Potential of fisheries restocking off the Algarve coast using aquaculture produced marine fish

Tese de dout., Ciências do Mar, da Terra e do Ambiente (Ecologia Marinha), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2012

Avaliação do potencial da Salicornia ramosissima para saladas frescas ou em pó (sal verde)

Dissertação de mest., Tecnologia dos Alimentos , Instituto Superior de Engenharia, Univ. do Algarve, 2013

Avaliação da espectroscopia de infravermelho para determinação de características físico-químicas da carne suína

Dissertação de mest., Tecnologia de Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2013

Interacção do vanádio com a miosina de músculo esquelético de coelho

Relatório de projecto de licenciatura, Bioquímica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2009

Avaliação da qualidade da água das ribeiras Odelouca, Odeleite e Foupana (Algarve, Portugal): uso de macroinvertebrados bentónicos como bioindicadores

São várias as perturbações antropogénicas que afectam os rios e as ribeiras em Portugal, razão pela qual é urgente a implementação de medidas que visem a sua protecção. Neste contexto, foi criada em 2000 a directiva quadro da água (DQA) que visa atingir o bom estado das massas de água até 2015. Este trabalho teve como objectivos: contribuir para o conhecimento da comunidade de macroinvertebrados nas ribeiras do Algarve; verificar a existência de diferenças na comunidade de macroinvertebrados entre ribeiras e em função dos habitats; comparar duas metodologias (INAG e “Habitats”) de amostragem para avaliação da qualidade ecológica da água; avaliar o estado ecológico (através de índices bióticos: IBMWP and IPtIs) das ribeiras de Odelouca, Foupana e Odeleite utilizando para este fim os macroinvertebrados. Estes índices são baseados no grau de tolerância à poluição e abundância de cada família. Os macroinvertebrados bentónicos (bioindicadores) foram recolhidos com uma rede de arrasto manual (malha 500μm), sendo o esforço de amostragem de 6 arrastos/ponto. Não se encontraram diferenças em termos de índices ecológicos e da estrutura da comunidade de macroinvertebrados (ao nível das famílias) quer entre ribeiras como entre habitats. A análise da estrutura da comunidade não diferiu para ambas as metodologias testadas, sugerindo que na metodologia INAG não existe perda de informação relacionada com a insuficiência de amostragens em determinados habitats. No entanto, verifica-se uma tendência para valores dos índices bióticos apresentarem sempre valores iguais ou superiores no caso da metodologia “Habitats. Os valores dos índices de qualidade da água permitiram classificar na generalidade a qualidade da água nas ribeiras em estudo como boa. A informação relativa aos índices bióticos deve ser complementadas com análises físico-químicas, ou com métodos como o River Habitat Survey (RHS), que permite determinar o índice “Habitat Quality Assessment” (HQA), que expressa as características naturais nos rios importantes para os organismos (número de zonas de corrente rápida, árvores), e “Habitat Modification Score” (HMS), que quantifica as alterações ocorridas nas ribeiras (ocupação do solo em torno dos rios: agricultura, pontes).

Differential requirements of aPKC activity within different epithelial tissues

Epithelial tissues are essential during morphogenesis and organogenesis. During development, epithelial tissues undergo several different remodeling processes, from cell intercalation to cell change shape. An epithelial cell has a highly polarized structure, which is important to maintain tissue integrity. The mechanisms that regulate and maintain apicobasal polarity and epithelial integrity are mostly conserved among all species and in different tissues within the same organism. aPKC-PAR complex localizes in the apical domain of polarized cells, and its function is essential for apicobasal polarization and epithelial integrity. In this work we characterized two novel alleles of aPKC: a temperature sensitive allele (aPKCTS), which has a point mutation on a kinase domain, and another allele with a point mutation on a highly conserved amino acid within the PB1 domain of aPKC (aPKCPB1). Analysis of the aPKCTS mutant phenotypes, lead us to propose that during development different epithelial tissues have differential requirements of aPKC activity. More specifically, our work suggests de novo formation of adherens junctions (AJs) is particularly sensitive to sub-optimal levels of apkc activity. Analysis of the aPKCPB1 allele, suggests that aPKC is likely to have an apical structural function mostly independent of its kinase activity. Altogether our work suggests that although loss of aPKC function is associated to similar epithelial phenotypes (e.g., loss of apicobasal polarization and epithelial integrity), the requirements of aPKC activity within these tissues are nevertheless likely to vary.

Comportamento in vitro de formulações lipossomais de um aminoglicosídeo com actividade anti-parasitária: citotoxicidade e interacção lipossoma-célula

Os sistemas de veiculação de fármacos, presentemente, apresentam-se como uma alternativa viável às terapias convencionais. De entre os diversos sistemas de transporte passíveis de associar substâncias farmacologicamente activas, destacam-se os de base lipídica, em particular, os lipossomas, os quais constituem um dos sistemas mais estudados e com maior sucesso, comprovado pelo número de produtos em fase clínica ou já aprovados para utilização em humanos. Os lipossomas são estruturas constituídas por membranas lipídicas organizadas em bicamadas, fechadas e concêntricas, que permitem a encapsulação de moléculas hidrofílicas no espaço interno aquoso e hidrofóbicas na bicamada lipídica. No presente trabalho, foram desenvolvidas metodologias com vista à encapsulação em lipossomas do aminoglicosídeo, a Paromomicina (PRM). Este fármaco está indicado para o tratamento de doenças infecciosas nomeadamente parasitárias e bacterianas. Algumas das principais desvantagens resultantes da sua utilização em clínica são, o reduzido tempo de circulação na corrente sanguínea, rápida excreção renal e consequentemente insuficiente concentração intracelular do fármaco. Como forma de ultrapassar algumas das desvantagens apresentadas, foram desenvolvidas formulações lipossomais de PRM com vista a melhorar o desempenho deste antibiótico. Para tal, foram preparadas e caracterizadas diversas formulações lipossomais de PRM com vista à selecção daquelas que apresentem maiores valores de eficácia de encapsulação (E.E.), e superior estabilidade. Com as formulações seleccionadas foram realizados estudos in vitro de interacção lipossoma-célula, utilizando uma linha celular humana monocítica leucémica, THP-1. De entre as formulações desenvolvidas e seleccionadas para os estudos in vitro de a formulação DPPC:DPPG, foi uma das que apresentou uma E.E. superior a 80% e valores de internalização celular superiores a 90%.

Insights into molecular and cellular events involved in normal and pathological vertebral development and fusion using zebrafish as model organism

The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.

Study of sewage sludge aimed at agricultural soil amendment by thermogravimetric analysis and fourier-transform infrared spectroscopy

Thermal degradation and gaseous products evolving from the pyrolysis of sewage sludge, aimed at agricultural soil amendment, were investigated using Thermogravimetric Analysis in conjunction with Fourier Transform Infrared Analysis (TG-FTIR). The materials were studied in temperatures ranging from 30 to 800 ºC. Furthermore infrared spectra of sewage sludge samples were performed as a complementary technique. In parallel the sewage sludge was spiked with ibuprofen in order to test whether the mentioned techniques are able to detect the drug. Thermal analysis showed the range of 200-400ºC as the most characteristic for weight loss, corresponding with the organic matter volatilization, while the range of 500-800ºC was also characteristic and due to the volatilization of carbonates. On the other hand, ibuprofen-spiking tests identified at temperature range (150-250ºC) where the compound totally volatilizes, therefore, in this work, the detection of ibuprofen by TGA was established for concentrations higher than 0.5 g/kg sludge, concentration 102 times higher than the concentrations measured by other authors in regular sewage sludge (Martín, et al., 2010). A correlation has been found between the ibuprofen concentrations in the sludge and the intensity of the absorption bands, both for FT-IR spectra at the maximum emission temperature for ibuprofen (232ºC) as for the FT-IR spectra of the non-pyrolyzed samples.

Aplicação de embalagens ativas com agentes naturais na preservação de alimentos

Este trabalho teve como principal objetivo estudar o efeito de 3 tipos de filme compostos por quitosano, extrato de graínha de uva (EGU) e carvacrol em três matrizes alimentares. As amostras foram embaladas e armazenadas durante 15 dias a 10 ºC e 65% de umidade. A cor (parâmetros Lab), perfil de textura, pH, atividade da água e % de umidade foram avaliados. No salmão verificou-se que o filme com maior concentração de EGU (filme 1) mostrou ser mais eficaz na manutenção da cor laranja/avermelhada (a inicial fresco-9,6; a final filme 1-9,3). Os perfis de textura seguiram o mesmo comportamento para todas as amostras (p>0,05). O pHaumentou de 6,3 a 8,8 na amostra de salmão fresco. O filme 1 foi o que se destacou com valores depHde 7,10 nos dias 1, 2 e 4 (p<0,05). A umidade e atividade da água diminuíram (58-34,49)e aumentaram, respetivamente (0,957-0,974). No abacaxi os valores normalizados de luminosidade variaram entre 0,98 (dia 1) e 0,64 (15 dias de armazenamento). Os valores normalizados de pHforam semelhantes variando entre 0,84 e 1. O fruto perdeu a sua firmeza, contudo os filme 2 e 3 mantiveram as características mais próximas das iniciais. A umidade e a atividade da água não sofreram alterações significativas (p>0,05). No queijo, o filme 3 manteve a sua cor, com valores de ae bmuito próximos à amostra fresco do dia 0. O pHe a textura não apresentaram diferenças significativas (p>0,05) ao longo do tempo. A umidade e a atividade da água apresentaram valores muito próximos, com exceção do filme 2 que diminuiu ao fim dos 15 dias para 0,92. Em geral, verificou-se uma melhoria das amostras relativamente ao ao pH e à cor e uma perda de firmeza. Conclui-se que estes agentes naturais se apresentam, em alguns parâmetros com potencial para desenvolver embalagens ativas com vista ao aumento do tempo de vida útil dos alimentos.

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.

Study of influence of regulatory polymorphisms of expression in development of breast cancer

The human genome has millions of genetics variants that can affect gene expression. These variants are known as cis-regulatory variants and are responsible for intra-species phenotypic differences and individual susceptibility to disease. One of the diseases affected by cis-regulatory variants is breast cancer. Breast cancer is one of the most common cancers, with approximately 4500 new cases each year in Portugal. Breast cancer has many genes mutated and TP53 has been shown to be relevant for this disease. TP53 is one of the most commonly mutated genes in human cancer and it is involved in cell cycle regulation and apoptosis. Previous work by Maia et al has shown that TP53 has differential allelic expression (DAE), which suggests that this gene may be under the influence of cis-regulatory variants. Also, its DAE pattern is totally altered in breast tumours with normal copy number. We hypothesized that cis-regulatory variants affecting TP53 may have a role in breast cancer development and treatment. The present work aims to identify the cis-regulatory variants playing a role in TP53 expression, using in silico, in vitro and in vivo approaches. By bioinformatic tools we have identified candidate cis-regulatory variants and predicted the possible transcription factor binding sites that they affect. By EMSA we studied DNA-protein interactions in this region of TP53. The in silico analysis allowed us to identified three candidate cis-regulatory SNPs which may affect the binding of seven transcription factors. However, the EMSA experiments have not been conclusive and we have not yet confirmed whether any of the identified SNPs are associated with gene expression control of TP53. We will carry out further experiments to validate our findings.

Alterações da paisagem decorrentes da construção da barragem do Alqueva: cenários para 2025, 2050 e 2100

A construção da barragem do Alqueva deu origem ao maior lago artificial da Europa, um enorme reservatório hídrico criado mediante o intento de aposta do regadio como elemento primordial no desenvolvimento da região do Alentejo. A esta vasta albufeira corresponde uma extensa envolvente que, em virtude da nova disponibilidade hídrica, tem apresentado alterações na paisagem em termos de uso do solo assentes na irrigação, turismo, entre outros fatores. No entanto, estas alterações poderão acarretar consequências ainda não estudadas concertadamente, como é o caso da erosão do solo que por deposição de sedimentos poderá assorear a barragem antes da mesma atingir o seu período de vida útil, comprometendo, assim, um dos maiores investimentos públicos realizados em território nacional. Tendo em vista esta problemática, a presente dissertação reveste-se de objetivos que passam pela análise da paisagem da Envolvente à Albufeira do Alqueva e cenarização das alterações de uso do solo para o futuro (2025, 2050 e 2100), seja pela elaboração de cenários projetivos de tendências passadas, como através de cenários prospetivos (produção de biomassa para bioenergia, intensificação da agricultura por via do regadio, incremento do turismo rural e desenvolvimento de resorts de golf, e alterações climáticas). Estes cenários territoriais são a peça fundamental do trabalho dissertativo que se apresenta, os quais possibilitam ensaiar acerca das implicações das alterações de uso do solo na erosão do solo, descortinadas pela variação do fator C (fator relativo à cobertura do solo) da equação de estimativa de perda de solo: Revised Universal Soil Loss Equation (RUSLE).

Molecular characterization of Gla-rich protein (GRP) gene expression and function

Gla-rich protein (GRP) is a vitamin K-dependent protein related to bone and cartilage recently described. This protein is characterized by a large number of Gla (γ-carboxyglutamic acid) residues being the protein with the highest Gla content of any known protein. It was found in a widely variety of tissues but highest levels was found in skeletal and cartilaginous tissues. This small secreted protein was also expressed and accumulated in soft tissues and it was clearly associated with calcification pathologies in the same tissues. Although the biological importance of GRP remains to be elucidated, it was suggested a physiological role in cartilage development and calcification process during vertebrate skeleton formation. Using zebrafish, an accepted model to study skeletal development, we have described two grp paralog genes, grp1 and grp2, which exhibited distinct patterns of expression, suggesting different regulatory pathways for each gene. Gene synteny analysis showed that grp2 gene is more closely related to tetrapod grp, although grp1 gene was proposed to be the vertebrate ortholog by sequence comparison. In addition, we identified a functional promoter of grp2 gene and using a functional approach we confirmed the involvement of transcription factors from Sox family (Sox9b and Sox10) in the regulation of grp2 expression. In an effort to provide more information about the function of grp isoforms, we generated two zebrafish transgenic lines capable to overexpress conditionally grp genes and possible roles in the skeleton development were studied. To better understand GRP function a mammalian system was used and the analysis of knockout mice showed that GRP is involved in chondrocyte maturation and the absence of GRP is associated to proteoglycans loss in calcified articular cartilage. In addition, we detected differences in chondrogenesis markers in articular chondrocyte primary culture. Overall, our data suggest a main role for GRP on chondrocyte differentiation.

Identification and molecular characterization of bone-related micrornas: functional implications

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014