nanoemulsion leads to a progressive toxicity on the cells independently of the media’s pH. Among the studied formulations the ones stabilized with the phospholipids DSPC and DSPE, that have elevated phase transition temperatures, were much less cytotoxic in comparison with the formulations obtained with lecithin, DOPC and DOPE. This same trend was detected for the complexes formed with pDNA. Finally a preliminary study of gene transfer to Hep G2 cells was performed using real-time PCR technique. Among the different formulations tested, the major quantity of reporter DNA detected seems to be for the formulation obtained with the DSPC phospholipid. This shows the potentialities of the use of nanoemulsions as transfection reagents of pDNA in Hep G2 cells. In conclusion, the overall results show the effect of the phospholipids on physicochemical properties, complexation, stability, cytotoxicity and transfection of cationic nanoemulsions as delivery systems for pDNA.
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The Effects of Microfluidization and Homogenization on the Composition and Structure of Liposomal Aggregates from Whey Buttermilk and Commercial ButtermilkNguyen, Tracey Mai T 01 August 2013 (has links)
Milk derived ingredients from the production of cheese and butter can be used as vehicles for nutrients. Buttermilk is a nutritious product of milk that comes from the churning of cream into butter. One of the advantages of buttermilk is that it is enriched in milk fat globule components, such as phospholipids and forms emulsions with fat when treated with high shear. The objective of this work was to explore the effects of shear on regular buttermilk and whey buttermilk in terms of liposomal aggregate size and chemical composition. The effects of microfluidization at 2000 psi and homogenization at 2000 psi/500 psi on the particle size distribution of liposomal aggregates between whey buttermilk (WBM) at pH 4.6 and 6.8 and commercial sweet buttermilk (SBM) at pH 4.60 were compared with whey protein isolate (WPI) at pH 4.6. At pH 6.80, WPI and SBM are too soluble in water to measure particle size but WBM is not as soluble. From this investigation, the mean particle diameter of the SBM aggregates at pH 4.6 decreased after the first pass through the microfluidizer and the same is true, after homogenization. SBM aggregates at pH 4.6 had a significantly larger mean particle diameter before treatments in both shear processes compared to WPI at pH 4.6 and WBM at pH 4.6 and WBM at pH 6.8 (p < 0.0001). WPI at pH 4.6 and WBM at both pH showed no significant differences in their mean particle size in both homogenized and microfluidized treatments. WPI and SBM samples resulted in significant particle diameter differences vi from before to after homogenizing at pH 4.6. SBM at pH 4.6 had significantly larger average particle diameter than WBM at pH 4.6 (p < 0.0002), WPI at pH 4.6 (p < 0.0002) and WBM at pH 6.8 (p < 0.0045) before microfluidization at pass 0.
WBM and WPI across all treatments showed very similar tendencies in small particle size attributes and some similarities in protein composition. In addition, the small aggregate size of WBM is suggested to be influenced by the presence of phospholipids and thus, creating significantly smaller mean particles compared to SBM even before inducing high shear. In contrast, treated and untreated SBM differed from WBM in phospholipid composition in both homogenization and microfluidization techniques. WBM samples contained more phospholipids than SBM, whereas WPI samples contained very low concentrations of phospholipids. Through HPLC analysis, WPI, SBM, and WBM showed different profiling of the phospholipid classes. These differences may be due structural changes of the aggregates from shearing, initial thermal treatments or hydrophobic and/or protein-phospholipid interactions between the aggregates. SBM samples also exhibited different protein profiling than WBM and WPI samples. This study suggests that high shear and presence of phospholipids impact the size distribution of liposomal aggregates through structural alterations. The aggregates can be utilized as a novel ingredient and in the processing of dairy foods to deliver nutrition.
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Influence de la structuration de l'interface colloïdale sur la formulation et la biodisponibilité d'acides gras d'intérêt nutritionnel / Influence of colloidal interfacial structuration on the formulation and nutritional fatty acid bioavailabilityBourgeois, Christine 10 December 2018 (has links)
Les études épidémiologiques récentes montrent une consommation insuffisante d’acides gras polyinsaturés (AGPI) dans les pays occidentaux. Cependant, la sensibilité des AGPI à l’oxydation est l’une des premières causes de détérioration des qualités organoleptique et nutritionnelle dans les produits alimentaires. Parallèlement, les lipides présents dans les produits alimentaires se trouvent le plus souvent sous forme émulsionnée. Ainsi, les entreprises désirant formuler des produits alimentaires enrichis en AGPI adoptent des stratégies technologiques pour les stabiliser chimiquement et physiquement, tout en assurant leur biodisponibilité. Une de ces stratégie consiste à rechercher, dans la nature, des systèmes émulsifiés stables afin d’en extraire des molécules tensioactives d’intérêt et de mimer l’organisation des lipides. C’est dans ce contexte que des études portent, depuis quelques années, sur les corps lipidiques (Oil Bodies, OB), structures végétales naturelles de stockage des lipides des plantes oléagineuses, composées de phospholipides (PL) et de protéines (OBP).L’objectif général de ce projet de thèse vise à maitriser la formulation d’émulsions préparées uniquement à base de colza (huile, PL et OBP) par une meilleure connaissance des interactions PL : OBP, d’étudier la stabilité des émulsions d’un point de vue physicochimique en conditions de stockage et dans des conditions mimant les conditions gastro-intestinales et, enfin, d’évaluer l’influence de la composition de l’interface des émulsions sur la bioaccessibilité des acides gras insaturés in vivo chez le rat.Les études spectroscopiques des interactions PL modèles : OBP ont mis en évidence des interactions favorables à la stabilisation des émulsions entre les PL anioniques, les PL insaturés et les OBP. Ces résultats ont orienté le choix vers une lécithine de colza spécifique. Les interactions PL : OBP, modulées par le pH et le rapport PL : OBP, influencent la réalisation des émulsions, la quantité d’OBP adsorbées à l’interface et la stabilité physique des émulsions, avec un crémage prononcé pour les émulsions riches en protéines. La synergie PL : OBP à l’interface semble être un facteur décisif pour ralentir l’oxydation de l’huile de colza émulsifiée. Le comportement des émulsions, dans des conditions mimant celles du milieu gastro-intestinal, montre que la présence d’OBP à l’interface favorise la floculation des émulsions à pH acide (mimant celui de l’estomac), mais que cette floculation est réversible lorsque le pH est ramené à des valeurs proches de celle de l’intestin. La présence des OBP favorise l’activité de la lipase pancréatique in vitro. Finalement, l’interface composée de PL et d’OBP améliore la bioaccessibilité lymphatique des AGPI in vivo chez le rat.En conclusion, nous avons montré qu’il est possible de formuler des émulsions uniquement à base de colza. Elles pourraient présenter une alternative intéressante aux émulsions stabilisées par des émulsifiants d’origine synthétique (politique clean label) ou d’origine animale (alimentation végane). / Recent epidemiologic studies show an insufficient intake of polyunsaturated fatty acids (PUFA) in occidental countries. Besides, the sensibility of PUFA to oxidation is one of the major causes of organoleptic and nutritional quality deterioration in food products. Moreover, lipids in food products are often in an emulsified stage. Therefore, industrials that care to formulate PUFA enriched food products adopt technological strategies to protect and stabilize the lipids while improving their bioavailability. One of those strategies consists in looking for stable emulsified systems that already exist in nature, extract tensioactive molecules of interest and mimic the lipid state. In this context, several studies deal with oil bodies (OB), that are natural occurring structures for lipid storage in oleaginous plants, composed of proteins (OBP) and phospholipids (PL).Therefore, the main objective of this PhD work is to manage emulsion formulation only based on canola (oil, PL and OBP). This goes through: 1) an understanding of the interactions between PL and OBP; 2) a study of the stability of the emulsions under storage and gastrointestinal conditions, and finally, 3) an investigation of the influence of the emulsion interfacial composition on the bioavailability of PUFA in rats.The spectroscopic studies of the model PL:OBP interactions showed favorable interactions for stabilizing the emulsions based on anionic PL, unsaturated PL and OBP. These results allowed choosing an adequate canola lecithin. The PL:OBP interactions, modulated by the pH and the PL:OB ratio, influences the formation of the emulsions, the quantity of OBP adsorbed at the interface and the physical stability of emulsions with a pronounced creaming in emulsions rich in proteins. The PL:OBP synergy at the interface seems to be a decisive factor to slow down the oxidation of the emulsified canola oil. The emulsion behavior in conditions that mimic that of the gastrointestinal track, shows that the presence of OBP at the interface favored the emulsion flocculation at acid pH (mimicking that of the stomach). However, the flocculation was reversible when the pH was adjusted to a value close to that of the intestine. OBP also increased the activity of the pancreatic lipase in vitro. Finally, the presence of PL and OBP at the interface increased the lymphatic bioaccessibility of the PUFA in rats.On the whole, we showed that it is possible to manage emulsion formulation only based on canola products. This could be of peculiar interest for clean labeling or vegan nutrition by subtracting synthetic emulsifiers or emulsifiers from animal origin, respectively.
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Fatty acid metabolism in HepG2 cells: Limitations in the accumulation of docosahexaenoic acid in cell membranesPortolesi, Roxanne, roxanne.portolesi@flinders.edu.au January 2007 (has links)
The current dietary recommendations for optimal health are designed to increase our intake of two bioactive omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), abundant naturally in fatty fish such as salmon. Health authorities recommend that the general population consume two to three fatty fish meals per week (1) for optimal health and for the prevention of cardiovascular disease. However, some modern Western societies consume only modest amounts of fish and seafood (2;3). Land based vegetable oils may provide an alternative to meet these needs. Linseed and canola oils are rich in alpha-linolenic acid (ALA, 18:3n-3) (4). ALA can be converted endogenously to EPA and DHA and suggests that increasing the dietary intake of ALA may increase the conversion and accumulation of DHA in tissues and plasma. However, elevated dietary intakes of ALA in animals and humans results in an increased level of EPA in tissues yet there is little or no change in the level of DHA (5-7). The current consensus is that the synthesis of DHA from ALA in humans is limited yet the mechanisms involved in regulating the accumulation of DHA in tissues are poorly understood.
The reputed rate-limiting enzyme in the conversion of fatty acids is delta 6 desaturase (D6D). ALA is a substrate for D6D and undergoes a series of desaturation and elongation reactions to yield n-3 long chain polyunsaturated fatty acids (LCPUFA). The final step in the synthesis of DHA from ALA involves translocation of its immediate fatty acid precursor, 24:6n-3 from the endoplasmic reticulum to the peroxisome to be partially beta-oxidised to yield DHA. The involvement of multiple enzymes in the desaturation-elongation pathway, and the integration of other pathways, such as phospholipid biosynthesis, suggests there are various steps that may regulate the accumulation of DHA in cell membranes. This thesis aimed to examine the possible regulatory steps in the conversion of fatty acids to LCPUFA, particularly in the synthesis of DHA from n-3 fatty acid precursors.
The human hepatoma cell line, HepG2, was used as an in vitro cell system to examine the accumulation of individual fatty acids and their metabolites in isolation from other competing fatty acid substrates. The accumulation of linoleic acid (LA, 18:2n-6) and ALA in HepG2 cell phospholipids following supplementation with increasing concentrations of each respective fatty acid correlated with that described in vivo, as was the accumulation of their conversion products. The accumulation of DHA in cells supplemented with ALA reached a plateau at concentrations above 5 micro g/ml and paralleled the accumulation of 24:6n-3 in cell phospholipids, suggesting that the delta 6 desaturation of 24:6n-3 was prevented by increasing concentrations of ALA, thereby limiting the accumulation of DHA. The accumulation of DHA in cells supplemented with eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic acid (DPA, 22:5n-3) was significantly greater than the level of DHA that accumulated in cells supplemented with ALA. However, regardless of substrate, the level of DHA in cell membranes reached a plateau at substrate concentrations above 5 micro g/ml.
This thesis further aimed to examine the effect of fatty acid supplementation on the mRNA expression of D6D in HepG2 cells. The expression and activity of D6D mRNA is subject to nutritional and hormonal regulation. The mRNA expression of D6D in HepG2 cells following supplementation with oleic acid (OA, 18:1n-9), LA, ALA, arachidonic acid (AA, 20:4n-6) or EPA was examined by real time RT PCR. The expression of D6D mRNA was reduced by up to 50% in cells supplemented with OA, LA, ALA , AA or EPA compared with control cells and suggests that fatty acids modulate the expression of the key enzyme involved in the conversion of fatty acids.
The effect of fatty acid co-supplementation on the fatty acid composition of HepG2 cell phospholipids was also examined in an attempt to gain insights into the role of D6D and the enzymes involved in peroxisomal beta-oxidation on the accumulation of DHA from n-3 fatty acid precursors. The reduction in the accumulation of DHA in cells co-supplemented with DPA and docosatetraenoic acid (DTA, 22:4n-6) was greater than in cells co-supplemented with DPA and LA, suggesting that peroxisomal beta-oxidation may have a greater role in determining the accumulation of DHA from DPA than the activity of D6D. Further investigation should be directed towards understanding the role that peroxisomal beta-oxidation may play in the synthesis of DHA from precursor fatty acids.
The fatty acid composition of cell membranes in vivo is a result of several physiological processes including dietary intake, phospholipids biosynthesis and fatty acid conversion as well as catabolic processes. This thesis demonstrates that a greater understanding of the regulation of the conversion of fatty acids will help to define dietary approaches that enhance the synthesis of n-3 LCPUFA from n-3 fatty acid precursors to lead to improved outcomes for health.
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Modulation of avian metabolism by dietary fatty acidsNewman, Ronald Edward January 2000 (has links)
The role of dietary fatty acids and their subsequent effects on metabolism has received considerable attention in mammalian species. It is becoming increasingly clear that fatty acids have metabolic roles over and above their influence on energy density of the diet. Recent studies have linked changes in the fatty acyl composition of the plasma membrane, induced by the dietary fat profile, to alterations in both lipid and glucose metabolism. These dietary induced changes have profound effects on insulin action, glucose transport and enzyme activity that regulate triglyceride and fatty acid synthesis, factors that ultimately influence protein and lipid deposition of animals. Because of their high growth rate, broiler chickens have a high requirement for energy and the use of triglycerides as a major energy source has resulted in a fat carcass. A change in the glucose-insulin balance has been suggested as being the main reason for differences in adiposity between broilers selected for fatness or leanness. The hypotheses of this thesis is based on the finding that dietary polyunsaturated fatty acids (PUFA�s) increase the sensitivity of muscle tissue to insulin and this would presumably augment insulin-stimulated glucose uptake into muscle cells. Therefore, increasing the capacity of broiler muscle tissue to utilise glucose as its principal energy substrate would reduce the bird�s reliance on triglycerides and this inturn would result in a leaner carcass. The aims of this study are firstly to explore the role that dietary PUFA�s from the n-3 and n-6 series have on the growth and body composition of broiler chickens and secondly to determine the relationships between dietary fatty acid profile, tissue insulin sensitivity and lipid deposition. Because dietary fatty acids have been implicated in the modulation of hormones important for the growth and development of animals, a third aim of this thesis is to determine the effects of dietary n-3 and n-6 polyunsaturated fatty acids on pituitary and adrenal sensitivity. Since the modulation of metabolism by dietary fatty acids has been attributed to changes in the fatty acyl composition of the plasma membrane, the final aim of this study will be to investigate and characterise the molecular species of the breast muscle choline and ethanolamine phospholipids. Feeding either fish oil a source of n-3 PUFA�s or sunflower oil a source of n-6 PUFA�s fatty acids to broiler chickens resulted in a significant (P<0.01) reduction in the abdominal fat pad mass and a modest increase in breast muscle mass when compared to broilers fed edible tallow. Associated with the changes in carcass composition was an alteration in energy substrate utilisation. This was reflected by lower respiratory quotients and reduced triglyceride and insulin concentrations for the chickens fed the two PUFA diets. Coupled to the shift in energy metabolism was a significant (P<0.05) increase in the proportion of PUFA�s incorporated into the abdominal fat pad and breast muscle. The dietary fat supplements resulted in the incorporation of specific fatty acid subtypes. Feeding fish oil significantly increased the proportion of long-chain n-3 PUFA�s whereas feeding sunflower oil significantly increased the proportion of long-chain n-6 PUFA�s compared to tallow feeding whose tissues were dominated by a higher proportion of saturated fatty acids. It was further shown that dietary n-3 and n-6 PUFA�s enhanced glucose /insulin action. Feeding either fish oil or sunflower oil to broiler chickens increased insulin action when examined by an intravenous glucose tolerance test. The maximal insulin release in response to the glucose infusion was higher in the tallow fed group compared to either the sunflower oil or fish oil groups. To estimate the disappearance rate of glucose from the plasma and its incorporation into tissues, 2-deoxy-D-3H glucose was infused into each chicken. There were no significant differences in the clearance rate of 2-deoxy-D-3H glucose from the plasma. However, when measured under steady state conditions, the labelled glucose incorporation into the breast muscle was greater in birds fed fish oil compared to either tallow or sunflower oil feeding. The dietary fatty acid induced increase in insulin action suggests that the sensitivity of muscle cells to insulin was enhanced. This modulation of tissue sensitivity by dietary fatty acids was also shown to occur at the level of the pituitary. To provide an estimate of pituitary sensitivity, bolus GnRH and GHRH infusions were given on different days to chickens fed the three dietary treatments. Feeding sunflower oil (n-6 PUFA�s) increased the level of GH that was released in response to the GHRH infusion when compared to birds fed either tallow or fish oil (n-3 PUFA�s). This dietary fatty acid modulation appears to be specific to certain pituitary cell types as there was no effect on LH secretion following the GnRH infusion. Dietary fatty acid modulation of endocrine gland sensitivity is particular to the gland type. Although the dietary treatments mediated a distinct pattern in pituitary sensitivity to GHRH infusion, these same three diets did not influence adrenal sensitivity, as there was no difference in the corticosterone profile following either ACTH or CRF infusion. The previously observed physiological changes for the three dietary groups was expected to be positively correlated to an alteration of the plasma membrane phospholipids induced by the dietary fatty acids. Supplementation with fish oil (n-3 PUFA�s) significantly increased levels of both eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) into the choline (PC) and ethanolamine (PE) breast muscle phospholipids compared to either sunflower oil (n-6 PUFA�s) or tallow supplementation. The increase in n-3 PUFA incorporation was associated with a corresponding decrease in the proportion of arachidonic acid (AA; 20:4n-6) an event that would presumably alter substrate availability for the 1- and 2-series eicosanoids. However, feeding sunflower oil or tallow gave a molecular species profile that was remarkably similar in both fatty acid subtype and proportion. This suggests that the plasma membrane dynamics would be similar for these two dietary groups. Therefore, it is appears that factors other than a change in the fatty acyl- composition of the plasma membrane may be responsible for modifying the physiology of the broiler.
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Antioxidant Distribution and Effectiveness in a Model Muscle SystemBallesteros, Ann Theodore 01 February 2009 (has links)
Gallic acid esters (GAE) of varying alkyl chain length were used to determine how antioxidant physical location and partitioning influence hemoglobin-catalyzed lipid oxidation. Specific GAE used were propyl gallate (PG), octyl gallate (OG), and lauryl gallate (LG). GAE partitioning experiments were performed with either isolated cod muscle membranes or washed cod muscle, which primarily contain polar membrane lipids and myofibrillar proteins. Canola oil was used in some experiments to determine how neutral lipids impact partitioning behavior. GAE distribution was determined spectrophotometrically in the recovered membranes, aqueous phase, and oil layer after employing differential centrifugation. Oxidation was monitored by measuring thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides development.When GAE were added to the membrane suspensions, significant differences (p < 0.05) in GAE partitioning were observed in the aqueous phase and membrane sediment, where increases in GAE alkyl chain length corresponded with a decrease in aqueous phase concentrations and increases in the membranes. GAE partitioning in the oil fraction did not show significant differences. Also, increases in GAE alkyl chain correlated with increases in GAE membrane detection when GAE were added to the washed fish muscle (p < 0.05).Adding GAE to the washed cod muscle before the canola oil was the most effective sequence of addition for extending the storage time before lipid oxidation was detected. Among the three GAE tested, PG showed the greatest inhibition against lipid oxidation. The effectiveness of the GAE in the washed cod-canola oil system follows the order, PG > OG > LG, which corresponds with decreasing hydrophobicity.The conclusions of this study are twofold. First, GAE partitioning into the muscle membranes was not the primary factor for delaying the onset of lipid oxidation. Rather, solubility in the aqueous phase showed the greatest impact on extending storage time. Secondly, the order in which GAE and canola oil were added to the washed cod (WC) muscle system influenced hemoglobin-catalyzed lipid oxidation behavior. Adding GAE before the neutral oil may have allowed the GAE to partition more easily into the polar regions of the washed muscle, which in turn provided the most effective protection against oxidation.
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