Effect of polyunsaturated fatty acids (PUFAs) on the proliferation and extracellular matrix mineralization and gene expression of gilthead seabream skeletal cell lines

The aquaculture industry aims at replacing significant amounts of marine fish oil by vegetable oils in fish diet. Dietary lipids have been shown to alter the fatty acid composition of bone compartments, which would impact the local production of factors controlling bone formation. Knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is however scarce in fish. Two in vitro bone-derived cell systems developed from seabream (an important species for aquaculture in the Mediterranean region) vertebra, capable of in vitro mineralization and exhibiting prechondrocyte (VSa13) and pre-osteoblast (VSa16) phenotype, were used to assess the effect of certain polyunsaturated fatty acids (PUFAs; arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) on cell proliferation, extracellular matrix (ECM) mineralization and gene expression. While all PUFAs promoted morphological changes in both cell lines, VSa16 cell proliferation appeared to be stimulated by PUFAs in a dose dependent manner until 100M, whereas proliferation of VSa13 cells was impaired at concentrations above 10M. AA, EPA and DHA inhibited VSa13 ECM mineralization, alone and in combination, while VSa16 ECM mineralization was only inhibited by AA and EPA. DHA had the opposite effect, increasing mineralization almost by 2 fold. When EFAs were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. Expression of marker genes for bone and lipid metabolisms has been investigated by qPCR and shown to be regulated in pre-osteoblasts exposed to individual PUFAs. Our results show that PUFAs are effectors of fish bone cell lines, altering cell morphology, proliferation and mineralization when added to culture medium. This work also demonstrates the suitability of our in vitro cell systems to get insights into mineralization-related effects of PUFAs in vivo and to evaluate the replacement of fish oils by vegetable oil sources in fish feeds.

A proteomic study of the effects of nutrient restriction in utero on postnatal metabolism

It is widely recognized that protein restriction in utero may cause metabolic and endocrine adaptations, which may be of benefit to the neonate on a short-term basis but may cause adverse long-term conditions such as obesity, Type 2 diabetes, metabolic syndrome, hypertension and cardiovascular diseases. Adequate foetal and early post natal nutrient and energy supply is therefore essential for adult animal health, performance and life span. In this project it was investigated the progressive adaptations of the hepatic proteome in male mink offspring exposed to either a low protein (FL) or an adequate protein (FA) diet in utero fed either on a low protein (LP) or on an adequate (AP) diet from weaning until sexual maturity. Specifically, the aim was to determine the metabolic adaptations at selected phases of the animal’s first annual cycle and establish the metabolic priorities occurring during those phases. The three different morphological stages studied during the first year of development included, end of bone growth at 4 months of age, maximal fat accretion at 6 months of age and sexual maturity at 12 months of age. A reference proteome of mink liver coming from these different animal groups were generated using 2D electrophoresis coupled to MALDI-TOF analysis and the way in which dietary treatment affect their proteome was established. Approximately 330 proteins were detected in the mink liver proteome. A total of 27 comparisons were carried out between all different animal groups which resulted in 20 differentially expressed proteins. An extensive survey was conducted towards the characterization of these proteins including their subcellular localization, the biological processes in which they are involved and their molecular functions. This characterization allowed the identification of proteins in various processes including the glycolysis and fatty acid metabolism. The detailed analysis of the different dietary treatment animal groups was indicative of differences in metabolism and also to changes associated with development in mink.

A incorporação das preferências do utilizador no desenvolvimento de software para dados biomédicos: uma contribuição da análise conjunta nas tecnologias de informação da saúde

O aumento significativo de patologias relacionadas com a alimentação, designadamente a obesidade, considerada pela Organização Mundial de Saúde como a “epidemia do século XXI”, remete o profissional da área da nutrição para a necessidade de uma intervenção cada vez mais eficiente. O objectivo deste estudo é avaliar a necessidade do desenvolvimento de novas tecnologias que possibilitem o auxílio aos processos de apoio nutricional, assim como medir as preferências no desenvolvimento de software. Para isso, utilizou-se a Análise Conjunta, na qual os resultados agregados foram hierarquizados mediante uma Análise Categórica de Componentes Principais, para posterior segmentação dos individuos, mediante uma Análise de Clusters Hierárquicos. Neste estudo, pode verificar-se que não existe preferências no desenvolvimento de software genérico, havendo uma grande receptividade dos profissionais de saúde ao desenvolvimento da Telemedicina na área da nutrição. Por fim, existem diferenças, estatisticamente significativas, entre os profissionais da área da nutrição, o que permite a sua caracterização.

Interacções tróficas entre sardinha (S. pilchardus) e cavala (S. colias)

A sardinha Ibero-Atlântica (Sardina pilchardus) e a cavala (Scomber colias) são importantes espécies pelágicas dos ecossistemas marinhos do Atlântico, quer do ponto de vista ecológico, quer do ponto de vista económico. Esta tese tem por objectivo estudar a alimentação de sardinha e de cavala e possíveis interacções tróficas entre ambas as espécies visto que estas partilham espaço e recursos alimentares. Estudou-se a dieta das duas espécies, capturadas na costa sul portuguesa entre Junho e Setembro, através da análise de presas nos conteúdos estomacais. Verificou-se que as principais presas nas suas dietas foram crustáceos (maioritariamente copépodes) pertencentes aos mesmos grupos taxonómicos, não se tendo verificado uma partição do zooplâncton com base no tamanho para as duas espécies. Os dados obtidos apontaram para a existência de uma sobreposição das dietas e potencial para competição. A sardinha apresentou um coeficiente de vacuidade elevado que foi mais acentuado quando em presença da cavala e observou-se a existência de uma alteração na dieta de cavala passando a alimentar-se de itens de maiores dimensões à medida que aumentava de tamanho. Os resultados constatam a existência provável de interacções tróficas entre estas duas espécies, sardinha e cavala.

Nutrition and farming practices as modulators of quality in gilthead seabream (Sparus aurata)

Gilthead seabream is the most important farmed species in the Mediterranean, and knowledge on how common farming practices impact its quality is limited. As such, this Thesis aimed to evaluate how gilthead seabream flesh quality is affected by some of these practices. In Chapter 2, the influence of nutritional factors was evaluated, specifically the high replacement of traditional marine-derived ingredients, both fishmeal and fish oil, with vegetable sources. We have seen that the vegetable-based diets tested did not greatly impact seabream flesh quality, although some alterations were seen in the fatty acid profile of the muscle. However, and despite having caused no alterations in flesh texture, vegetable ingredients reduced the amount of sulphated glycosaminoglycans in the extracellular matrix, affected muscle pH and reduced the activity of proteolytic enzymes. Throughout this Thesis, we measured for the first time the activity of proteolytic enzymes in seabream muscle, and cathepsin B was found to play a pivotal role in post-mortem muscle degradation. In Chapter 3, we evaluated the effect of harvesting and slaughter stress on seabream quality, and contrary to what is seen in most farmed species, our results show that gilthead seabream muscle structure is highly resistant to changes caused by stressful events. Nonetheless, considering that welfare is an increasingly important quality criterion, the use of a zero-withdrawal anaesthetic as a rested harvest technique or even slaughter method could prove valuable to the industry. In Chapter 4, we used maslinic acid as a dietary supplement, to modulate the muscle’s energetic status pre-mortem. As a finishing strategy, maslinic acid failed to increase levels of glycogen and ATP in the muscle. However, supplementation resulted in higher muscle fibre diameter and lower cathepsin B activity, and maslinic acid is likely to be useful to promote growth in this species. In general our Thesis has generated new knowledge to a major challenge facing the aquaculture industry, which is to find a compromise between the trends towards intensive rearing and consumer demand for healthy, high quality seafood being ethically acceptable and having a low impact on the environment.

A influência do perfeccionismo parental, dos estilos parentais e dos padrões de vinculação no desenvolvimento do perfeccionismo em jovens adultos

As conceções clássicas sobre as origens do perfeccionismo e os principais modelos explicativos do seu desenvolvimento assentam no pressuposto de que a relação parental constitui um importante fator na compreensão deste traço de personalidade nos filhos. Apesar do suporte teórico e conceptual, os resultados da investigação empírica são parciais e pouco conclusivos. Neste contexto, definimos como objetivos principais analisar a influência do Perfeccionismo parental no desenvolvimento do Perfeccionismo dos filhos jovens adultos e compreender o papel dos Estilos Parentais e dos Padrões de Vinculação. Procurou-se ainda averiguar se o efeito destas variáveis era moderado pelo sexo do progenitor ou pelo sexo do filho. Os resultados encontrados permitem um conhecimento mais aprofundado sobre o papel destes fatores parentais. A perceção de um Estilo Parental Autoritário e de uma Vinculação pouco Segura aos pais parece contribuir marcadamente para o desenvolvimento do Perfeccionismo Desadaptativo. Por outro lado, um Estilo Parental Democrático e uma Vinculação Segura parecem apenas influenciar uma parte do Perfeccionismo o Adaptativo. O estudo do contributo diferencial de cada um dos progenitores no desenvolvimento do perfeccionismo em função do sexo do filho permitiu testar as duas hipóteses descritas na literatura sobre o papel moderador do sexo. Os dados sugerem que os filhos desenvolvem tendências perfeccionistas pela identificação com o progenitor do mesmo sexo (hipótese do cuidador do mesmo sexo), enquanto a componente desadaptativa do perfeccionismo parece desenvolve-se tanto nos filhos do sexo masculino como nas filhas quando a mãe manifesta também níveis elevados de Perfeccionismo Desadaptativo (hipótese do cuidador principal). Os resultados apoiam ainda o Modelo da Aprendizagem Social e o Modelo das Expectativas Sociais formulados por Flett, Hewitt, Oliver e Macdonald (2002). Por último, são discutidas as implicações destes resultados na prevenção e na intervenção psicológica com pais e filhos e apontadas algumas das limitações do presente trabalho que poderão ser colmatadas em futuras investigações.

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.

Estrutura de comunidades de peixes, amostradas por SVSI - sistema de vídeo subaquático iscado, na Costa Sul de Portugal

Desenvolveu-se um Sistema de Vídeo Submerso Iscado (SVSI) viavelmente adaptado para caracterizar comunidades de peixes do substrato rochoso subtidal (forma não-destrutiva e não-invasiva), entre 5 e 50 metros, na costa sul de Portugal (Algarve). Entre Junho e Julho de 2013, amostraram-se 15 pontos na zona Greta (Praia de Faro), divididos em 3 Amostragens (A1, A2, A3) com isco sardinha, mexilhão e sem isco. Captaram-se foto-sequências de intervalo 1 segundo durante 60 minutos e as imagens resultantes processadas qualitativa e quantitativamente para obtenção de resultados. Registaram-se 32 espécies no total. Os tempos de entrada do 1º indivíduo de cada espécie permitiram aferir 30 minutos máximos de filmagem SVSIs para resultados viáveis, enquanto abundâncias por número de indivíduos comprovaram o isco sardinha como mais eficaz e com maior poder atrativo (do que mexilhão). As espécies Coris julis, Diplodus vulgaris, Diplodus cervinus, Diplodus sargus e Serranus cabrilla foram as mais interventivas no estudo, cujo intervalo de tempo entre contagens se adequou em 30 segundos. O estudo de índices de abundâncias e Nmax mostraram não existirem diferenças entre as comunidades ictiológicas de cada tipo de iscagem, contudo existem diferenças dentro da comunidade geral em si do substrato rochoso. A análise alimentar pelo menor tempo de 1ª alimentação indicou Coris julis, Diplodus vulgaris, Serranus cabrilla e Octopus vulgaris como as espécies mais rápidas a morder os iscos. No geral, 69% das espécies preferiram isco sardinha. Comportamentos como a passagem esporádica, territorialidade e interações com a câmara foram as ações dominantes na análise comportamental. Esta técnica e respetiva metodologia foram o primeiro estudo do género nesta região, que de forma simples, se mostrou uma boa ferramenta metodológica para estudos marinhos sobre comunidades, suas preferências alimentares, abundâncias e tempos de reação, aconselhando-se o seu desenvolvimento tecnológico e melhoramento metodológico analítico dos resultados no futuro.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

The Rathbone Hotel: internship

The main objective of the internship is to complete the requirement of the Master program as prescribed by the University of the Algarve. An intern has to prepare an internship report at the end of the internship period but the main objective of the internship is to get experience of the real world organisation. The internship was concluded with the objective of getting practical knowledge in all departments at The Rathbone Hotel. The first responsibility I was assigned during these 12 months of internship period was to assist the Housekeeping Department. Secondly, I was allotted the work of F&B, Accounts, Reservations, Front Office, Back Office and assisting the Assistant Manager was the last task I was assigned during my Internship program. As an intern, I realised that I was successful to gather a lot of significant learning experiences which would be helpful in my future career. Work in all departments of The Rathbone Hotel offered me ample space and opportunities, not only to learn but also to exhibit my skills as a Rathbone team member. I could use my theoretical knowledge in real practice while participating in many discussions. I was actively involved in the revenue meetings where I shared my knowledge and views regarding the performance in Reservations of The Rathbone Hotel. I successfully completed all the assigned duties and passed them over to the supervisor at the end of the internship. I thoroughly enjoyed the challenges that came along every single day. These lessons that I have learned will be a valuable one for my future activities as well.

A pintura rupestre do centro de Portugal: antropização simbólica da paisagem pelas primeiras sociedades agro-pastoris

Esta dissertação tem como objectivo principal tentar compreender os mecanismos conceptuais que levaram à marcação de determinados locais, através de pinturas rupestres esquemáticas, por grupos humanos durante o Neolítico e Calcolítico. Foram analisados diversos núcleos de abrigos com pinturas rupestres localizados no centro do território português: o Abrigo do Ribeiro das Casas (Almeida), Abrigo de Segura (Idanha-a-Nova), Abrigos do Pego da Rainha (Mação), Abrigo do Lapedo (Leiria), Lapa dos Coelhos (Torres Novas), Lapa dos Louções (Arronches), Igreja dos Mouros (Arronches) e Abrigo Pinho Monteiro (Arronches). A análise das características formais e técnicas de execução (incluindo uma abordagem arqueométrica aplicada aos pigmentos) permitiram tecer considerações sobre a metodologia utilizada no momento de realização das pinturas. Através do estudo das características de implantação de cada sítio foi possível estabelecer quatro padrões de localização dos abrigos pintados, que, juntamente com a descrição sistemática dos motivos representados e sua interligação permitiram a criação de uma periodização em dois períodos distintos. Numa primeira fase, temos a iconografia de transição das comunidades recolectoras para os primeiros grupos agro-pastoris, que exibe ainda pormenores da imaginética paleolítica, surgindo zoomorfos de grandes dimensões e antropomorfos com caracteres formais, correspondendo assim a uma arte pré-esquemática. Com a consolidação do sistema agro-pastoril, a sedentarização e a complexificação económico-social, os esquemas de antropização da paisagem e do mundo conceptual colectivo alteram-se profundamente, originando representações reduzidas aos seus elementos mais básicos, tornando-se totalmente esquemáticas. Ocorre uma transmutação dos motivos em ideogramas, dando origem ao segundo período intitulado de arte esquemática ideográfica, cujo enquadramento cronológico abarca o período compreendido entre o Neolítico Final e o início da Idade do Bronze. Segundo muitos autores, a arte esquemática peninsular deverá ser vista como um processo muito heterogéneo resultante das próprias dinâmicas das comunidades que a produziram, e deste modo apenas passível de ser analisada num âmbito regional alargado. Por estas razões a periodização estabelecida deverá, por agora, ser aplicada apenas à área geográfica em estudo na presente dissertação

Resposta fisiológica e bioquímica ao jejum na dourada (sparus aurata)

Dissertação de Mestrado, Aquacultura, Unidade de Ciências e Tecnologias dos Recursos Aquáticos, Universidade do Algarve, 1997

Antioxidant and phenolic compounds in sweet potato peels and leaves: food applications and health benefits

Tese de doutoramento, Ciências Biotecnológicas (Biotecnologia Alimentar), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Sistemas informáticos em saúde para a qualidade dos cuidados de enfermagem, revisão sistemática

Dissertação de mestrado, Gestão de Unidades de Saúde, Faculdade de Economia, Universidade do Algarve, 2015

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015