Compósitos CNTs/biocerâmico para a estimulação elétrica óssea in situ

The present thesis aims to develop a biocompatible and electroconductor bone graft containing carbon nanotubes (CNTs) that allows the in situ regeneration of bone cells by applying pulsed external electrical stimuli. The CNTs were produced by chemical vapor deposition (CVD) by a semi-continuous method with a yield of ~500 mg/day. The deposition parameters were optimised to obtain high pure CNTs ~99.96% with controlled morphologies, fundamental requisites for the biomedical application under study. The chemical functionalisation of CNTs was also optimised to maximise their processability and biocompatibility. The CNTs were functionalised by the Diels-Alder cycloaddition of 1,3-butadiene. The biological behaviour of the functionalised CNTs was evaluated in vitro with the osteoblastic cells line MG63 and in vivo, by subcutaneous implantation in rats. The materials did not induce an expressed inflammatory response, but the functionalised CNTs showed a superior in vitro and in vivo biocompatibility than the non-functionalised ones. Composites of ceramic matrix, of bioglass (Glass) and hydroxyapatite (HA), reinforced with carbon nanotubes (CNT/Glass/HA) were processed by a wet approach. The incorporation of just 4.4 vol% of CNTs allowed the increase of 10 orders of magnitude of the electrical conductivity of the matrix. In vitro studies with MG63 cells show that the CNT/Glass/HA composites guarantee the adhesion and proliferation of bone cells, and stimulate their phenotype expression, namely the alkaline phosphate (ALP). The interactions between the composite materials and the culture medium (α-MEM), under an applied electrical external field, were studied by scanning vibrating electrode technique. An increase of the culture medium electrical conductivity and the electrical field confinement in the presence of the conductive samples submerged in the medium was demonstrated. The in vitro electrical stimulation of MG63 cells on the conductive composites promotes the increase of the cell metabolic activity and DNA content by 130% and 60%, relatively to the non-stimulated condition, after only 3 days of daily stimulation of 15 μA for 15 min. Moreover, the osteoblastic gene expression for Runx2, osteocalcin (OC) and ALP was enhanced by 80%, 50% and 25%, after 5 days of stimulation. Instead, for dielectric materials, the stimulus delivering was less efficient, giving an equal or lower cellular response than the non-stimulated condition. The proposed electroconductive bone grafts offer exciting possibilities in bone regeneration strategies by delivering in situ electrical stimulus to cells and consequent control of the new bone tissue formation rate. It is expected that conductive smart biomaterials might turn the selective bone electrotherapy of clinical relevance by decreasing the postoperative healing times.

A educação intercultural na formação contínua de professores de línguas

Situado entre o discurso investigativo e o profissional da Didática de Línguas, o presente estudo assenta: num entendimento da educação em línguas como um processo valorizador da diversidade linguística e cultural, tendo como fim último a promoção da intercompreensão e do diálogo intercultural, dentro dos pressupostos de uma didática das línguas e do plurilinguismo; na conceção do professor de línguas como um dos principais atores na educação de cidadãos / comunicadores interculturais, vendo-se, portanto, a braços com novas exigências, para as quais, muitas vezes, não se sente preparado; e nos pressupostos de que a identidade profissional condiciona fortemente a forma como o professor desempenha a sua ação didática, sendo este processo de se tornar professor contínuo e dependente, quer do sujeito-professor e dos seus percursos profissionais e formativos, quer do contexto (profissional, local, nacional, global) em que este se insere. Pretende-se, com este estudo, contribuir para que a educação intercultural seja uma realidade nas nossas escolas, potenciando a sua migração contextualizada dos documentos orientadores das políticas linguísticas e educativas nacionais e transnacionais e dos discursos da investigação em Didática de Línguas. Para o efeito, desenvolvemos um programa de investigação/formação denominado O Professor Intercultural, durante o ano letivo 2006/2007, com professores de línguas (materna e estrangeiras) de três escolas básicas e secundárias do distrito de Aveiro. Este programa integrava um curso (25 horas) e uma oficina (50 horas), ambas as ações de formação acreditadas pelo Conselho Científico-Pedagógico da Formação Contínua. Do ponto de vista formativo, com este programa pretendíamos levar as professoras em formação a desenvolver competências pessoais e profissionais que lhes permitissem gerir a diversidade nos seus contextos profissionais, tendo em vista o desenvolvimento nos seus alunos de uma competência de comunicação intercultural (CCI). Do ponto de vista investigativo, não só pretendíamos compreender as representações dos sujeitos relativamente à educação intercultural em geral e à CCI em particular; como também identificar princípios e estratégias de formação potenciadores do desenvolvimento de competências profissionais docentes para trabalhar a CCI, a partir das perspetivas dos próprios sujeitos. Trata-se, portanto, de um estudo de caso de cariz qualitativo e interpretativo / fenomenológico, com potencialidades heurísticas, que pretende evidenciar os sujeitos, as suas representações, as interações consigo e com os outros e a forma como conceptualizam a identidade profissional docente e as suas dinâmicas de desenvolvimento. Como instrumentos de recolha de dados, privilegiámos os Portefólios Profissionais que foram sendo construídos ao longo do percurso de formação; a sessão “Entre Línguas e Culturas” da plataforma Galanet (www.galanet.eu), recurso de formação no âmbito da oficina (entre fevereiro e maio de 2007); o “Diário do Investigador”; e as “entrevistas narrativas e de confrontação” efetuadas sensivelmente um ano após o final do programa de investigação/formação. Os resultados da análise de conteúdo revelam que os sujeitos consideram a CCI uma competência multidimensional e complexa, reconhecendo três componentes: afetiva (domínio do saber ser e saber viver com o outro), cognitiva (domínio do saber) e praxeológica (domínio do saber-fazer). A componente afetiva constitui, de acordo com os resultados, o motor de arranque do desenvolvimento desta competência, que, posteriormente, é alargada em dinâmicas de informação-(inter)ação-reflexão. Por outro lado, dada a grande pertinência que atribuem à abordagem intercultural e à urgência com que veem a sua integração escolar, os sujeitos consideram a CCI uma das competências inerentes à competência profissional docente, elemento integrador da identidade profissional, numa forte ligação com a missão ética e política que cada vez mais é associada ao docente (de línguas). Para além disso, percecionam o seu desenvolvimento profissional docente como um processo que os acompanha ao longo da vida, fruto das idiossincrasias e predisposições do próprio indivíduo, mas também das dinâmicas da sua formação, das caraterísticas dos contextos em que se movimenta e da colaboração com o Outro, no seu espaço profissional ou fora dele. Importa salientar que este desenvolvimento profissional é potenciado, segundo os nossos resultados, por propostas de formação assentes numa abordagem acional e reflexiva, articulando dinâmicas investigação-ação-reflexão como as propostas no nosso programa de formação, nomeadamente no âmbito da oficina. Neste quadro, concluímos o presente estudo, indicando alguns caminhos possíveis para a formação de professores de línguas para a educação intercultural.

Phthalocyanines : interaction with carbon structures and as PDT agents

This dissertation describes the synthesis and characterization of different phthalocyanine (Pc) derivatives, as well as some porphyrins (Pors), for supramolecular interaction with different carbon nanostructures, to evaluate their potential application in electronic nanodevices. Likewise, it is also reported the preparation and biological evaluation of interesting phthalocyanine conjugates for cancer photodynamic therapy (PDT) and microorganisms photodynamic inactivation (PDI). The phthalonitrile precursors were prepared from commercial phthalonitriles by nucleophilic substitution of -NO2, -Cl, or -F groups, present in the phthalonitrile core, by thiol or pyridyl units. After the synthesis of these phthalonitriles, the corresponding Pcs were prepared by ciclotetramerization using a metallic salt as template at high temperatures. A second strategy involved the postfunctionalization of hexadecafluorophthalocyaninato zinc(II) through the adequate substituents of mercaptopyridine or cyclodextrin units on the macrocycle periphery. The different compounds were structurally characterized by diverse spectroscopic techniques, namely 1H, 13C and 19F nuclear magnetic resonance spectroscopies (attending the elemental composition of each structure); absorption and emission spectroscopy, and mass spectrometry. For the specific photophysical studies were also used electrochemical characterization, femtosecond and raman spectroscopy, transmission electron and atomic force microscopy. It was highlighted the noncovalent derivatisation of carbon nanostructures, mainly single wall carbon nanotubes (SWNT) and graphene nanosheets with the prepared Pc conjugates to study the photophysical properties of these supramolecular nanoassemblies. Also, from pyridyl-Pors and ruthenium phthalocyanines (RuPcs) were performed Por-RuPcs arrays via coordination chemistry. The results obtained of the novel supramolecular assemblies showed interesting electron donor-acceptor interactions and might be considered attractive candidates for nanotechnological devices. On the other hand, the amphiphilic phthalocyanine-cyclodextrin (Pc-CD) conjugates were tested in biological trials to assess their ability to inhibit UMUC- 3 human bladder cancer cells. The results obtained demonstrated that these photoactive conjugates are highly phototoxic against human bladder cancer cells and could be applied as promising PDT drugs.

Comunidades de prática online e a construção de competências para o ensino ativo

O presente estudo foi realizado no âmbito da formação continuada de professores através das interações numa Comunidade de Prática online (CoP online). Nas CoPs ocorrem a partiha de dúvidas e problemas comuns ao trabalho dos docentes. Quando as interações são mediadas por computadores, sejam estas síncronas ou assíncronas, ultrapassam as barreiras de espaço e tempo, se adequando ao cotidiano dos professores, invariavelmente tomado por múltiplas atividades. Nesta investigação delineamos os seguintes objetivos: i) Construir uma CoP online ancorada no software social Facebook, como espaço virtual de interação entre os participantes; ii) Comprender as interações através duma CoP online como etapa na construção de competências; iii) Criar e desenvolver um programa de formações presenciais e online com vistas à desenvover as competências de Questionamento e Argumentação; e, iv) Avaliar os impactos da formação através da CoP online no desenvolvimento das competências do Questionamento e da Argumentação enquanto estratégia de promoção do ensino ativo. Como estratégia, optamos em promover as interações através do Facebook por não ocasionar custos aos participantes e por atender ao modelo de presença virtual já existente. Participaram das interações professores em exercício da Autarquia do Ensino Superior de Garanhuns (AESGA), situada no interior de Pernambuco, Brasil. As interações decorreram no período de Abril de 2012 até Março de 2013, numa comunidade fechada, de livre adesão através de convite. Para solidificar a aprendizagem através da CoP online promovemos dois ciclos de formação, um presencial e um online. Em ambas as intervenções visamos o desenvolvimento das competências do Questionamento e da Argumentação por entendermos que estas são competências instrumentais para o desenvolvimento do Pensamento Crítico e da reflexão essenciais ao docente na revisão constante de suas práticas. Nas sessões online, realizadas com o apoio do Skype e concretizadas no software ArguQuest para dinamizar atividades práticas no desenvolvimento das competências do Questionamento e da Argumentação do professor, oportunizando a este a construção coletiva de estratégias alternativas de ensino e de aprendizagem, fundamentadas no ensino ativo. O estudo foi realizado através dum estudo de caso único, alicerçado no paradigma naturalista e de abordagem qualitativa. Os dados de cariz numérico fundamentaram as análises qualitativas, promovendo também a triangulação dos dados. As interações entre os professores foram analisadas através do modelo IAM (Interaction Analysis Model) proposto por Gunawardena, Lowe e Anderson (1997), que tem como objetivo determinar se o conhecimento foi cosntruído através das interações e qual o grau de mudança do entendimento decorrente das interações. Para verificar esta construção do conhecimento analisamos os excertos de episódios interativos com apoio do software de análise qualitativa WebQDA (Neri de Souza, Costa & Neri de Souza, 2012). A análise das situações de aprendizagem planejadas e desenvolvidas demonstraram que as TIC, nomeadamente os softwares sociais, podem ser aplicados com eficiência no desenvolvimento de competências dos professores em exercício, apresentando-se como alternativa para a promoção da formação continuada de docentes. Em relação ao desenvolvimento das competências do Questionamento e da Argumentação, percebemos que os docentes foram sensibilizados para trabalhar com uma metodologia interativa, promovendo um ambiente favorável à aprendizagem ativa. Através da aplicação adaptada do modelo de construção de conhecimento preconizado no ArguQuest, foi possível acompanhar a prática do docente junto aos seus alunos, ampliando o seu repertório didático, e, sobretudo, implantando uma forma de aprender fundamentada na construção compartilhada de Questionamentos e Argumentos.

Interação intercultural telecolaborativa em português língua não materna

No âmbito do tema Multimédia em Educação, optámos por analisar uma sequência pedagógica em Português Língua Não Materna, contextualizada em interações interculturais telecolaborativas, mediadas pela plataforma Adobe Connect Pro. Para o efeito, foram transcritas as gravações dessas interações, submetidas a análise de conteúdo com o software de abordagem qualitativa webQDA, tendo em conta as seguintes dimensões: (i) dimensão operacional das literacias digitais online; (ii) atitude perante as tecnologias online; (iii) negociação de sentido, (iii) enunciados não relacionados com o projeto; (iv) atividades reflexivas sobre a língua por parte dos aprendentes; (v) enunciados dos participantes falantes nativos em interação com os aprendentes. Após a análise e discussão de resultados, é possível afirmar que a realização de interações interculturais telecolaborativas contribui para a reformulação de conhecimentos (Lankshear e Knobel, 2006) em termos de: competência tecnológica e literacia online; perceção de como as limitações técnicas podem condicionar a realização de um projeto; tomada de consciência do desenvolvimento (e vantagens) de uma atitude 2.0; aprofundamento da competência na língua-alvo e do conhecimento de realidades culturais através da comunicação com falantes nativos. Ainda assim, reconhecemos a pertinência e urgência do desenvolvimento de um trabalho com acuidade na área da formação intercultural telecolaborativa quer no âmbito da formação de professores (inicial e contínua) quer com os alunos durante as aulas de língua.

Interactions of cetaceans with Spanish and Portuguese fisheries in Atlantic waters : costs, benefits and implications for management

With the aim to provide new insights into operational cetacean-fishery interactions in Atlantic waters, this thesis assesses interactions of cetaceans with Spanish and Portuguese fishing vessels operating in Iberian and South West Atlantic waters. Different opportunistic research methodologies were applied, including an interview survey with fishers (mainly skippers) and onboard observations by fisheries observers and skippers, to describe different types of interactions and to identify potential hotspots for cetacean-fishery interactions and the cetacean species most involved, and to quantify the extent and the consequences of these interactions in terms of benefits and costs for cetaceans and fisheries. In addition, the suitability of different mitigation strategies was evaluated and discussed. The results of this work indicate that cetaceans interact frequently with Spanish and Portuguese fishing vessels, sometimes in a beneficial way (e.g. cetaceans indicate fish schools in purse seine fisheries), but mostly with negative consequences (depredation on catch, gear damage and cetacean bycatch). Significant economic loss and high bycatch rates are, however, only reported for certain fisheries and associated with particular cetacean species. In Galician fisheries, substantial economic loss was reported as a result of bottlenose dolphins damaging artisanal coastal gillnets, while high catch loss may arise from common dolphins scattering fish in purse seine fisheries. High cetacean bycatch mortality arises in trawl fisheries, mainly of common dolphin and particularly during trawling in water depths below 350 m, and in coastal set gillnet fisheries (mainly common and bottlenose dolphins). In large-scale bottom-set longline fisheries in South West Atlantic waters, sperm whales may significantly reduce catch rates through depredation on catch. The high diversity of cetacean-fishery interactions observed in the study area indicates that case-specific management strategies are needed to reduce negative impacts on fisheries and cetaceans. Acoustic deterrent devices (pingers) may be used to prevent small cetaceans from approaching and getting entangled in purse seines and set gillnets, although possible problems include cetacean habituation to the pinger sounds, as well as negative side effects on non-target cetaceans (habitat exclusion) and fisheries target species (reduced catch rates). For sardine and horse mackerel, target species of Iberian Atlantic fisheries, no aversive reaction to pinger sounds was detected during tank experiments conducted in the scope of this thesis. Bycatch in trawls may be reduced by the implementation of time/area restrictions of fishing activity. In addition, the avoidance of fishing areas with high cetacean abundance combined with the minimization of fishery-specific sound cues that possibly attract cetaceans, may also help to decrease interactions. In large-scale bottom-set longline fisheries, cetacean depredation on catch may be reduced by covering hooked fish with net sleeves ("umbrellas") provided that catch rates are not negatively affected by this gear modification. Trap fishing, as an alternative fishing method to bottom-set gillnetting and longlining, also has the potential to reduce cetacean bycatch and depredation, given that fish catch rates are similar to the rates obtained by bottom-set gillnets and longlines, whereas cetacean by-catch is unlikely. Economic incentives, such as the eco-certification of dolphin-safe fishing methods, should be promoted in order to create an additional source of income for fishers negatively affected by interactions with cetaceans, which, in turn, may also increase fishers’ willingness to accept and adopt mitigation measures. Although the opportunistic sampling methods applied in this work have certain restrictions concerning their reliability and precision, the results are consistent with previous studies in the same area. Moreover, they allow for the active participation of fishers that can provide important complementary ecological and technical knowledge required for cetacean management and conservation.

As comunidades virtuais no Facebook no setor do vinho: estudo de caso

A Internet tem conseguido mudar os limites das interações sociais e económicas, por fornecer uma infraestrutura robusta para comunicação sem constrangimento de distâncias no tempo e no espaço, que pode ser usada em diversas formas para acumulação e arquivo de recursos de conhecimento. Esta revolução da Internet levou a uma difusão sem precedentes de comunidades virtuais em que os consumidores participam e expressam as suas opiniões sobre os produtos e serviços que consomem ou pretendem consumir. O presente trabalho pretende explorar a temática das comunidades virtuais como fator de inovação, tendo como base o estudo de caso de uma comunidade virtual ligada ao setor do vinho, procurando compreender até que ponto as comunidades virtuais podem contribuir para a evolução dos produtos ou mesmo para a criação de novos produtos. O trabalho inicia-se com a revisão da literatura em torno do conhecimento, internet, comunidades virtuais, lead users e o processo de inovação, crowdsourcing e a cocriação no processo de desenvolvimento de novos produtos. Conclui-se que as empresas podem construir estratégias que permitam uma canalização da informação proporcionada pelos consumidores e Lead Users, procurando, por exemplo, utilização de ferramentas de crowdsourcing. Segue-se a aplicação de uma estratégia de investigação qualitativa, o estudo de caso, à página no Facebook Vinhos do Alentejo, em que se analisa uma comunidade de consumidores de vinhos do Alentejo que proporciona ideias de melhoria do produto ou da experiência de consumo. A recolha dos dados dessa página e a análise de conteúdo foram apoiadas pelo NVivo 10. Como principais resultados destacam-se a utilização desta página como ferramenta de marketing, na divulgação dos vinhos, da gastronomia, da região do Alentejo e ainda a obtenção de experiências preferidas de consumo dos vinhos.

A intencionalidade educativa do educador de infância num contexto de pedagogia-em-participação: avaliação e qualidade da intervenção educativa na educação de infância

A presente investigação enquadra-se na avaliação e qualidade da intervenção educativa na Educação de Infância, assumindo neste estudo um relevante enfoque as questões da intencionalidade educativa. Tendo como suporte um estudo de caso qualitativo, realizado num contexto de Jardim de Infância que desenvolve a Pedagogia-em-Participação enquanto perspetiva educacional sustentadora da intervenção educativa, procurou-se compreender quais eram as intencionalidades que os educadores de infância detinham e de que forma é que estas se manifestavam, a três níveis: i) o que o profissional pensava relativamente à sua intervenção educativa; ii) como é que organizava a sua intervenção e como intervinha no contexto onde estava inserido; iii) o que procurava observar, registar e documentar, enquanto forma de dar visibilidade às aprendizagens das crianças e às suas próprias intencionalidades. Enquanto forma de atribuição de uma sustentação teórica a esta investigação e enquanto estratégia de definição dos procedimentos do traçado investigativo, procedeu-se ao estudo do pensamento de alguns pedagogos e à sua relação com a questão da intencionalidade educativa. Realizou-se, equitativamente, um aprofundamento de compreensões em torno de modelos pedagógicos de cariz participativo. Procedeu-se à análise e compreensão teórica e empírica do objeto de estudo, tendo subjacente dimensões da pedagogia da infância referenciadas no quadro teórico desta investigação, a saber: tempo pedagógico, espaços e materiais pedagógicos, interações, atividades e projetos, observação, planificação e registo, avaliação e documentação pedagógica. A fase de investigação empírica desenvolveu-se entre setembro de 2010 e julho de 2012 e considerou quatro subfases: subfase 0 – Negociação do estudo e acesso ao campo; subfase I – Familiarização com o contexto; subfase II – Observação sistemática e registo de notas de campo; subfase III – Análise dos portefólios de aprendizagem das crianças; subfase IV – Inquérito por entrevista aos educadores de infância.Os resultados do estudo apontam para a expressão da intencionalidade a diversos níveis. A intencionalidade evidenciou-se tanto nas ações e pensamentos dos intervenientes no processo educativo, como no ambiente educativo e na documentação pedagógica. Tanto as crianças como os educadores foram protagonistas da ação e participaram na definição, concretização e avaliação de intencionalidades. Apesar de emergirem de sujeitos diversos e em circunstâncias diferentes do quotidiano educativo, as intencionalidades dos sujeitos harmonizaram-se com os propósitos pedagógicos delineados pela Pedagogia-em-Participação para os tempos da rotina educativa, que tanto as crianças como os educadores conheciam. Os resultados do estudo clarificam que, para se identificar e interpretar a intencionalidade educativa do educador de infância, há necessidade de se atentar para a organização do ambiente educativo (espaços e materiais), para a sua dinâmica (tempo pedagógico) e para os propósitos subjacentes a esta mesma dinâmica. Para além disso, o ambiente educativo materializa e facilita a emergência das intencionalidades tanto do educador como das crianças. Os resultados do estudo revelam que a documentação assume-se um espelho de evidências dos propósitos dos sujeitos: espelho da forma como pensam, observam, sentem, valorizam e representam a realidade experienciada. Uma visão integradora e holística em torno dos resultados permite, por fim, evidenciar a necessidade de se proceder a mais estudos no âmbito desta temática, nomeadamente, no domínio das pedagogias participativas.

Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules

The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.

Single and joint effects of natural and chemical stressors to the terrestrial isopod Porcellionides pruinosus

Environmental contamination and climate changes constitute two of the most serious problems affecting soil ecosystems in agricultural fields. Agriculture is nowadays a highly optimized process that strongly relies on the application of multiple pesticides to reduce losses and increase yield production. Although constituting, per se, a serious problem to soil biota, pesticide mixtures can assume an even higher relevance in a context of unfavourable environmental conditions. Surprisingly, frameworks currently established for environmental risk assessments keep not considering environmental stressors, such as temperature, soil moisture or UV radiation, as factors liable to influence the susceptibility of organisms to pesticides, or pesticide mixtures, which is raising increasing apprehension regarding their adequacy to actually estimate the risks posed by these compounds to the environment. Albeit the higher attention received on the last few years, the influence of environmental stressors on the behaviour and toxicity of chemical mixtures remains still poorly understood. Aiming to contribute for this discussion, the main goal of the present thesis was to evaluate the single and joint effects of natural stressors and pesticides to the terrestrial isopod Porcellionides pruinosus. The first approach consisted on evaluating the effects of several abiotic factors (temperature, soil moisture and UV radiation) on the performance of P. pruinosus using several endpoints: survival, feeding parameters, locomotor activity and avoidance behaviour. Results showed that these stressors might indeed affect P. pruinosus at relevant environmental conditions, thus suggesting the relevance of their consideration in ecotoxicological assays. At next, a multiple biomarker approach was used to have a closer insight into the pathways of damage of UV radiation and a broad spectrum of processes showed to be involved (i.e. oxidative stress, neurotoxicity, energy). Furthermore, UV effects showed to vary with the environment medium and growth-stage. A similar biomarker approach was employed to assess the single and joint effects of the pesticides chlorpyrifos and mancozeb to P. pruinosus. Energy-related biomarkers showed to be the most differentiating parameters since age-classes seemed to respond differently to contamination stress and to have different metabolic costs associated. Finally, the influence of temperature and soil moisture on the toxicity of pesticide mixtures was evaluated using survival and feeding parameters as endpoints. Pesticide-induced mortality was found to be oppositely affected by temperature, either in single or mixture treatments. Whereas chlorpyrifos acute toxicity was raised under higher temperatures the toxicity of mancozeb was more prominent at lower temperatures. By the opposite, soil moisture showed no effects on the pesticide-induced mortality of isopods. Contrary to survival, both temperature and soil moisture showed to interact with pesticides to influence isopods’ feeding parameters. Nonetheless, was however the most common pattern. In brief, findings reported on this thesis demonstrated why the negligence of natural stressors, or multiple stressors in general, is not a good solution for risk assessment frameworks.

Microbe-mediated recovery of salt marshes contaminated with oil hydrocarbons

Salt marshes are highly productive intertidal habitats that serve as nursery grounds for many commercially and economically important species. Because of their location and physical and biological characteristics, salt marshes are considered to be particularly vulnerable to anthropogenic inputs of oil hydrocarbons. Sediment contamination with oil is especially dangerous for salt marsh vegetation, since low molecular weight aromatic hydrocarbons can affect plants at all stages of development. However, the use of vegetation for bioremediation (phytoremediation), by removal or sequestration of contaminants, has been intensively studied. Phytoremediation is an efficient, inexpensive and environmental friendly approach for the removal of aromatic hydrocarbons, through direct incorporation by the plant and by the intervention of degrading microbial populations in the rhizosphere (microbe-assisted phytoremediation). Rhizosphere microbial communities are enriched in important catabolic genotypes for degradation of oil hydrocarbons (OH) which may have a potential for detoxification of the sediment surrounding the roots. In addition, since rhizosphere bacterial populations may also internalize into plant tissues (endophytes), rhizocompetent AH degrading populations may be important for in planta AH degradation and detoxification. The present study involved field work and microcosms experiments aiming the characterization of relevant plant-microbe interactions in oilimpacted salt marshes and the understanding of the effect of rhizosphere and endosphere bacteria in the role of salt marsh plants as potential phytoremediation agents. In the field approach, molecular tools were used to assess how plant species- and OH pollution affect sediment bacterial composition [bulk sediment and sediment surrounding the roots (rhizosphere) of Halimione portulacoides and Sarcocornia perennis subsp. perennis] in a temperate estuary (Ria de Aveiro, Portugal) chronically exposed to OH pollution. In addition, the 16S rRNA gene sequences retrieved in this study were used to generate in silico metagenomes and to evaluate the distribution of potential bacterial traits in different microhabitats. Moreover, a combination of culture-dependent and -independent approaches was used to investigate the effect of oil hydrocarbons contamination on the structure and function of endophytic bacterial communities of salt marsh plants.Root systems of H. portulacoides and S. perennis subsp. perennis appear to be able to exert a strong influence on bacterial composition and in silico metagenome analysis showed enrichment of genes involved in the process of polycyclic aromatic hydrocarbon (PAH) degradation in the rhizosphere of halophyte plants. The culturable fraction of endophytic degraders was essentially closely related to known OH-degrading Pseudomonas species and endophytic communities revealed sitespecific effects related to the level of OH contamination in the sediment. In order to determine the effects of oil contamination on plant condition and on the responses in terms of structure and function of the bacterial community associated with plant roots (rhizosphere, endosphere), a microcosms approach was set up. The salt marsh plant Halimione portulacoides was inoculated with a previous isolated Pseudomonas sp. endophytic degrader and the 2-methylnaphthalene was used as model PAH contaminant. The results showed that H. portulacoides health and growth were not affected by the contamination with the tested concentration. Moreover, the decrease of 2-methylnaphthalene at the end of experiment, can suggest that H. portulacoides can be considered as a potential plant for future uses in phytoremedition approaches of contaminated salt marsh. The acceleration of hydrocarbon degradation by inoculation of the plants with the hydrocarbon-degrading Pseudomonas sp. could not, however, be demonstrated, although the effects of inoculation on the structure of the endophytic community observed at the end of the experiment indicate that the strain may be an efficient colonizer of H. portulacoides roots. The results obtained in this work suggest that H. portulacoides tolerates moderate concentrations of 2-methylnaphthalene and can be regarded as a promising agent for phytoremedition approaches in salt marshes contaminated with oil hydrocarbons. Plant/microbe interactions may have an important role in the degradation process, as plants support a diverse endophytic bacterial community, enriched in genetic factors (genes and plasmids) for hydrocarbon degradation.

Surface reactivity enhancement of silica-based glass-ceramic scaffolds

A paradigm shift is taking place from using transplanting tissue and synthetic implants to a tissue engineering approach that aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates, guiding the growth of new tissue. The central focus of this thesis was to produce porous glass and glass-ceramic scaffolds that exhibits a bioactive and biocompatible behaviour with specific surface reactivity in synthetic physiological fluids and cell-scaffold interactions, enhanced by composition and thermal treatments applied. Understanding the sintering behaviour and the interaction between the densification and crystallization processes of glass powders was essential for assessing the ideal sintering conditions for obtaining a glass scaffolds for tissue engineering applications. Our main goal was to carry out a comprehensive study of the bioactive glass sintering, identifying the powder size and sintering variables effect, for future design of sintered glass scaffolds with competent microstructures. The developed scaffolds prepared by the salt sintering method using a 3CaO.P2O5 - SiO2 - MgO glass system, with additions of Na2O with a salt, NaCl, exhibit high porosity, interconnectivity, pore size distribution and mechanical strength suitable for bone repair applications. The replacement of 6 % MgO by Na2O in the glass network allowed to tailor the dissolution rate and bioactivity of the glass scaffolds. Regarding the biological assessment, the incorporation of sodium to the composition resulted in an inibition cell response for small periods. Nevertheless it was demonstrated that for 21 days the cells response recovered and are similar for both glass compositions. The in vitro behaviour of the glass scaffolds was tested by introducing scaffolds to simulated body fluid for 21 days. Energy-dispersive Xray spectroscopy and SEM analyses proved the existence of CaP crystals for both compositions. Crystallization forming whitlockite was observed to affect the dissolution behaviour in simulated body fluid. By performing different heat treatments, it was possible to control the bioactivity and biocompatability of the glass scaffolds by means of a controlled crystallization. To recover and tune the bioactivity of the glass-ceramic with 82 % crystalline phase, different methods have been applied including functionalization using 3- aminopropyl-triethoxysilane (APTES). The glass ceramic modified surface exhibited an accelerated crystalline hydroxyapatite layer formation upon immersion in SBF after 21 days while the as prepared glass-ceramic had no detected formation of calcium phosphate up to 5 months. A sufficient mechanical support for bone tissue regeneration that biodegrade later at a tailorable rate was achievable with the glass–ceramic scaffold. Considering the biological assessment, scaffolds demonstrated an inductive effect on the proliferation of cells. The cells showed a normal morphology and high growth rate when compared to standard culture plates. This study opens up new possibilities for using 3CaO.P2O5–SiO2–MgO glass to manufacture various structures, while tailoring their bioactivity by controlling the content of the crystalline phase. Additionally, the in vitro behaviour of these structures suggests the high potential of these materials to be used in the field of tissue regeneration.

Combined effects of chemical and natural stressors to soil organisms and plants

Nas últimas décadas, a Terra tem experimentado um aquecimento global e mudanças nos padrões de precipitação. Muitos estudos sobre a avaliação de risco de agrotóxicos em organismos não-alvo foram realizados com base em protocolos padronizados, com condições abióticas controladas. Mas, em campo, os organismos são expostos a flutuações de vários fatores ambientais, bem como a poluentes, que podem alterar os limites de tolerância dos organismos aos stressores naturais, bem como alterar a toxicidade ou biodisponibilidade do químico em causa. Considerando isso, o principal objetivo deste trabalho foi o de avaliar de que modo e em que medida os fatores ambientais (temperatura, humidade do solo e radiação UV) podem interagir uns com os outros ou afetar a toxicidade do carbaril para invertebrados do solo e plantas. Para isso, foram utilizadas quatro espécies padrão: Folsomia candida, Eisenia andrei, Triticum aestivum e Brassica rapa, e simulados diferentes cenários climáticos, com vários parâmetros letais e subletais analisados. A exposição combinada foi analisada utilizando, quando possível, a ferramenta MIXTOX, com base no modelo de referência de acção independente (IA) e possíveis desvios, assim como rácios sinergísticos/antagonísticos (a partir de valores de EC50/LC50), quando a dose-resposta de um dos stressores não foi obtida. Todos os fatores de stress aplicados isoladamente causaram efeitos significativos sobre as espécies testadas e sua exposição combinada com carbaril, apresentaram respostas diferenciadas: para as minhocas, a seca e temperaturas elevadas aumentaram os efeitos deletérios do carbaril (sinergismo), enquanto o alagamento e temperaturas baixas diminuíram sua toxicidade (antagonismo). Para os colêmbolos, o modelo IA mostrou ser uma boa ferramenta para prever a toxicidade do carbaril tanto para temperaturas altas como para as baixas. Para as duas espécies de plantas foram encontradas diferenças significativas entre elas: em termos gerais, as interações entre carbaril e os stressores naturais foram observadas, com sinergismo aparecendo como o padrão principal relacionado com a radiação UV, solos secos e temperaturas elevadas, enquanto o padrão principal relacionado com temperaturas baixas e stress de alagamento foi o antagonismo. Quando os efeitos de dois stressores naturais (radiação UV e humidade do solo) em plantas foram avaliados, uma interação significativa foi encontrada: a seca aliviou o efeito deletério da radiação UV em T. aestivum e o alagamento aumentou os seus efeitos, mas para B. rapa a adição de ambos os stresses de água causou um aumento (sinergismo) dos efeitos deletérios da radiação UV para todos os parâmetros avaliados. Portanto é necessário que as diferenças sazonais e latitudinais, bem como as mudanças climáticas globais, sejam integradas na avaliação de risco de contaminantes do solo.

A computational study of ionic liquids used in the production of fuels and biofuels

For the past decades it has been a worldwide concern to reduce the emission of harmful gases released during the combustion of fossil fuels. This goal has been addressed through the reduction of sulfur-containing compounds, and the replacement of fossil fuels by biofuels, such as bioethanol, produced in large scale from biomass. For this purpose, a new class of solvents, the Ionic Liquids (ILs), has been applied, aiming at developing new processes and replacing common organic solvents in the current processes. ILs can be composed by a large number of different combinations of cations and anions, which confer unique but desired properties to ILs. The ability of fine-tuning the properties of ILs to meet the requirements of a specific application range by mixing different cations and anions arises as the most relevant aspect for rendering ILs so attractive to researchers. Nonetheless, due to the huge number of possible combinations between the ions it is required the use of cheap predictive approaches for anticipating how they will act in a given situation. Molecular dynamics (MD) simulation is a statistical mechanics computational approach, based on Newton’s equations of motion, which can be used to study macroscopic systems at the atomic level, through the prediction of their properties, and other structural information. In the case of ILs, MD simulations have been extensively applied. The slow dynamics associated to ILs constitutes a challenge for their correct description that requires improvements and developments of existent force fields, as well as larger computational efforts (longer times of simulation). The present document reports studies based on MD simulations devoted to disclose the mechanisms of interaction established by ILs in systems representative of fuel and biofuels streams, and at biomass pre-treatment process. Hence, MD simulations were used to evaluate different systems composed of ILs and thiophene, benzene, water, ethanol and also glucose molecules. For the latter molecules, it was carried out a study aiming to ascertain the performance of a recently proposed force field (GROMOS 56ACARBO) to reproduce the dynamic behavior of such molecules in aqueous solution. The results here reported reveal that the interactions established by ILs are dependent on the individual characteristics of each IL. Generally, the polar character of ILs is deterministic in their propensity to interact with the other molecules. Although it is unquestionable the advantage of using MD simulations, it is necessary to recognize the need for improvements and developments of force fields, not only for a successful description of ILs, but also for other relevant compounds such as the carbohydrates.

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.