UV-induced stress in bacterioneuston : mechanisms and ecological implications

As bactérias desempenham um papel chave na reciclagem de energia e matéria nas teias tróficas aquáticas. No entanto, as suas pequenas dimensões, curto tempo de geração e o facto de os seus genomas constituírem uma grande porção do seu volume celular, tornam as bactérias mais suscetíveis às alterações ambientais que os organismos superiores. O aumento dos níveis de radiação UVB (280-320 nm) constitui uma ameaça particularmente importante para as comunidades bacterianas dos sistemas aquáticos, uma vez que a radiação consegue penetrar até profundidades consideráveis. No entanto, os mecanismos através dos quais a radiação causa danos nas bactérias ainda não são claros, o que impede a modelação precisa dos efeitos da radiação UV nas comunidades bacterianas naturais. O bacterioneuston habita a microcamada superficial (primeiro milímetro da coluna de água), estando naturalmente exposto a níveis de radiação UV superiores aos que o bacterioplâncton está exposto. Deste modo, a microcamada superficial pode ser vista como um nicho ecológico modelo para estudar as interações entre as bactérias e a radiação UV. Os objetivos deste trabalho foram (i) avaliar a influência do nível de exposição natural à radiação das comunidades bacterianas na sua sensibilidade à radiação UV, através da comparação das respostas fotobiológicas do bacterioneuston e bacterioplâncton; (ii) aprofundar o conhecimento acerca dos mecanismos através dos quais a radiação UV causa danos, bem como dos fatores que afetam a interação entre a radiação UV e as bactérias; e (iii) avaliar o potencial da proteína RecA, que medeia a resposta SOS das bactérias, para ser usada como marcador de danos induzidos por UV nas comunidades bacterianas. Verificou-se que o bacterioneuston é mais resistente à radiação UVB que o bacterioplâncton e recupera de modo mais eficiente dos danos induzidos por UV, particularmente em condições de escassez de nutrientes, indicando assim que o nível de exposição natural das comunidades bacterianas à radiação afeta a sua sensibilidade à radiação UV. Os resultados das análises independentes do cultivo revelaram o potencial da radiação UV para afetar a estrutura das comunidades bacterianas ao selecionar bactérias resistentes. A análise do perfil de utilização de fontes de carbono usando o sistema de Ecoplacas Biolog ® e a determinação das taxas de incorporação de leucina e timidina permitiu também verificar que a radiação UV modifica o funcionamento das comunidades bacterianas. Os resultados obtidos indicam a possibilidade do bacterioneuston conter um conjunto de estirpes resistentes a UV que, mediante as condições meteorológicas apropriadas, podem ser selecionadas aquando da exposição à radiação.

Toxicogenomics of natural and anthropogenic effectors in eleutheroembryos

The introduction of chemicals into the environment by human activities may represent a serious risk to environmental and human health. Environmental risk assessment requires the use of efficient and sensitive tools to determine the impact of contaminants on the ecosystems. The use of zebrafish for the toxicity assessment of pharmaceuticals, drugs, and pollutants, is becoming well accepted due to zebrafish unique advantages for the screening of compounds for hazard identification. The aim of the present work is to apply toxicogenomic approaches to identify novel biomarkers and uncovered potential modes of action of classic and emergent contaminants able to disrupt endocrine systems, such as the Retinoic Acid Receptor, Retinoid X Receptor and the Aryl Hydrocarbon Receptor. This study relies on different nuclear and cytosolic protein receptors and other conditional (ligand- or stress- activated) transcriptional factors that are intimately involved in the regulation of defensome genes and in mechanisms of chemical toxicity. The transcriptomic effects of organic compounds, endogenous compounds, and nanoparticles were analysed during the early stages of zebrafish development. Studying the gene expression profiles of exposed and unexposed organisms to pollutants using microarrays allowed the identification of specific gene markers and to establish a "genetic code" for the tested compounds. Changes in gene expression were observed at toxicant concentrations that did not cause morphological effects. Even at low toxicant concentrations, the observed changes in transcript levels were robust for some target genes. Microarray responses of selected genes were further complemented by the real time quantitative polymerase chain reaction (qRT-PCR) methodology. The combination of bio-informatic, toxicological analyses of differential gene expression profiles, and biochemical and phenotypic responses across the treatments allowed the identification of uncovered potential mechanisms of action. In addition, this work provides an integrated set of tools that can be used to aid management-decision making by improving the predictive capability to measure environmental stress of contaminants in freshwater ecosystems. This study also illustrates the potential of zebrafish embryos for the systematic, large-scale analysis of chemical effects on developing vertebrates.

DACA: architecture to implement dynamic access control mechanisms on business tier components

Access control is a software engineering challenge in database applications. Currently, there is no satisfactory solution to dynamically implement evolving fine-grained access control mechanisms (FGACM) on business tiers of relational database applications. To tackle this access control gap, we propose an architecture, herein referred to as Dynamic Access Control Architecture (DACA). DACA allows FGACM to be dynamically built and updated at runtime in accordance with the established fine-grained access control policies (FGACP). DACA explores and makes use of Call Level Interfaces (CLI) features to implement FGACM on business tiers. Among the features, we emphasize their performance and their multiple access modes to data residing on relational databases. The different access modes of CLI are wrapped by typed objects driven by FGACM, which are built and updated at runtime. Programmers prescind of traditional access modes of CLI and start using the ones dynamically implemented and updated. DACA comprises three main components: Policy Server (repository of metadata for FGACM), Dynamic Access Control Component (DACC) (business tier component responsible for implementing FGACM) and Policy Manager (broker between DACC and Policy Server). Unlike current approaches, DACA is not dependent on any particular access control model or on any access control policy, this way promoting its applicability to a wide range of different situations. In order to validate DACA, a solution based on Java, Java Database Connectivity (JDBC) and SQL Server was devised and implemented. Two evaluations were carried out. The first one evaluates DACA capability to implement and update FGACM dynamically, at runtime, and, the second one assesses DACA performance against a standard use of JDBC without any FGACM. The collected results show that DACA is an effective approach for implementing evolving FGACM on business tiers based on Call Level Interfaces, in this case JDBC.

Nanosensor for assessing the risk of a cardiovascular disease

The incidence of cardiovascular diseases (CVD) has been increasing according to the European and global statistics. Thus, the development of new analytical devices, such as biosensors for assessing the risk of CVD could become a valuable approach for the improvement of healthcare service. In latest years, the nanotechnology has provided new materials with improved electronic properties which have an important contribution in the transduction mechanism of biosensors. Thus, in this thesis, biosensors based on field effect transistors with single-walled carbon nanotubes (NTFET) were developed for the detection of C-reactive protein (CRP) in clinical samples, that is, blood serum and saliva from a group of control patients and a group of CVD risk patients. CRP is an acute-phase protein, which is commonly known as the best validated biomarker for the assessment of CVD, the single-walled carbon nanotubes (SWCNT) were applied as transduction components, and the immunoreaction (interaction between the CRP antigen and the antibodies specific to CRP) was used as the mechanism of molecular recognition for the label-free detection of CRP. After the microfabrication of field effect transistors (FET), the screening of the most important variables for the dispersion of SWCNT, the assemblage of NTFET, and their application on standard solutions of CRP, it was found that NTFET respond accurately to CRP both in saliva and in serum samples, since similar CRP levels were found with the NTFET and the traditional methodology (ELISA technique). On the other hand, a strong correlation between salivary and serum CRP was found with NTFET, which means that saliva could be used, based on non-invasive sampling, as an alternative fluid to blood serum. It was also shown that NTFET could discriminate control patients from CVD risk patients, allowing the determination of a cut-off value for salivary CRP of 1900 ng L-1, which corresponds to the well established cut-off of 3 mg L-1 for CRP in serum, constituting an important finding for the possible establishment of a new range of CRP levels based on saliva. According to the data provided from the volunteer patients regarding their lipoprotein profile and lifestyle factors, it was concluded that the control and the CVD risk patients could be separated taking into account the various risk factors established in literature as strong contributors for developing a CVD, such as triglycerides, serum CRP, total cholesterol, LDL cholesterol, body mass index, Framingham risk score, hypertension, dyslipidemia, and diabetes mellitus. Thus, this work could provide an additional contribution to the understanding of the association of biomarkers levels in serum and saliva samples, and above all, cost-effective, rapid, label-free, and disposable NTFET were developed, based on a noninvasive sampling, for the assessment of CVD risk, thus constituting a potential point-of-care technology.

Tecnologias de informação e comunicação e o cidadão sénior : estudo sobre o impacto em variáveis psicossociais e a conceptualização de serviços com e para o cidadão sénior

Perante uma sociedade em célere envelhecimento demográfico e permanente avanço tecnológico, justifica-se a aposta em estudos que potenciem a ação comunicativa e a diminuição do isolamento social decorrente das perdas biopsicossociais associadas à idade sénior. Esta tese possui quatro objetivos de estudo: i) pretende-se investigar qual é o impacto da utilização das Tecnologias de Informação e Comunicação (TIC) no autoconceito (AC), no ânimo e na qualidade de vida (QV) de um grupo de seniores; ii) perceber se existe e qual a relação entre as variáveis independentes sexo, idade, estado civil, escolaridade, profissão, IPSS, regime de frequência, tempo na IPSS, orientação para frequentar a IPSS, visita de familiares e visita de amigos e as variáveis dependentes AC, ânimo, QV e respetivos fatores e domínios, nos momentos de pré e pós-teste; iii) perceber se a sua participação no processo de conceptualização de um serviço de comunicação assíncrona, email, influencia a sua usabilidade ao nível das componentes eficácia, eficiência e satisfação; iv) e sugerir a componente política da comunidade online sénior em desenvolvimento no âmbito do Projeto SEDUCE. Para o desenvolvimento do estudo estabeleceram-se parcerias com quatro Instituições Particulares de Segurança Social do concelho de Aveiro, integradas no âmbito do projeto SEDUCE. Os instrumentos utilizados para a avaliação do autoconceito, do ânimo e da qualidade de vida foram o Inventário Clínico de Auto-Conceito, a Escala de Ânimo do Centro Geriátrico de Philadelphia e a Escala de Qualidade de Vida da Organização Mundial de Saúde WHOQOL-Bref, respetivamente. No processo de conceptualização do serviço de email e da componente política da comunidade online utilizou-se a observação participativa e o contextual design. O estudo envolveu a participação de 42 seniores distribuídos por duas condições experimentais: 22 seniores do grupo experimental utilizaram as TIC duas vezes por semana (em sessões de 90 minutos cada, num total de 80 sessões) e 19 seniores do grupo de controlo passivo não experimentaram qualquer intervenção. Para a avaliação das variáveis psicossociais realizaram-se dois momentos de avaliação, antes e depois de 11 meses de intervenção, de Agosto de 2011 a Julho de 2012. Ao longo das sessões de envolvimento com as TIC observou-se que os seniores apresentam, continuamente, dificuldades em: manipular o rato e percecionar a sua ação no monitor; fazer a distinção entre teclas (enter, spacebar, delete, caps lock entre outras); em utilizar duplas teclas para colocar pontuação e acentuação; iniciar atividades no Microsoft Office Word; selecionar a informação disponibilizada em motores de pesquisa; perceber quais as zonas clicáveis; falta de confiança em efetivar ações; receio em iniciar nova atividades, pela falta de conhecimento e pelo medo de errar; memorizar endereços de email e passwords; e dar continuidade às tarefas. Na utilização do serviço de email consideram importante receber resposta quando enviam uma mensagem, assim como responder sempre aos remetentes; raramente colocam assunto nas mensagens; e expressam grande satisfação ao receber mensagens de familiares e/ou amigos. O processo de desenvolvimento de serviços com a participação ativa dos seniores revela-se exequível mas é necessário adaptar as práticas: os processos devem ser iterativos; evitar linguagem formal; clarificar o objetivo; deixar os seniores pensar em voz alta; dar-lhes tempo; mantê-los focados e não conduzi-los nas tarefas. Os resultados sugerem que houve aumento significativo do domínio físico da qualidade de vida do grupo experimental. Os participantes que exprimiram maiores níveis de satisfação ao utilizar as TIC apresentam uma perspetiva mais positiva sobre a maturidade psicológica e menos solidão e insatisfação. No grupo experimental e no grupo de controlo passivo verificam-se relações entre as variáveis independentes e as variáveis dependentes, quer no momento de pré-teste como de pós-teste. Conclui-se que a participação dos seniores no processo de conceptualização do serviço de email permitiu fomentar a componente eficácia da usabilidade mas não a satisfação ao utilizar o mesmo. Os resultados sobre a eficiência são inconclusivos. Sobre a componente política os seniores validam a existência de termos de utilização que orientem o comportamento de todos os utilizadores, assim como de uma política de privacidade. A área de registo proposta é adequada ao utilizador sénior.

Effect assessment of nanoparticles toxicity in the terrestrial compartment

Over 11 million tons of nanomaterials (NMs) have been produced in 2012 and predictions point the increase in production. Despite predictions and extended usage via consumer products and industry, the understanding of the potential impact of these materials on the environment is virtually absent. The main aim of this thesis is to understand how a selected group of nanomaterials (metal based particles) may impact soil invertebrates, with special focus on the mechanisms of response. Since a case-by-case Environmental Risk Assessment (ERA) of all the emerging contaminants (particularly NMs) is impossible, among others due to time and cost reasons, to gain understanding on the mechanism of action and response is very important to reach a common paradigm. Understanding the modes of action provides predictive characters in cross particle extrapolation. Besides, it also provides insight for the production of new and sustainable materials. Overall, the effects of the selected NMs (Copper and Silver, Titanium and Zirconium oxides) and the respective salt forms, were investigated at the gene expression (using high-throughput tools, microarray and qPCR technology), biochemical (using enzymatic assays for analysis of oxidative stress markers) and organism (survival and reproduction as in OECD test guidelines) levels, this using standard soil species (Enchytraeus albidus, Enchytraeus crypticus, Eisenia fetida). Gene expression analysis provided valuable information on the mechanisms affected by each of the NMs. The gene expression profile highlighted a (nano)material signature and the effect of the duration of exposure. The functional analyses integrated with the biochemical and organism data, revealed a good understanding power. The biochemical parameters (oxidative stress related) were distinct across the materials and also influenced by duration of exposure and concentration. The standardized organismal responses differed the least between the various materials. The overall outcome is that, in this context of NMs effect assessment, gene expression and enzymatic assays introduced a very important knowledge gap, which could not had been achieved by the standard organismal effects alone. A reoccurring issue with some metal based NMs is the possible dissolution and subsequent release of ions that then causes toxicity e.g. Cu-NPs or Ag-NPs release Cu2+ or Ag+. The oxidation state of the particles was investigated, although this was not the focus of the thesis. The study of fate, e.g. dissolution of NPs, is also only in its beginning and the appropriate techniques are currently being developed. The results showed a specific nanoparticle effect. The UV exposure with titanium dioxide nanoparticles increased its effect.

Artefactos tangíveis e adaptáveis no ambiente doméstico

Recentemente assistimos a uma evolução da relação do Homem com a tecnologia, em larga medida acompanhada por novos modelos de interacção que modificam a forma de conceber os artefactos e constroem novos contextos de uso. Procuramos investigar, na presente tese, uma das abordagens emergentes, o universo dos media tangíveis, articulando a perspectiva do design da tecnologia orientada para a Human-Computer Interation (HCI), com a dimensão social, cultural e estética no uso da tecnologia. Os media tangíveis, ao contrário do que sucede com os conteúdos digitais convencionais, têm espessura e expressão física e, porque são dotados de um corpo que habita o espaço das disposições físicas, estão sujeitos à acção do mundo cultural e das práticas sociais que regem os demais objectos físicos que podemos encontrar no nosso quotidiano. Esta nova relação com a tecnologia digital obrigará as disciplinas que se encontram mais próximas do desenvolvimento tecnológico, tais como o Design de Interacção e a HCI, a abrirem-se aos contributos e abordagens das ciências humanas. Admitindo que a natureza subjacente ao processo da adaptabilidade no ambiente doméstico altera o equilíbrio da relação entre o design e o uso da tecnologia, julgamos ser essencial o desenvolvimento de uma fenomenologia da interação. Por outro lado, a adaptabilidade dos media tangíveis apresenta um conjunto de dificuldades, não apenas de ordem técnica, mas também de natureza conceptual, que têm dificultado o desenvolvimento e a implementação no terreno de tecnologias personalizáveis. Um dos objectivos da presente tese consiste em investigar um quadro conceptual capaz de enquadrar o fenómeno da adaptabilidade dos media tangíveis, e desenvolver uma tecnologia que possa servir de objecto a um estudo empírico com base numa abordagem etnográfica.

Modelo para a valorização partilhada da marca: a comunicação mediada por computador como suporte ao envolvimento dos recursos humanos na cocriação da marca organizacional

No atual contexto de disseminação e uso das tecnologias da comunicação para a geração de conteúdo não especializado, igualmente partilhado e utilizado, torna-se pertinente sublinhar a participação dos recursos humanos na cocriação da marca organizacional. Esta participação, todavia, encontra alguns constrangimentos e necessidades, tais como a falta de orientação no uso devido da marca gráfica, e a adequação das ferramentas comunicacionais e de gestão do conhecimento de que estes indivíduos são portadores. Neste enquadramento, pretende-se validar um modelo heurístico que explique, satisfatoriamente, de que forma as organizações podem envolver os seus recursos humanos em atividades de valorização da marca, em contextos de informação e comunicação tecnologicamente mediados, e que possa assumirse como um impulsionador da adoção destas práticas. A abordagem metodológica é de natureza exploratória, iterativa e qualitativa, assentando na Grounded Theory e, portanto, num processo indutivo de produção de conhecimento, reconhecendo a existência de momentos de descoberta e, aproximando-se do paradigma construtivista. O estudo que conduz à proposta final de um modelo de comunicação mediada por computador para a valorização da marca assenta, essencialmente, na revisão da literatura através da pesquisa bibliográfica, e num estudo de caso, que assume como procedimentos técnicos os inquéritos por entrevista, para uma primeira verificação dos pressupostos do modelo, a construção de um protótipo não-funcional de uma Central Participativa de Marca (CPM), capaz de expressar a componente tecnológica presente no modelo, e a realização de grupos focais, com o intuito de alcançar a sua validação não-experimental. O estudo de caso realizado debruça-se sobre a Universidade de Aveiro (UA) e sobre o uso e apropriação da sua marca, por parte dos membros da sua comunidade docente e não-docente. Os resultados das entrevistas exploratórias, para as quais se recorre a uma amostra por conveniência composta por entidades relevantes no que à promoção da marca da UA concerne, são combinados com os conteúdos extraídos do levantamento bibliográfico, de modo a serem contemplados tanto na aferição dos domínios de interação que informam o modelo, como na conceção do protótipo de uma plataforma de colaboração online, especificamente orientada à marca, apresentado a cinco grupos focais que neles integram a população-alvo estudada. Este estudo empírico permite uma primeira validação do modelo heurístico proposto, confirmando que a participação na cocriação de artefactos de marca tende a acontecer de forma não oficial, por falta de iniciativas organizacionais que valorizem o capital humano, e que a orientação para o uso e apropriação da marca é fundamental na concretização deste tipo de contributos, dados por indivíduos não especializados em branding, design, comunicação ou estratégia de marketing. Os resultados permitem validar ainda a configuração e a estrutura de interação propostas para o sistema subjacente ao protótipo da CPM, assim como a generalidade das suas funcionalidades, permitindo a proposta de uma heurística focada na experimentação da marca no seio organizacional. Finalmente, são identificadas linhas de investigação futura nesta área, decorrentes dos resultados alcançados e das limitações do estudo realizado.

Pregnancy and newborns disorders followed by urine metabolomics

Chapter 1 introduces the scope of the work by identifying the clinically relevant prenatal disorders and presently available diagnostic methods. The methodology followed in this work is presented, along with a brief account of the principles of the analytical and statistical tools employed. A thorough description of the state of the art of metabolomics in prenatal research concludes the chapter, highlighting the merit of this novel strategy to identify robust disease biomarkers. The scarce use of maternal and newborn urine in previous reports enlightens the relevance of this work. Chapter 2 presents a description of all the experimental details involved in the work performed, comprising sampling, sample collection and preparation issues, data acquisition protocols and data analysis procedures. The proton Nuclear Magnetic Resonance (NMR) characterization of maternal urine composition in healthy pregnancies is presented in Chapter 3. The urinary metabolic profile characteristic of each pregnancy trimester was defined and a 21-metabolite signature found descriptive of the metabolic adaptations occurring throughout pregnancy. 8 metabolites were found, for the first time to our knowledge, to vary in connection to pregnancy, while known metabolic effects were confirmed. This chapter includes a study of the effects of non-fasting (used in this work) as a possible confounder. Chapter 4 describes the metabolomic study of 2nd trimester maternal urine for the diagnosis of fetal disorders and prediction of later-developing complications. This was achieved by applying a novel variable selection method developed in the context of this work. It was found that fetal malformations (FM) (and, specifically those of the central nervous system, CNS) and chromosomal disorders (CD) (and, specifically, trisomy 21, T21) are accompanied by changes in energy, amino acids, lipids and nucleotides metabolic pathways, with CD causing a further deregulation in sugars metabolism, urea cycle and/or creatinine biosynthesis. Multivariate analysis models´ validation revealed classification rates (CR) of 84% for FM (87%, CNS) and 85% for CD (94%, T21). For later-diagnosed preterm delivery (PTD), preeclampsia (PE) and intrauterine growth restriction (IUGR), it is found that urinary NMR profiles have early predictive value, with CRs ranging from 84% for PTD (11-20 gestational weeks, g.w., prior to diagnosis), 94% for PE (18-24 g.w. pre-diagnosis) and 94% for IUGR (2-22 g.w. pre-diagnosis). This chapter includes results obtained for an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) study of pre-PTD samples and correlation with NMR data. One possible marker was detected, although its identification was not possible. Chapter 5 relates to the NMR metabolomic study of gestational diabetes mellitus (GDM), establishing a potentially predictive urinary metabolic profile for GDM, 2-21 g.w. prior to diagnosis (CR 83%). Furthermore, the NMR spectrum was shown to carry information on individual phenotypes, able to predict future insulin treatment requirement (CR 94%). Chapter 6 describes results that demonstrate the impact of delivery mode (CR 88%) and gender (CR 76%) on newborn urinary profile. It was also found that newborn prematurity, respiratory depression, large for gestational age growth and malformations induce relevant metabolic perturbations (CR 82-92%), as well as maternal conditions, namely GDM (CR 82%) and maternal psychiatric disorders (CR 91%). Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the value of maternal or newborn urine metabolomics for pregnancy monitoring and disease prediction, towards the development of new early and non-invasive diagnostic methods.

Analysis of the organic matter associated to sea salt : definition of potential molecular markers based on the volatile composition and presence of glycosidic derivatives and polysaccharides

Sea salt is a natural product obtained from the evaporation of seawater in saltpans due to the combined effect of wind and sunlight. Nowadays, there is a growing interest for protection and re-valorisation of saltpans intrinsically associated to the quality of sea salt that can be evaluated by its physico-chemical properties. These man-made systems can be located in different geographical areas presenting different environmental surroundings. During the crystallization process, organic compounds coming from these surroundings can be incorporated into sea salt crystals, influencing their final composition. The organic matter associated to sea salt arises from three main sources: algae, surrounding bacterial community, and anthropogenic activity. Based on the hypothesis that sea salt contains associated organic compounds that can be used as markers of the product, including saltpans surrounding environment, the aim of this PhD thesis was to identify these compounds. With this purpose, this work comprised: 1) a deep characterisation of the volatile composition of sea salt by headspace solid phase microextraction combined with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (HS-SPME/GCGC–ToFMS) methodology, in search of potential sea salt volatile markers; 2) the development of a methodology to isolate the polymeric material potentially present in sea salt, in amounts that allow its characterisation in terms of polysaccharides and protein; and 3) to explore the possible presence of triacylglycerides. The high chromatographic resolution and sensitivity of GC×GC–ToFMS enabled the separation and identification of a higher number of volatile compounds from sea salt, about three folds, compared to unidimentional chromatography (GC–qMS). The chromatographic contour plots obtained revealed the complexity of marine salt volatile composition and confirmed the relevance of GC×GC–ToFMS for this type of analysis. The structured bidimentional chromatographic profile arising from 1D volatility and 2D polarity was demonstrated, allowing more reliable identifications. Results obtained for analysis of salt from two locations in Aveiro and harvested over three years suggest the loss of volatile compounds along the time of storage of the salt. From Atlantic Ocean salts of seven different geographical origins, all produced in 2007, it was possible to identify a sub-set of ten compounds present in all salts, namely 6-methyl-5-hepten-2-one, 2,2,6-trimethylcyclohexanone, isophorone, ketoisophorone, β-ionone-5,6-epoxide, dihydroactinidiolide, 6,10,14-trimethyl-2-pentadecanone, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate, 2,4,4-trimethylpentane-1,3-diyl bis(2-methylpropanoate), and 2-ethyl-1-hexanol. These ten compounds were considered potential volatile markers of sea salt. Seven of these compounds are carotenoid-derived compounds, and the other three may result from the integration of compounds from anthropogenic activity as metabolites of marine organisms. The present PhD work also allowed the isolation and characterisation, for the first time, of polymeric material from sea salt, using 16 Atlantic Ocean salts. A dialysis-based methodology was developed to isolate the polymeric material from sea salt in amounts that allowed its characterisation. The median content of polymeric material isolated from the 16 salts was 144 mg per kg of salt, e.g. 0.014% (w/w). Mid-infrared spectroscopy and thermogravimetry revealed the main occurrence of sulfated polysaccharides, as well as the presence of protein in the polymeric material from sea salt. Sea salt polysaccharides were found to be rich in uronic acid residues (21 mol%), glucose (18), galactose (16), and fucose (13). Sulfate content represented a median of 45 mol%, being the median content of sulfated polysaccharides 461 mg/g of polymeric material, which accounted for 66 mg/kg of dry salt. Glycosidic linkage composition indicates that the main sugar residues that could carry one or more sulfate groups were identified as fucose and galactose. This fact allowed to infer that the polysaccharides from sea salt arise mainly from algae, due to their abundance and composition. The amino acid profile of the polymeric material from the 16 Atlantic Ocean salts showed as main residues, as medians, alanine (25 mol%), leucine (14), and valine (14), which are hydrophobic, being the median protein content 35 mg/g, i.e. 4,9 mg per kg of dry salt. Beside the occurrence of hydrophobic volatile compounds in sea salt, hydrophobic non-volatile compounds were also detected. Triacylglycerides were obtained from sea salt by soxhlet extraction with n-hexane. Fatty acid composition revealed palmitic acid as the major residue (43 mol%), followed by stearic (13), linolenic (13), oleic (12), and linoleic (9). Sea salt triacylglycerides median content was 1.5 mg per kg of dry salt. Both protein and triacylglycerides seem to arise from macro and microalgae, phytoplankton and cyanobacteria, due to their abundance and composition. Despite the variability resulting from saltpans surrounding environment, this PhD thesis allowed the identification of a sea salt characteristic organic compounds profile based on volatile compounds, polysaccharides, protein, and triacylglycerides.

Contributos para a eficácia do clustering usando o tagging social

Nos últimos anos temos vindo a assistir a uma mudança na forma como a informação é disponibilizada online. O surgimento da web para todos possibilitou a fácil edição, disponibilização e partilha da informação gerando um considerável aumento da mesma. Rapidamente surgiram sistemas que permitem a coleção e partilha dessa informação, que para além de possibilitarem a coleção dos recursos também permitem que os utilizadores a descrevam utilizando tags ou comentários. A organização automática dessa informação é um dos maiores desafios no contexto da web atual. Apesar de existirem vários algoritmos de clustering, o compromisso entre a eficácia (formação de grupos que fazem sentido) e a eficiência (execução em tempo aceitável) é difícil de encontrar. Neste sentido, esta investigação tem por problemática aferir se um sistema de agrupamento automático de documentos, melhora a sua eficácia quando se integra um sistema de classificação social. Analisámos e discutimos dois métodos baseados no algoritmo k-means para o clustering de documentos e que possibilitam a integração do tagging social nesse processo. O primeiro permite a integração das tags diretamente no Vector Space Model e o segundo propõe a integração das tags para a seleção das sementes iniciais. O primeiro método permite que as tags sejam pesadas em função da sua ocorrência no documento através do parâmetro Social Slider. Este método foi criado tendo por base um modelo de predição que sugere que, quando se utiliza a similaridade dos cossenos, documentos que partilham tags ficam mais próximos enquanto que, no caso de não partilharem, ficam mais distantes. O segundo método deu origem a um algoritmo que denominamos k-C. Este para além de permitir a seleção inicial das sementes através de uma rede de tags também altera a forma como os novos centróides em cada iteração são calculados. A alteração ao cálculo dos centróides teve em consideração uma reflexão sobre a utilização da distância euclidiana e similaridade dos cossenos no algoritmo de clustering k-means. No contexto da avaliação dos algoritmos foram propostos dois algoritmos, o algoritmo da “Ground truth automática” e o algoritmo MCI. O primeiro permite a deteção da estrutura dos dados, caso seja desconhecida, e o segundo é uma medida de avaliação interna baseada na similaridade dos cossenos entre o documento mais próximo de cada documento. A análise de resultados preliminares sugere que a utilização do primeiro método de integração das tags no VSM tem mais impacto no algoritmo k-means do que no algoritmo k-C. Além disso, os resultados obtidos evidenciam que não existe correlação entre a escolha do parâmetro SS e a qualidade dos clusters. Neste sentido, os restantes testes foram conduzidos utilizando apenas o algoritmo k-C (sem integração de tags no VSM), sendo que os resultados obtidos indicam que a utilização deste algoritmo tende a gerar clusters mais eficazes.

Trophic plasticity in the cnidarian-dinoflagellate symbiosis

Coral reefs are of utmost ecological and economical importance but are currently in global decline due to climate change and anthropogenic disturbances. Corals, as well as other cnidarian species, live in symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium. This relationship provides the cnidarian host with alternative metabolic pathways, as the symbionts translocate photosynthetic carbon to the animal. Besides this autotrophic nutrition mode, symbiotic cnidarians also take up organic matter from the environment (heterotrophy). The nutritional balance between auto- and heterotrophy is critical for the functioning, fitness and resilience of the cnidariandinoflagellate symbiosis. New methodological approaches were developed to better understand the role of auto- and heterotrophy in the ecophysiology of cnidarians associated with Symbiodinium, and the ecological implications of this trophic plasticity. Specifically, the new approaches were developed to assess photophysiology, biomass production of the model organism Aiptasia sp. and molecular tools to investigate heterotrophy in the cnidarian-dinoflagellate symbiosis. Using these approaches, we were able to non-invasively assess the photophysiological spatial heterogeneity of symbiotic cnidarians and identify spatial patterns between chlorophyll fluorescence and relative content of chlorophyll a and green-fluorescent proteins. Optimal culture conditions to maximize the biomass production of Aiptasia pallida were identified, as well as their implications on the fatty acid composition of the anemones. Molecular trophic markers were used to determine prey digestion times in symbiotic cnidarians, which vary between 1-3 days depending on prey species, predator species and the feeding history of the predator. This method was also used to demonstrate that microalgae is a potential food source for symbiotic corals. By using a stable isotope approach to assess the trophic ecology of the facultative symbiotic Oculina arbuscula in situ, it was possible to demonstrate the importance of pico- and nanoplanktonic organisms, particularly autotrophic, in the nutrition of symbiotic corals. Finally, we showed the effects of functional diversity of Symbiodinium on the nutritional plasticity of the cnidarian-dinoflagellate symbiosis. Symbiont identity defines this plasticity through its individual metabolic requirements, capacity to fix carbon, quantity of translocated carbon and the host’s capacity to feed and digest prey.

Metabolic signature of lung cancer: a metabolomic study of human tissues and biofluids

This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.

The effects of chemicals in isopods : a multi-organizational evaluation

The global aim of this thesis was to evaluate and assess the effects of a pesticide (dimethoate) and a metal (nickel), as model chemicals, within different organization levels, starting at the detoxification pathways (enzymatic biomarkers) and energy costs associated (energy content quantification, energy consumption and CEA) along with the physiological alterations at the individual and population level (mortality), leading to a metabolomic analysis (using liquid 1H-NMR) and finally a gene expression analysis (transcriptome and RT-qPCR analysis). To better understand potential variations in response to stressors, abiotic factors were also assessed in terrestrial isopods such as temperature, soil moisture and UV radiation. The evaluation performed using biochemical biomarkers and energy related parameters showed that increases in temperature might negatively affect the organisms by generating oxidative stress. It also showed that this species is acclimated to environments with low soil moisture, and that in high moisture scenarios there was a short gap between the optimal and adverse conditions that led to increased mortality. As for UV-R, doses nowadays present have shown to induce significant negative impact on these organisms. The long-term exposure to dimethoate showed that besides the neurotoxicity resulting from acetylcholinesterase inhibition, this stressor also caused oxidative stress. This effect was observed for both concentrations used (recommended field dose application and a below EC50 value) and that its combination with different temperatures (20ºC and 25ºC) showed different response patterns. It was also observed that dimethoate’s degradation rate in soils was higher in the presence of isopods. In a similar study performed with nickel, oxidative stress was also observed. But, in the case of this stressor exposure, organisms showed a strategy where the energetic costs necessary for detoxification (biomarkers) seemed to be compensated by positive alterations in the energy related parameters. In this work we presented for the first time a metabolomic profile of terrestrial isopods exposed to stressors (dimethoate and niquel), since until the moment only a previous study was performed on a metabolomic evaluation in nonexposed isopods. In the first part of the study we identify 24 new metabolites that had not been described previously. On the second part of the study a metabolomic profile variation of abstract non-exposed organism throughout the exposure was presented and finally the metabolomic profile of organisms exposed to dimethoate and nickel. The exposure to nickel suggested alteration in growth, moult, haemocyanin and glutathione synthesis, energy pathways and in osmoregulation. As for the exposure to dimethoate alterations in osmoregulation, energy pathways, moult and neurotransmission were also suggested. In this work it was also presented the first full body transcriptome of a terrestrial isopod from the species Porcellionides pruinosus, which will complement the scarce information available for this group of organisms. This transcriptome also served as base for a RNA-Seq and a RT-qPCR analysis. The results of the RNA-Seq analysis performed in organisms exposed to nickel showed that this stressor negatively impacted at the genetic and epigenetic levels, in the trafficking, storage and elimination of metals, generates oxidative stress, inducing neurotoxicity and also affecting reproduction. These results were confirmed through RT-qPCR. As for the impact of dimethoate on these organisms it was only accessed through RT-qPCR and showed oxidative stress, an impact in neurotransmission, in epigenetic markers, DNA repair and cell cycle impairment. This study allowed the design of an Adverse Outcome Pathway draft that can be used further on for legislative purposes.

Occurrence and fate of estrogens and antibiotics in the environment evaluated by low-cost analytical methodologies

Endocrine disruptors and pharmaceuticals are considered to be concerning environmental contaminants. During the last two decades, studies dealing with the occurrence and fate of these emerging contaminants in the aquatic environment have raised attention and its number is constantly increasing. The presence of these contaminants in the environment is particularly important since they are known to induce adverse effects in the ecosystems even at extremely low concentrations. Estrogens and antibiotics, in particular, are identified as capable of induce endocrine disruption and contribute for the appearance of multi-resistant bacteria, respectively. A better assessment and understanding of the real impact of these contaminants in the aquatic environment implies the evaluation of their occurrence and fate, which is the main aim of this Thesis. Two estrogens (17-estradiol and 17-ethinylestradiol) and an antibiotic (sulfamethoxazole) were the contaminants under study and their occurrence in surface and waste waters was assessed by the implementation of enzyme linked immunosorbent assays (ELISAs). The assays were optimized in order to accomplish two important aspects: to analyze complex water samples, giving special attention to matrix effects, and to increase the sensitivity. Since the levels of these contaminants in the environment are extremely low, a pre-concentration methodology was also object of study in this Thesis. Dispersive liquid-liquid microextraction (DLLME) was developed for the preconcentration of E2 and EE2, subsequently quantified by either highperformance liquid chromatography (HPLC) and the previously optimized ELISAs. Moreover, the use of anthropogenic markers, i.e. indicators of human presence or activity, has been discussed as a tool to track the origin and type of contamination. An ELISA for the quantification of caffeine, as an anthropogenic marker, was also developed in order to assess the occurrence of human domestic pollution in Portuguese surface waters. Finally, photodegradation is considered to be one of the most important pathways contributing for the mitigation of pollutants’ presence in the aquatic environment. Both direct and indirect photodegradation of E2 and EE2 were evaluated. Since the presence of humic substances (HS) is known to have a noticeable influence on the photodegradation of pollutants and in order to mimic the real aquatic environment, special attention was given to the influence of the presence and concentration of different fractions of HS on the photodegradation of both hormones.