Genotoxicity assessment of water soluble fractions of biodiesel and its diesel blends using the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay

The designation of biodiesel as an environmental-friendly alternative to diesel oil has improved its commercialization and use. However, most biodiesel environmental safety studies refer to air pollution and so far there have been very few literature data about its impacts upon other biotic systems, e.g. water, and exposed organisms. Spill simulations in water were carried out with neat diesel and biodiesel and their blends aiming at assessing their genotoxic potentials should there be contaminations of water systems. The water soluble fractions (WSF) from the spill simulations were submitted to solid phase extraction with C-18 cartridge and the extracts obtained were evaluated carrying out genotoxic and mutagenic bioassays [the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay]. Mutagenic and genotoxic effects were observed, respectively, in the Salmonella/microsome preincubation assay and the in vitro MN test carried out with the biodiesel WSF. This interesting result may be related to the presence of pollutants in biodiesel derived from the raw material source used in its production chain. The data showed that care while using biodiesel should be taken to avoid harmful effects on living organisms in cases of water pollution. (C) 2011 Elsevier Ltd. All rights reserved.

Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray

Abstract Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process.

MARC 21 e XML como ferramentas para a consolidação da catalogação cooperativa automatizada: Uma revisão de literatura

The cooperation and the sharing of information cataloguing and bibliographical in environment automated, this was only possible with the creation and adoption of interchange format MARC21. But due to the progresses of the technologies of information and communication, of the crescent use of Internet and of the databases and databanks, there were the need of the creation and development of tools that optimize the organization activities, retrieval and interchange of information. XML is one of those developments that have as purpose to facilitate the management, storage and transmission of data through Internet. Before that, it was proposed through a literature revision, to analyze Interchange Format MARC21 and Markup Language XML as tools for the consolidation of the Automated Cooperative Cataloguing, your differences of storage flexibilities, organization, retrieval and interchange of data through Internet. This research made possible the divulgation to the community librarian, through a literature revision, that has been discussed internationally on MARC21 and XML

Biochemical and histological characterization of tomato mutants

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H2O2) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.

Troca e criação de valor: possibilidades competitivas advindas da estratégia de redes

O compartilhamento de custos e de conhecimento, traduzidos neste trabalho por troca e criação de valor, foi objeto de estudo em duas redes empresariais do varejo alimentício, localizadas na região central do estado do Rio Grande do Sul. O objetivo foi investigar se essas possibilidades competitivas, advindas da estratégia de rede, estão sendo exploradas pelos gestores das empresas associadas. A pesquisa foi conduzida pelo método de estudo de caso, cuja coleta de dados processou-se por meio de visitas in loco acompanhadas de entrevistas com os principais executivos das redes e de questionários para os gestores das empresas associadas. Como resultado, pôde-se perceber que ambas exploram com maior ênfase as possibilidades de troca de valor, principalmente nas relações conjuntas de aquisição e relacionamento com clientes, em detrimento dos outros componentes da cadeia de valor. Por outro lado, em termos de criação de valor, verificou-se que existe um ambiente de intensa aprendizagem em ambas as redes, mas evidenciaram-se dificulda- des de efetivar a criação de valor com ações conscientes.

Spatio-temporal variability of sugarcane yield efficiency in the state of São Paulo, Brazil

The objective of this work was to assess the spatial and temporal variability of sugarcane yield efficiency and yield gap in the state of São Paulo, Brazil, throughout 16 growing seasons, considering climate and soil as main effects, and socioeconomic factors as complementary. An empirical model was used to assess potential and attainable yields, using climate data series from 37 weather stations. Soil effects were analyzed using the concept of production environments associated with a soil aptitude map for sugarcane. Crop yield efficiency increased from 0.42 to 0.58 in the analyzed period (1990/1991 to 2005/2006 crop seasons), and yield gap consequently decreased from 58 to 42%. Climatic factors explained 43% of the variability of sugarcane yield efficiency, in the following order of importance: solar radiation, water deficit, maximum air temperature, precipitation, and minimum air temperature. Soil explained 15% of the variability, considering the average of all seasons. There was a change in the correlation pattern of climate and soil with yield efficiency after the 2001/2002 season, probably due to the crop expansion to the west of the state during the subsequent period. Socioeconomic, biotic and crop management factors together explain 42% of sugarcane yield efficiency in the state of São Paulo.

Natural regeneration in abandoned fields following intensive agricultural land use in an Atlantic Forest Island, Brazil

The time required to regrowth a forest in degraded areas depends on how the forest is removed and on the type of land use following removal. Natural regeneration was studied in abandoned old fields after intensive agricultural land use in areas originally covered by Brazilian Atlantic Forests of the Anchieta Island, Brazil in order to understand how plant communities reassemble following human disturbances as well as to determine suitable strategies of forest restoration. The fields were classified into three vegetation types according to the dominant plant species in: 1) Miconia albicans (Sw.) Triana (Melastomataceae) fields, 2) Dicranopteris flexuosa (Schrader) Underw. (Gleicheniaceae) thickets, and 3) Gleichenella pectinata (Willd.) Ching. (Gleicheniaceae) thickets. Both composition and structure of natural regeneration were compared among the three dominant vegetation types by establishing randomly three plots of 1 x 3 m in five sites of the island. A gradient in composition and abundance of species in natural regeneration could be observed along vegetation types from Dicranopteris fern thickets to Miconia fields. The gradient did not accurately follow the pattern of spatial distribution of the three dominant vegetation types in the island regarding their proximity of the remnant forests. A complex association of biotic and abiotic factors seems to be affecting the seedling recruitment and establishment in the study plots. The lowest plant regeneration found in Dicranopteris and Gleichenella thickets suggests that the ferns inhibit the recruitment of woody and herbaceous species. Otherwise, we could not distinguish different patterns of tree regeneration among the three vegetation types. Our results showed that forest recovery following severe anthropogenic disturbances is not direct, predictable or even achievable on its own. Appropriated actions and methods such as fern removal, planting ground covers, and enrichment planting with tree species were suggested in order to restore the natural forest regeneration process in the abandoned old fields.

Distribuição vertical dos sistemas de polinização bióticos em áreas de cerrado sentido restrito no Triângulo Mineiro, MG, Brasil

Alguns fatores podem influenciar a distribuição dos recursos florais e sistemas de polinização bióticos nos ecossistemas, como por exemplo, o clima, a altitude, a região geográfica, fragmentação de áreas naturais e as diferenças na composição florística na estratificação vertical. Este estudo teve por objetivo avaliar a distribuição dos sistemas de polinização bióticos na estratificação vertical em fragmentos de cerrado sentido restrito no Triângulo Mineiro. Não houve diferença significativa (χ²0,05,9 =14,17; p = 0,12) na riqueza florística geral entre os fragmentos, nem quando comparada em separado para cada estrato (arbóreo, arbustivo, herbáceo e liana), estando o estrato arbustivo mais bem representado. Da mesma forma, não houve diferenças significativas entre fragmentos quanto aos sistemas de polinização (χ²0,05,21 =13,80; p = 0,8778), sendo a polinização por abelhas mais comum, correspondendo ao menos 85% das espécies de plantas em cada fragmento. Em termos relativos, as plantas polinizadas por abelhas foram dominantes em todos os estratos, chegando a 100% das lianas e herbáceas em alguns fragmentos. Neste estudo, com base na composição florística e distribuição dos sistemas de polinização na estratificação vertical, podemos caracterizar um mosaico vertical no cerrado sentido restrito, que tem implicações na sustentabilidade das comunidades no cerrado, assim como os mosaicos horizontais de fitofisionomias.

Effect of Prior Photodegradation on the Biodegradation of Polypropylene/Poly(3-hydroxybutyrate) Blends

In this work, the effect of blend composition and previous photodegradation on the biodegradation of polypropylene/ poly(3-hydroxybutyrate) (PP/PHB) blends was studied. The individual polymers and blends with or without the addition of poly(ethylene-co-methyl acrylate- co-glycidyl methacrylate) [P(E-MA-GMA)] as a compatibilizer (in the case of 80/20 blend) were exposed to UV light for 4 weeks and their biodegradation was evaluated. The biodegradation of PHB phase within the blends was hindered as PHB was the dispersed phase and PP fibrous particles were observed at the surface of the blend samples after biodegradation. Previous photodegradation lessened PHB biodegradation but enhanced the biodegradation of PP and the blends within the biodegradation time studied. Photodegradation resulted in cracks at the surface of PP and the blends, which probably facilitated the biotic reactions due to an easier access of the enzymes to deeper polymer layers. It also resulted in a decrease of molecular weight of PP phase and formation of carbonyl and hydroxyl groups which were consumed during biodegradation. Size exclusion chromatography analysis revealed that only the short chains of PP were consumed during biodegradation.

Adaptation and Constraint in the Plant Reproductive Phase

Conservatism is a central theme of organismic evolution. Related species share characteristics due to their common ancestry. Some concern have been raised among evolutionary biologists, whether such conservatism is an expression of natural selection or of a constrained ability to adapt. This thesis explores adaptations and constraints within the plant reproductive phase, particularly in relation to the evolution of fleshy fruit types (berries, drupes, etc.) and the seasonal timing of flowering and fruiting. The different studies were arranged along a hierarchy of scale, with general data sets sampled among seed plants at the global scale, through more specific analyses of character evolution within the genus Rhamnus s.l. L. (Rhamnaceae), to descriptive and experimental field studies in a local population of Frangula alnus (Rhamnaceae). Apart from the field study, this thesis is mainly based on comparative methods explicitly incorporating phylogenetic relationships. The comparative study of Rhamnus s.l. species included the reconstruction of phylogenetic hypotheses based on DNA sequences. Among geographically overlapping sister clades, biotic pollination was not correlated with higher species richness when compared to wind pollinated plants. Among woody plants, clades characterized by fleshy fruit types were more species rich than their dry-fruited sister clades, suggesting that the fleshy fruit is a key innovation in woody habitats. Moreover, evolution of fleshy fruits was correlated with a change to more closed (darker) habitats. An independent contrast study within Rhamnus s.l. documented allometric relations between plant and fruit size. As a phylogenetic constraint, allometric effects must be considered weak or non-existent, though, as they did not prevail among different subclades within Rhamnus s.l. Fruit size was correlated with seed size and seed number in F. alnus. This thesis suggests that frugivore selection on fleshy fruit may be important by constraining the upper limits of fruit size, when a plant lineage is colonizing (darker) habitats where larger seed size is adaptive. Phenological correlations with fruit set, dispersal, and seed size in F. alnus, suggested that the evolution of reproductive phenology is constrained by trade-offs and partial interdependences between flowering, fruiting, dispersal, and recruitment phases. Phylogenetic constraints on the evolution of phenology were indicated by a lack of correlation between flowering time and seasonal length within Rhamnus cathartica and F. alnus, respectively. On the other hand, flowering time was correlated with seasonal length among Rhamnus s.l. species. Phenological differences between biotically and wind pollinated angiosperms also suggested adaptive change in reproductive phenology.

Marine Seaweed Invasions : the Ecology of Introduced Fucus evanescens

Biological invasions are an important issue of global change and an increased understanding of invasion processes is of crucial importance for both conservation managers and international trade. In this thesis, I have studied the invasion of the brown seaweed Fucus evanescens, to investigate the fate and effect of a perennial, habitat-forming seaweed introduced to a coastal ecosystem. A long-term study of the spread of F. evanescens in Öresund (southern Sweden) showed that the species was able to expand its range quickly during the first 20 years after the introduction, but that the expansion has been slow during the subsequent 30 years. Both in Öresund and in Skagerrak, the species is largely restricted to sites where native fucoids are scarce. Laboratory experiments showed that the restricted spread of F. evanescens cannot be explained by the investigated abiotic factors (wave exposure and salinity), although salinity restricts the species from spreading into the Baltic Sea. Neither did I find evidence for that herbivores or epibiota provide biotic resistance to the invader. On the contrary, F. evanescens was less consumed by native herbivores, both compared to the native fucoids and to F. evanescens populations in its native range, and little overgrown by epiphytes. Instead, the restricted spread may be due to competition from native seaweeds, probably by pre-occupation of space, and the establishment has probably been facilitated by disturbance. The studies provided little support for a general enemy release in introduced seaweeds. The low herbivore consumption of F. evanescens in Sweden could not be explained by release from specialist herbivores. Instead, high levels of chemical anti-herbivore defence metabolites (phlorotannins) could explain the pattern of herbivore preference for different fucoids. Likewise, the low epibiotic colonisation of F. evanescens plants could be explained by high resistance to epibiotic survival. This shows that colonisation of invading seaweeds by native herbivores and epibionts depends on properties of the invading species. The large differences between fucoid species in their quality as food and habitat for epibionts and herbivores imply that invasions of such habitat-forming species may have a considerable effect on a number of other species in shallow coastal areas. However, since F. evanescens did not exclude other fucoids in its new range, its effect on the recipient biota is probably small.

Does depth and sedimentation interact with sea urchings to affect algal assemblage patterns on eastern atlantic reefs?

[EN]A range of factors may affect the composition and abundance of macroalgae on subtidal rocky reefs. We experimentally determined the interactive effect of the occurrence of the long-spine sea urchin, Diadema antillarum, depth and sedimentation levels on macroalgal assemblage structure on eastern Atlantic rocky reefs. Specifically, we manipulated sea urchin densities (removal of all individuals vs. untouched controls at natural densities) on rocky reefs devoid of erect vegetation, and predicted (1) that removal of sea urchins would differently affect macroalgal assemblage structure between deep (16-18 m) and shallow (8-9 m) reef strata, and that (2) the effect of sea urchin removal on macroalgae would be altered under different scenarios of sedimentation (ambient vs. enhanced). Experimental circular plots (2 m in diameter) were set up at 3 locations at Gran Canaria (Canarian Archipelago), and were maintained and monitored every 4 wk for 1 y. At the end of the experimental period, the structure of the algal assemblages differed between urchin treatments and depth strata, with a larger cover of turf and bushlike algae where urchins were removed and at the shallow reef stratum. More important, differences in algal assemblage structure between urchin treatments were irrespective of sedimentation levels, but shifted from the shallow to the deep stratum. This interactive effect was, in turn, observed for bushlike algae, as a result of a larger magnitude of response (i.e., larger cover) in the shallow stratum relative to the deep stratum, but was not detected for either turf or crustose coralline algae. These results highlight the importance of sorne physical conditions (here, differences in depth) to interact with biotic processes (here, urchin abundance) to create patterns in the organization of subtidal and benthic assemblages

Effects of habitat characteristics on cryptic fish assemblages

Habitat structure is known to influence the abundance of fishes on temperate reefs. Biotic interactions play a major role in determining the distribution and abundance of species. The significance of these forces in affecting the abundance of fishes may hinge on the presence of organisms that either create or alter habitat. On temperate reefs, for example, macroalgae are considered autogenic ecosystem engineers because they control resource availability to other species through their physical structure and provide much of the structure used by fish. On both coral and temperate reefs, small cryptic reef fishes may comprise up to half of the fish numbers and constitute a diverse community containing many specialized species. Small cryptic fishes (<100 mm total length) may be responsible for the passage of 57% of the energy flow and constitute ca. 35% of the overall reef fish biomass on coral reefs. These benthic fish exploit restricted habitats where food and shelter are obtained in, or in relation to, conditions of substrate complexity and/or restricted living space. A range of mechanisms has been proposed to account for the diversity and the abundance of small fishes: (1) lifehistory strategies that promote short generation times, (2) habitat associations and behaviour that reduce predation and (3) resource partitioning that allows small species to coexist with larger competitors. Despite their abundance and potential importance within reef systems, little is known of the community ecology of cryptic fishes. Specifically on habitat associations many theories suggested a not clear direction on this subject. My research contributes to the development of marine fish ecology by addressing the effects of habitat characteristics upon distribution of cryptobenthic fish assemblages. My focus was on the important shallow, coastal ecosystems that often serve as nursery habitat for many fish and where different type of habitat is likely to both play important roles in organism distribution and survival. My research included three related studies: (1) identification of structuring forces on cryptic fish assemblages, such as physical and biological forcing; (2) macroalgae as potential tools for cryptic fish and identification of different habitat feature that could explain cryptic fish assemblages distribution; (3) canopy formers loss: consequences on cryptic fish and relationship with benthos modifications. I found that: (1) cryptic fish assemblages differ between landward and seaward sides of coastal breakwaters in Adriatic Sea. These differences are explained by 50% of the habitat characteristics on two sides, mainly due to presence of the Codium fragile, sand and oyster assemblages. Microhabitat structure influence cryptic fish assemblages. (2) Different habitat support different cryptic fish assemblages. High heterogeneity on benthic assemblages reflect different fish assemblages. Biogenic components that explain different and diverse cryptic fish assemblages are: anemonia bed, mussel bed, macroalgal stands and Cystoseira barbata, as canopy formers. (3) Canopy forming loss is not relevant in structuring directly cryptic fish assemblages. A removal of canopy forming algae did not affect the structure of cryptic fish assemblages. Canopy formers algae on Conero cliff, does not seem to act as structuring force, probably due to its regressive status. In conclusion, cryptic fish have been shown to have species-specific associations with habitat features relating to the biological and non biological components afforded by fish. Canopy formers algae do not explain cryptic fish assemblages distribution and the results of this study and information from the literature (both from the Mediterranean Sea and elsewhere) show that there are no univocal responses of fish assemblages. Further exanimations on an non regressive status of Cystoseira canopy habitat are needed to define and evaluate the relationship between canopy formers and fish on Mediterranean sea.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Determinants of specific food consumption in the Canary Islands (Spain)

The consumption of specific functional foods (FF) and some determinants of FF item selection were assessed using a questionnaire administered to 1112 individuals in the Canary Islands (Spain). Food items considered were Milk products: easily digestible milk (or milk low in lactose), milk enriched with vitamins and/or minerals, skimmed milk with soluble fiber, milk with royal jelly, milk with modified fatty acids (omega 3), milk products low in fat, pro-biotic foods (yoghurt and fermented milk) and yoghurt with phytosterols; Cereals: fortified breakfast cereals, wholemeal cereals and energy bars; Drinks: juices and enriched drinks, stimulating drinks and isotonic drinks; DHA-enriched, low cholesterol eggs; Meat products: low salt sausages and cooked low fat ham; Fats: enriched margarine, margarine rich in phytosterols and