Megalin is an endocytotic receptor belonging to the low-density lipoprotein family. It has often been viewed only as merely a scavenger receptor of absorptive and secretory epithelia. Recent work has revealed that the megalin intracellular domain contains several motifs potentially binding proteins involved in signal transduction. To find potential intracellular proteins binding to megalin, a yeast two-hybrid screening was initiated with the intracellular tail of megalin as the bait. A partial clone encoding the scaffolding protein postsynaptic protein 95 (PSD-95) was found to bind to megalin with its second PDZ-domain. Co-localization experiments in HEK-293 cells and kidney, placenta and parathyroid tissue confirmed this interaction. The PSD-95 related proteins PSD-93 and SAP102 were also confirmed to bind megalin with their PDZ2-domains, but the corresponding domain from SAP97 did not bind. Mutation analysis revealed that an amino acid residue change Ala to Thr was the cause of this. Megalin has within the central nervous system (CNS) been shown to be expressed only in the ependymal cells and choroid plexus. Nothing has been known about megalin expression in the spinal cord. To study spatio-temporal expression of megalin in the spinal cord, extensive staining of prenatal and postnatal mouse spinal cord was undertaken. Megalin expression was found in the dorsal part of the embryonic spinal cord. Most of these cells also expressed vimentin, suggesting that megalin has a role in the normal development of astrocytes. In the postnatal mouse, megalin seems to be expressed in oligodendrocytes only in the spinal cord white matter, and co-incident with myelination. This suggests that megalin is involved in the formation and maintenance of myelin along long spinal pathways. Megalin staining was clearly seen in the nucleus of these cells, indicating that megalin works in a notch-like signalling pathway. Uptake of retinol to the retina pigment epithelium (RPE) has long been thought to be a diffusion process. Staining for megalin in RPE revealed strong expression, and uptake experiments with 3H-retinol bound to retinol-binding protein and blocking with the LDL-receptor family specific antagonist receptor-associated protein (RAP) showed that megalin is a receptor for uptake of retinol to the RPE.

Los parásitos nematodos producen diversas proteínas solubles que unen lípidos (LBPs) estructuralmente distintas a las del huésped. Las funciones que cumplen se desconocen pero se hipotetiza que estarían involucradas en las funciones típicas internas de organismos multicelulares, como la utilización y transporte de compuestos no solubles, y en externas especializadas. Algunas de estas proteínas participarían en la modificación del entorno local en el tejido del huésped, posibilitando la modulación y la evasión de la respuesta inmune. Entre las LBPs producidas por nematodos se encuentran las FAR (Fatty Acid and Retinol binding proteins), una clase novedosa de proteínas que unen ácidos grasos y retinol. Tienen un tamaño aproximado de 19 kDa y sus estructuras que parecen ser ricas en alfa-hélices aún no han sido completamente dilucidadas. La comprensión del rol que cumple esta familia de proteínas tiene gran interés fisiopatológico ya que podrían desempeñar funciones relevantes en la biología de los parásitos que las producen y dadas las diferencias estructurales que presentarían con respecto a las LBPs de sus huéspedes, servirían como potenciales blancos para el diseño de nuevas terapias antiparasitarias. Con la finalidad de contribuir a la caracterización de las proteínas FAR y avanzar de este modo en la determinación de su función biológica, se llevaron a cabo estudios biofísicos y estructurales que permitieron resolver la estructura de Na-FAR-1 en solución por espectroscopía de resonancia magnética nuclear. Determinándose que consta de once hélices que conforman una cavidad interna de gran tamaño, donde podrían ubicarse ligandos hidrofóbicos. La estequiometría de unión de los complejos formados por Na-FAR-1 estaría dada por cuatro moléculas de ácido oleico por molécula de proteína, pero se limitaría a una única molécula de ligando en el caso del retinol y de los análogos fluorescentes de ácidos grasos empleados para su estudio. A su vez se evidenció que además de los ligandos esperados como ácidos grasos y retinol, esta proteína es capaz de unir fosfolípidos y diacilglicéridos. La amplia diversidad de unión a ligandos, sumada a su localización en el intestino del nematodo, indicarían que podría participar en el direccionamiento hacia los distintos tejidos de los lípidos ingeridos.

This thesis deals with the qualitative and quantitative representation of vitamins in the meat-vegetables based infant food. The work is divided into the theoretical and the experimental part. The theoretical part describes a child nutrition and the vitamins. In the chapters, dealing with vitamins, is a description of particular vitamins, their biological functions in the body, the recommended daily dose and their stability during food processing. These chapters concern fat-soluble vitamins (vitamin A, D, E and K) and water-soluble vitamins (B-group vitamins and vitamin C) as well. The work deals with nutrition of infants, toddlers and young children under age of 10 years too. For the each age group there is given the daily recommended dose of vitamins. Experimental part deals with the quantification of vitamins (retinol, ascorbic acid, A-tocopherol and B-carotene) by the HPLC analysis in the samples of the meat-vegetables based infant food. The influence of storage time and conditions (exposure to daylight vs storage in the dark) was measured on concetration of the examined vitamins in the samples. The results were statistically analyzed using software Statistica 12 (Wilcoxon signed-rank test, linear regression, p<0,05) and showed, that all monitored vitamins degrades during storage. The concetration of vitamins in the product is also influenced by the storage conditions (the faster degradation of vitamins occurred in samples exposed to daylight).

Resumo: O efeito da vitamina A sobre os parâmetros de desempenho da tilápia do Nilo foi avaliado em um experimento com duração de 130 dias. Trezentos e vinte alevinos de tilápia do Nilo (7,43 ± 0,19g) foram aleatoriamente distribuídos em 40 aquários de fibra de vidro com volume total de 250L e alimentados com dieta prática contendo níveis crescentes de vitamina A (0, 200, 400, 800, 1.600, 3.200, 6.400, 12.800 UI de vit. A/kg da dieta). A fonte de vitamina utilizada foi o acetato de retinol (Rovimix A 1000). Ao final do período experimental os peixes foram pesados e a ração quantificada para a avaliação do ganho de peso médio, conversão alimentar aparente (CAA), consumo diário de ração, porcentagem de sobrevivência, taxa de crescimento específico (TCE), taxa de eficiência protéica (TEP) e taxa de retenção protéica (TRP). Foi observado efeito quadrático (P<0,01) para todas as variáveis com o aumento do nível de vitamina A nas dietas com exceção da CAA e TEP. Os peixes alimentados com as dietas não suplementadas apresentaram reduzida TEP e piores índices de CAA. Sinais de deficiência de vitamina A observados neste estudo foram: apatia, natação errática, exoftalmia, petéquias hemorrágicas localizadas na base da nadadeira caudal e superfície corporal, e presença de ascite na cavidade abdominal. A presença de níveis adequados de vitamina A é essencial para o adequado crescimento da tilápia do Nilo e a exigência com base no ganho de peso e taxa de retenção protéica foi de 4.704 e 5.300 UI de vit. A/ kg da dieta, respectivamente. / Abstract: A 130-day feeding trial was undertaken to evaluate the effect of vitamin A on growth performance of sex reversed Nile tilapia (Oreochromis niloticus). 320 Nile tilapia fingerlings with 7.49 ± 0.19g weight (mean±SD) were randomly stocked into 40 250Laquaria and fed practical diets containing graded levels of vitamin A (0; 200; 400; 800; 1,600; 3,200; 6,400; 12,800 IU vit. A/kg diet). Vitamin A source was retinol acetate (Rovimix A 1000). At the end of the experimental period fish and diets were weighed to evaluate weight gain (WG), feed conversion ratio (FCR), survival (SUR), specific growth rate (SGR), protein efficiency ratio (PER) and protein retention (PR). All gowth parameters showed quadratic effect (P<0.01) according to vitamin A level regardless the FCR and PER. Fish fed the non-supplemented diet showed reduced PER and higher FCR. Clinical signs of vitamin A deficiency observed in this study were resting and abnormal swimming behavior, exophthalmia, hemorrhages at the base of fins and on skin and serous fluids in abdominal cavity. Adequate vitamin A levels are necessary for normal Nile tilapia growth and vitamin A requirement based on WG and PR is 4,704 and 5,300 IU vit. A/kg diet, respectively. / Orientador: Margarida Maria Barros / Coorientador: Ricardo de Oliveira Orsi / Banca: Fabiana Pilarski / Banca: José Fernando Machado Menten / Banca: Maria José Tavares Ranzani de Paiva / Banca: Dirlei Antonio Berto / Doutor

Vitamin A deficiency (VAD) continues to be a major health problem in developing countries. In South Africa, in 1999, one out of three children under the age of six years in the country had poor/ marginal vitamin A status. Limpopo Province was one of the provinces that most seriously affected by VAD. The study aim was to explore and describe the consumption patterns of vitamin A-rich foods of 10-13 year old children living in a rural area in Venda, and consequently making recommendations on nutrition education in this regard. A survey was conducted in Vyeboom Village in Limpopo Province, at Makhado local municipality in the Vhembe District. About 155 school children aged 10-13 years (boys and girls) participated in this study, using convenience, random and stratified sampling to draw the sample from three primary schools (Avhatondwi, Tshirunzanani and Thomas Ntshavheni). Data was collected during winter of 2006 using a socio-demographic questionnaire, the 24-hour recall and non-quantitative food-frequency questionnaires. The data was divided into four subsections regarding the 10-13 year old children that participated in this study, namely: the demographic information, food habits, food consumption patterns, and foods consumed rich in vitamin A. The SAS statistical analysis (version 8.2) software was used to analyze the data from the questionnaire by means of descriptive statistics (percentages, frequencies, means and summary of the tables). Inferential statistics (two way tables and chi-square tests) were used to test the associations between two categorical variables. Mothers were the caretakers in the families, because she mostly played a major role in the decision-making, preparation and serving of the food that were consumed by these children. Social cultural factors influenced the children’s food intake, because some of these foods were especially low in vitamin A and high in sugar content (e.g. sweets), compared to those foods that children were forbidden to eat (e.g. liver) which were high in vitamin A and other nutrients. The foods that children were mostly forced to eat (dark green leafy vegetables) were very nutritious and given in order to prevent hunger and wastage (left-over in the morning). The general meal pattern of these children was three meals a day with two, one or no snacks in between meals. The mostly consumed foods during these meals were bread, tea, stiff and soft maize meal porridge, dark green leafy vegetables and meat (chicken). Dark green leafy vegetables played an important role in supplying beta-carotene to these children, while the intake of retinol-rich foods was poor because they are expensive and labeled as highly allergenic, whilst other foods were forbidden. Intake of fortified foods was very high because porridge was the staple food and commonly consumed during lunch and supper. The results provided insight regarding the food habits, consumption patterns and the intake of vitamin A rich foods and were used to substantiate recommendations aimed at the improvement of the intake vitamin A rich foods among the children. / Dissertation (MConsSci (General))--University of Pretoria, 2008. / Consumer Science / unrestricted

L'identification et l'amélioration du potentiel pro-vitaminique A des sauces accompagnant les aliments de base pourraient constituer une voie alimentaire de lutte contre la carence en vitamine A (CVA) chez les jeunes enfants au Bénin. La qualité nutritionnelle et la rétention en provitamines A des sauces lors des procédés traditionnels de préparation ont été évaluées au moyen d'une démarche itérative terrain et laboratoire. Une enquête de consommation alimentaire par pesées et mesures anthropométriques auprès de 420 enfants a permis d'évaluer l'état nutritionnel des sujets et l'adéquation des apports en fer, zinc et vitamine A (VA) et d'identifier les principales sources de VA dans l'alimentation. La mangue, les œufs, l'huile de palme rouge (HPR) ou diverses sauces légumes-feuilles (LF) constituent les principaux aliments locaux riches en VA consommés (34,2% des enfants enquêtés). Lorsqu'elles sont consommées, les sauces participent à la couverture de 71 à 129% des apports journaliers recommandés en VA des jeunes enfants. Le suivi au niveau ménage des procédés traditionnels de préparation des sauces les plus prometteuses notamment les sauces LF-amarante à base d'HPR ou de jus de noix de palme (NP), a permis d'identifier les traitements thermiques appliqués aux LF, aux NP ou à l'HPR comme des étapes critiques. Le chauffage de l'HPR à 180-200°C apparaît comme l'étape la plus préjudiciable à la VA. Elle réduit de plus de 70%, et en moins de 3 min, les teneurs en α-carotène, en β-carotène et en activité équivalent rétinol (AER). La violaxanthine, caroténoïde non pro-VA est le seul composé significativement affecté lors du blanchiment des feuilles d'amarante (100°C) mais l'AER reste élevée avec ou sans ajout de potasse traditionnelle. Les sauces formulées à base des ingrédients LF-amarante, NP ou HPR présentent une bonne acceptabilité, une AER élevée et leur consommation pourrait être promue avantageusement dans le cadre d'approches alimentaires de lutte contre la CVA. / The identification and the improvement of the potential in pro-vitamins A of sauces accompanying staple foods could constitute a food-based approach for combating vitamin A deficiency (VAD) among young children in Benin. The nutritional quality and the retention of pro-vitamins A in sauces during traditional home processing has been assessed by using a field-laboratory iterative approach. Anthropometric measurements were used to appreciate subjects' nutritional status. A food consumption survey of 420 young children was conducted to assess the adequacy of iron, zinc and vitamin A (VA) intakes, and to identify the main VA-rich foods eaten by children using the weighed food record method. Mangoes, eggs, red palm oil (RPO), various leafy vegetables (LV) and palm nut juice sauces appeared to be the main VA-rich foods consumed by 34.2% investigated children. When consumed, these LV sauces containing RPO or palm nut juice (PNJ) contributed to the meeting of 71-129% of the recommended VA intake of young children. The traditional processing method of the most promising sauces such as amaranth leaf sauces based on RPO or PNJ was monitored step by step during home visits and allowed identifying the thermal treatments applied to LV, to palm nut or to RPO as critical steps. Heating the RPO at 180-200°C appears as the more prejudicial step to VA. It decreased more than 70% and in less than 3 min, α-carotene, β-carotene and Retinol Activity Equivalent (RAE) contents. Violaxanthin, a non pro-VA carotenoid, was the only compound to be significantly affected by the thermal treatment (100°C) of amaranth leaves. RAE remained high after blanching even when alkaline traditional potash was added. The formulated sauces on the basis of the ingredients LV-amaranth, palm nut or RPO present a good acceptability, a high RAE and their consumption could be promoted favorably in food-based strategies to alleviate VAD.

No description available.

No description available.

Résumé : L'interaction entre la β-Lactoglobuline (β-Lg) et le rétinol ou entre les micelles de phosphocaséines (PC) et le rétinol à un pH proche de la neutralité, a été étudiée à pression atmosphérique et sous pression isostatique jusqu'à 400 MPa. Les constantes de dissociation et le nombre de sites de liaison ont été calculés indiquant des différences d'affinité en fonction de la structure protéique. A 25°C, des pressions inférieures à 150 MPa favorisent l'association β-Lgrétinol (rapport molaire β-Lg/rétinol : 1/1). A ≥ 150 MPa le complexe se dissocie. A 350 MPa, la β-Lg est dénaturée et le complexe irréversiblement dissocié. Le complexe PCrétinol (rapport molaire PC/rétinol : 1/1) reste au contraire formé après un traitement à 400 MPa et 25°C, bien que la pression induise des phénomènes de dissociation/réassociation des assemblages micellaires à ≥ 100 MPa. L'interaction PCrétinol stabiliserait plutôt les micelles de PC vis-à-vis de la pression, de même qu'une température de pressurisation modérée (35°C) comparativement à une température plus basse (15°C). Un isolat protéique de lactosérum (IPL) en dispersion dans l'eau à 10% (p/p) de protéines (pH 6,5) et en présence d'acétate de rétinol (AcRet) (rapport molaire β-Lg/AcRet : 10/1) a été traité par (i) haute-pression isostatique (HP) (350 MPa, 25°C, 15 min), (ii) traitement thermique de courte durée (TTCD) (75°C, 4 s) ou (iii) homogénéisation à ultra-haute pression (UHPH) (300 MPa, Tin = 24°C). Les trois traitements permettent de former des agrégats de β-Lg capables de retenir l'acétate de rétinol, mais avec une efficacité différente dépendant probablement des mécanismes d'agrégation induit par le chauffage (TTCD), la pression isostatique (HP) ou dynamique (UHPH). Des dispersions à 2,38% (p/p) en phosphocaséines (pH 6,6) en présence d'acétate de rétinol (rapport molaire PC/AcRet : 5/1) ont été traitées par (i) HP (300 MPa, 14°C ou 34°C, 15 min), ou (ii) UHPH (300 MPa, Tin = 14°C). Ces deux traitements favorisent la rétention de l'acétate de rétinol par les micelles de PC pouvant ainsi servir de cargo pour véhiculer des molécules bioactives. Mots clefs : haute pression isostatique, haute pression dynamique, homogénéisation à ultra-haute pression, fluorescence, β-Lactoglobuline, micelles de phosphocaséines, rétinol, acétate de rétinol, agrégats protéiques, interaction protéine-ligand. / Abstract: The binding of retinol to native β-Lactoglobulin (β-Lg) or phosphocasein (PC) micelles at pH close to neutral was studied at atmospheric pressure or under isostatic high-pressure. The dissociation constants and number of binding sites were calculated indicating that difference in retinol affinity depended on protein structure. At 25°C, pressure level < 150 MPa promoted β-Lgretinol association (β-Lg/retinol molar ratio: 1/1). At ≥ 150 MPa, the complex dissociated. At 350 MPa, β-Lg was denatured and the complex irreversibly dissociated. PC and retinol (PC/retinol molar ratio: 1/1) remained associated after pressurisation at 400 MPa and 25°C, while pressure induced dissociation/reassociation phenomena of micelle assemblies. The binding of retinol to PC stabilised micelles towards pressure, as well as moderate temperature of pressurisation (35°C) compared to lower temperature (15°C).A whey protein isolate (WPI) dispersed in water at 10% (w/w) proteins (pH 6.5) in the presence of retinyl acetate (RetAc) (β-Lg/RetAc molar ratio: 10/1) was processed by (i) isostatic high-pressure (HP) (350 MPa, 25°C, 15 min), (ii) short-time thermal treatment (STTT) (75°C, 4 s) or (iii) ultra-high pressure homogenisation (UHPH) (300 MPa, Tin = 24°C). All processing produced β-Lg aggregates able to retain RetAc, but with different efficiency depending on aggregation mechanisms induced by heating (STTT), isostatic high-pressure (HP) or dynamic high-pressure (UHPH). Phosphocaseins dispersed at 2.38% (w/w) proteins (pH 6.6) in the presence of RetAc (PC/RetAc molar ratio: 5/1) were processed by (i) HP (300 MPa, 14°C or 34°C, 15 min), or (ii) UHPH (300 MPa, Tin = 14°C). Both treatments promoted RetAc retention by phosphocasein micelles that can be used as cargoes to transport bioactive molecules.Keywords: isostatic high-pressure, dynamic high-pressure, ultra-high pressure homogenisation, fluorescence, β-Lactoglobulin, phosphocasein micelles, retinol, retinyl acetate, protein aggregates, protein-ligand binding.Discipline: Biochimie, Chimie et Technologie des Aliments.Thèse préparée à : Université Montpellier 2 – Equipe de Biochimie et Technologie Alimentaire – UMR IATE 1208 – Pôle EVAP – Place E. Bataillon, 34095 Montpellier, France.

L'objet de cette thèse est l'étude de la pénétration des actifs cosmétiques dans la peau. Les axes d'investigation principaux ont concerné l'influence des propriétés physicochimiques des actifs et des ingrédients de la formule sur les mécanismes de pénétration. Les actifs cosmétiques choisis sont le rétinol, actif lipophile, et la caféine, actif hydrophile. Les formulations investiguées sont des émulsions de type huile dans eau, comparées aux solutions de tensioactifs correspondantes. Trois huiles cosmétiques ont été utilisées: Butylène glycol de cocoate, Octyldodecyl myristate et la Paraffine liquide, stabilisées en émulsion avec des tensioactifs ester de polyéthylène glycol (PEG20 et PEG6) possédant des longueurs de chaîne carbonées variables (C8, C12, C18 et C18:1). La pénétration percutanée a été mesurée quantitativement en utilisant la méthode des cellules de diffusion de Franz en fonctionnement statique et dynamique et qualitativement par la microscopie confocale Raman. Avec cette combinaison de techniques analytiques, il est possible, de mesurer la pénétration et d’évaluer l'impact de chaque composant de la formulation sur la pénétration cutanée d'un actif. Une corrélation a pu être établie entre l’effet fluidifiant d’une huile et l’augmentation de la pénétration du rétinol. Par ailleurs les tensioactifs, même s’ils ont montré un effet moindre en terme de fluidification conduisent également à une augmentation de la pénétration en raison d’une variation du coefficient de partage de l’actif entre la formule et la peau. Concernant la caféine, l’influence de la structure des tensioactifs et en particulier de la longueur de chaîne carbonée a été mise en évidence / The purpose of this thesis is to study the penetration of cosmetics actives into the skin. The main lines of investigation concerned the influence of actives and formulation components physicochemical properties on the penetration mechanisms. The selected cosmetic actives are retinol, lipophilic, and caffeine, hydrophilic. The investigated formulations are oil in water emulsions, compared to their corresponding surfactant solutions. Three cosmetic oils were used: Butylene glycol cocoate, Octyldodecyl myristate and liquid paraffin. Emulsions are stabilized with polyethylene glycol ester surfactants (PEG20 and PEG6) having variable carbon chain lengths (C8, C12, C18 and C18: 1). Percutaneous penetration was measured quantitatively using Franz diffusion cells in a static and dynamic way and qualitatively by confocal Raman microscopy. With this combination of analytical techniques, it is possible to measure the penetration and evaluate the impact of each formulation component on skin penetration of an active. A correlation could be established between the fluidizing effect of an oil and the increase in retinol penetration. Moreover, the surfactants, although they showed less effect in terms of fluidizing also lead to an increase in penetration due to a variation of the active partition coefficient between the formula and the skin. Regarding caffeine, the influence of the surfactant structure and in particular the carbon chain length has been pointed out