Electrospun fibrous mats for skin and abdominal wall repair

Esta tese centra-se no desenvolvimento de materiais biodegradáveis e nãodegradáveis produzidos por eletrofiação com aplicação na área biomédica. O poli(3-hidroxibutirato-co-3-hidroxivalerato) (PHBV), um poliéster biodegradável, foi selecionado como base dos materiais biodegradáveis, enquanto o poli(tereftalato de etileno) (PET), um polímero sintético, estável e biocompatível, foi selecionado para a produção das matrizes não degradáveis. Adicionou-se quitosana aos sistemas com o objetivo de melhorar o processo de eletrofiação e as propriedades morfológicas, físico-químicas e biológicas dos materiais resultantes. A composição química, bem como as características morfológicas e físicoquímicas dos materiais em estudo, foram manipuladas de modo a otimizar a sua performance como suportes celulares para engenharia de tecidos. Foram realizados estudos in vitro com cultura de fibroblastos L929 para avaliar o comportamento das células, i.e. viabilidade, adesão, proliferação e morte, quando cultivadas nas matrizes produzidas por eletrofiação. Adicionalmente foram realizados ensaios in vivo para investigar o potencial dos materiais em estudo na regeneração cutânea e como tela abdominal. Os principais resultados encontrados incluem: o desenvolvimento de novas matrizes híbridas (PHBV/quitosana) adequadas ao crescimento de fibroblastos e ao tratamento de lesões de pele; o desenvolvimento de um sistema de eletrofiação com duas seringas para a incorporação de compostos bioativos; diversas estratégias para manipulação das características morfológicas dos materiais de PHBV/quitosana e PET/quitosana produzidos por eletrofiação; uma melhoria do conhecimento das interações fibroblastos-suporte polimérico; a verificação de uma resposta inflamatória desencadeada pelos materiais nãodegradáveis quando utilizados no tratamento de defeitos da parede abdominal, o que sugere a necessidade de novos estudos para avaliar a segurança do uso de biomateriais produzidos por eletrofiação.

Phylogenetic and functional diversity of estuarine bacterioneuston

A micro-camada superficial da água (SML) é caracterizada pela ocorrência de grandes quantidades de compostos orgânicos, pela acumulação de contaminantes antropogênicos e é submetida a uma intensa radiação solar, extrema mudança de temperatura e, no caso dos estuários, flutuação de salinidade. Estas propriedades físico-químicas estão, provavelmente, a modular a comunidade bacteriana (bacterioneuston) com propriedades filogenéticas e funcionais específicas. Neste estudo, as abordagens dependentes e independentes do cultivo foram aplicadas para avaliar a estrutura e dinâmica das comunidades bacterioneuston e bacterioplâncton em três localizações geográficas ao longo do estuário da Ria de Aveiro. Além disso, comparámos a diversidade filogenética de grupos específicos (Aeromonas, Pseudomonas e Psychrobacter) presentes em bacterioneuston e bacterioplâncton. Finalmente, as duas comunidades foram comparadas em termos de prevalência e diversidade de bactérias resistentes aos antibióticos e respetivos genes de resistência. Bactérias heterotróficas cultiváveis foram enriquecidas em SML. Eletroforese em gel de gradiente desnaturante (DGGE) permitiu a identificação de filotipos específicos em SML. Além disso, a análise de agrupamento dos perfis de DGGE de ambas as comunidades revelou uma ligeira tendência de agrupamento de acordo com a camada amostrada. As diferenças entre as duas comunidades variaram de acordo com factores espaciais e temporais. Em termos de diversidade filogenética de grupos específicos, não foram identificadas diferenças consistentes entre SML e UW com relação às comunidades de Aeromonas. Com relação ao género Pseudomonas, uma unidade operacional taxonómica cultivável foi consistentemente hiper-representada nas amostras de SML. Metodologias dependentes e independentes do cultivo revelaram a presença de populações de Psychrobacter complexas e muito estáveis em todos os sítios e datas de amostragens, com diferenças significativas entre as comunidades de Psychrobacter presentes em SML e UW. Estirpes representativas de prováveis novas espécies também foram cultivadas. Em termos de resistência aos antibióticos, a prevalência de bactérias resistentes em SML foi alta sugerindo selecção pelas condições presentes em SML. É preciso enfatizar que a resistência aos antibióticos foi incomum entre as bactérias estuarinas e os mecanismos de resistência foram, predominantemente, intrínsecos. Pela combinação de abordagens inovadoras dependentes e independentes do cultivo, este estudo forneceu novas e consistentes informações com relação às diferenças em ambas as comunidades bacterianas e em relação a alguns dos fatores que contribuem para a sua formação.

Sustainable viticulture in Bairrada appellation: vineyard and harvest year effects on grapes oenological potential

The sustainable viticulture of a region passes, among other aspects, for maximizing the varieties potential minimizing subsequent interventions during winemaking, which should contribute to the production of quality wines maintaining their typicity and rationalizing costs. The detailed knowledge of each Appellation specificities, namely vineyard parcel (soil type and topographical peculiarities) and harvest climatic conditions is crucial for sustainability in this sector. Thus, in line with this current trend, the starting point for the development of this PhD thesis was to evaluate the oenological potential of different varieties cultivated throughout Bairrada Appellation (Portugal). During maturation several changes in grape varieties occur, namely berries become sweeter, less acidic, and they develop flavour, aroma and colour properties. The development of these characteristics is essential to define grapes oenological potential, i.e. to estimate the possibility of their usage to produce specific wines. A three years sampling plan was designed to evaluate the effect of harvest year and parcel characteristics on V. vinifera cv. Arinto, Bical, Sauvignon Blanc, Baga, Castelão, Touriga Nacional, and Sousão grapes composition. For each variety, 3 parcels with different characteristics were selected. Several physicochemical parameters were evaluated, during maturation: berry weight, pH, titratable acidity, sugar and phenolic contents, antiradical activity, and volatile composition (free fraction). Special attention was devoted to grapes at technologic maturity, since, besides these parameters, glycosidically-linked fraction was also considered. By using the results obtained at technologic maturity, a comprehensive approach was applied to identify the significance of harvest and parcel characteristics effects on each variety composition. Considering all the parameters under study, it may be highlighted some significant differences. According to the obtained results determined during maturation, it was possible to conclude that Arinto, Bical and Sauvignon Blanc grapes from parcels with clay-sandy and clay-calcareous soils have higher phenolic content and antiradical activity. Otherwise, Sauvignon Blanc presented similar volatile composition for grapes cultivated in the 3 parcels, while Arinto and Bical exhibited higher volatile content in grapes from claysandy and clay-calcareous soils. For Baga, Castelão and Touriga Nacional red varieties, grapes with higher phenolic content, antiradical activity, and volatile content were obtained from clayey and clay-calcareous soils. Furthermore, for Touriga Nacional, parcels altitude seems also to modulate grapes composition. Beyond parcel effect, harvest year conditions also influence grapes composition: 2011 harvest was related with lower phenolic and volatile contents, as well as lower antiradical activity.For grapes collected at technologic maturity, analysis of variance-simultaneous component analysis (ASCA) was applied combining all the parameters under study, in order to assess the influence of harvest and parcel characteristics on each variety oenological potential. The results obtained using this comprehensive approach is closely related with those observed during maturation and revealed that harvest was the main factor that influenced grapes composition (53% to 68% of the total data set variance) followed by parcel characteristics, explaining ca. 15-19% of the total data set variance. The oenological potential of each variety may be different from one parcel to another, i.e., clay-sandy and clay-calcareous related-environments seem to favour Arinto and Bical white grapes composition, but for the red varieties, grapes composition was favoured by clayey and clay-calcareous soils. Besides, also higher altitude seems to favour Touriga Nacional grapes composition. Sauvignon Blanc seems to be a variety well adapted to the different parcel characteristics. In order to go forward in the valuation of these varieties, the aroma properties of 6 monovarietal wines were studied based on an aroma network-approach, linking molecular data related to volatile composition and aroma data about the key odor active molecules. This approach allowed to identify different wine aroma properties and to infer about the consumer’s sensory perception. It was found that aroma properties differ from one wine variety to another: while Arinto and Sauvignon Blanc wine exhibited higher tree fruity, sweety and flowery aromas, related essentially with ester compounds and C13 norisoprenoids, the opposite was obtained for Bical wine, corroborating the aroma sensory perceptions of the trained panel. Sauvignon Blanc also exhibited higher toasted aromas (related with thiols, mainly with 2-methyl-3-furanthiol). Touriga Nacional red wine exhibited higher tree, tropical, and berry fruits notes (sensory described as sweet fruits), toasted and flowery aromas, while these are similar for the other red wines under study. Besides Portuguese Bairrada wines, this aroma network approach is a tool that can be used to explain the aroma properties of wines worldwide. The grape and wine data generated under the present PhD thesis, in the context of Bairrada Appellation, shows the unique character of each variety, and may be used by growers and wine producers as a support for decisionmaking based on objective criteria, increasing the sustainability in this sector. For instance, it is possible to take advantage of the natural resources and produce products with different characteristics obtained from the same variety, minimizing costs during the winemaking process.

Lipidomic analysis of mesenchymal cells candidates for cell therapy

Mesenchymal stromal cells are adult stem cells found mostly in the bone marrow. They have immunosuppressive properties and they have been successfully applied as biological therapy in several clinical trials regarding autoimmune diseases. Despite the great number of clinical trials, MSCs’ action is not fully understand and there are no identified markers that correlate themselves with the immunomodulatory power. A lipidomic approach can solve some of these problems once lipids are one of the major cells’ components. Therefore, in this study cells’ lipidome was analysed and its deviations were evaluated according to the medium of culture and to the presence of pro-inflammatory stimuli, mimicking physiological conditions in which these cells are used. This was the first study ever made that aimed to analyse the differences in the phospholipid profile between mesenchymal stromal cells non-stimulated and stimulated with proinflammatory stimulus. This analysis was conducted in both cells cultured in medium supplemented with animal serum and in cells cultured in a synthetic medium. In cells cultured in the standard medium the levels of phosphatidylcholine (PC) species with shorter fatty acids (FAs) acyl chains decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of lysoPC (LPC)(18:0) - an anti-inflammatory LPC - observed in cells subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN- γ also enhanced the levels of phosphatidylethanolamine PE(40:6) and decreased the levels of PE(38:6). Higher expression of phosphatidylserine PS(36:1) and sphingomyelin SM(34:0) along with a decrease in PS(38:6) levels were observed. However, in cells cultured in a synthetic medium, TNF-α and IFN-γ only enhanced the levels of PS(36:1). These results indicate that lipid metabolism and signaling is modulated during mesenchymal stromal cells action.