Botany origin and protein content of homemade honeys from Galicia hives (NW Spain)

The botanic origin and the protein content of 15 honeys from small bee farms exploitations of Galicia, for family consume, were studied; the aim is to check if the protein wealth and the pollen wealth are dependent parameters. Seven honeys resulted to be Rhamnus frangula unifloral (pollen patterns with low diversity), two Castanea sativa Miller unifloral, other one heather unifloral, and five was multifloral honeys of various pollen patterns (four Castanea predominant and one Rhamnus frangula predominant). Their pollen wealth was low; eight honeys classified in the Maurizio Class I, 3 in Class II, 2 in Class III, and one in Maurizio Class IV. There has been a wide variability in its protein content (0.09- 4.83 mg prot./g honey). The relative amount of pollen from different taxa has a direct or inverse proportionality to wealth protein.

Indoor air quality in elementary schools of Lisbon and Aveiro

Tem havido uma preocupação crescente com a qualidade do ar interior (QAI) nas escolas em muitos países. Muitos estudos epidemiológicos têm encontrado diferenças regionais entre ambientes interiores. Apesar da elevada incidência de asma e rinite na população infantil, praticamente nada se sabia sobre a QAI em escolas portuguesas. A percepção dos problemas de QAI é crucial para avaliar os riscos para a saúde e rendimento dos estudantes, e para sugerir meios de reduzir a exposição a poluentes indesejáveis. Neste estudo procurou-se obter as concentrações de poluentes de interesse em estabelecimentos de ensino do 1º ciclo de Lisboa e Aveiro, estimar o estado atual de casos de asma e rinite em escolas primárias da capital, avaliar a influência de diferentes materiais das salas de aula/construção e hábitos escolares na QAI, identificar potenciais fontes de poluentes nos interiores e exteriores das salas de aula e propor medidas mitigadoras. Catorze escolas de Lisboa foram visitadas para obter a caracterização física das construções em termos de estrutura, ventilação, materiais de acabamento, produtos de limpeza, densidade de ocupação e potenciais fontes interiores de poluição. Os estudantes foram questionados sobre os seus hábitos e sintomas respiratórios através de inquéritos do modelo ISAAC (International Study of Asthma and Allergies in Childhood). Durante a primavera, outono e inverno (2008-2010), nas salas de aula e pátios, foram monitorizados, por amostragem passiva, compostos orgânicos voláteis (COVs), carbonilos e dióxido de azoto (NO2). Foram também medidos parâmetros de conforto e níveis de microrganismos. Duas escolas localizadas, uma no centro da cidade e outra na região suburbana, em Aveiro foram estudadas em 2010. Parâmetros de conforto, microrganismos, COVs, NO2, material particulado (PM10) foram medidos no interior e no exterior de ambas escolas. Os iões solúveis, carbono orgânico e elementar (OC e EC), e compostos orgânicos presentes no material particulado foram subsequentemente analisados em laboratório. Uma medida mitigadora - fitoremediação - foi avaliada na escola do centro da cidade de Aveiro em 2011. Os resultados do estudo mostraram que a QAI é pior do que a do ar exterior. Em geral, os níveis de CO2 e dos bioaerosóis excederam os níveis máximos aceitáveis para o conforto dos ocupantes estipulado pelas regulamentações portuguesas. Quase todos os COVs e carbonilos identificados mostraram razões interior/exterior (I/E) maiores que uma unidade, o que demonstra a importante contribuição de fontes interiores em todas as escolas. As razões I/E das concentrações de NO2 nunca excederam a unidade. Os níveis interiores diários de PM10 foram sempre maiores que os exteriores, exceto nos fins de semana. Após a colocação de plantas numa das salas de aula, observou-se uma redução estatisticamente significativa nos níveis de CO2, COVs, carbonilos, PM10, OC, e dos iões nitrato, sulfato, amónia, cálcio e carbonato. A possível redução dos níveis de poluentes no interior após a colocação de plantas pode representar uma solução de baixo custo para reduzir a exposição a muitos compostos, melhorar o rendimento e aumentar o bem estar dos alunos e professores em sala de aula.

Photoinactivation of viruses by porphyrins

A inativação fotodinâmica tem sido usada com sucesso na inativação de microorganismos. Diversos aspetos da inativação fotodinâmica foram já estudados para diferentes microrganismos, contudo, existe ainda pouca informação disponível no que diz respeito à inativação de bacteriófagos por processos fotodinâmicos. Este trabalho pretendeu elucidar e avaliar vários aspetos da fotoinativação de vírus, em particular de bacteriófagos, incluindo (i) o efeito de diversos parâmetros de luz utilizados na fotoinativação de bacteriófagos; (ii) a eficiência da inativação fotodinâmica de diferentes tipos de bacteriófagos (fagos do tipo DNA e RNA); (iii) o principal mecanismo através do qual a inativação fotodinâmica tem lugar; (iv) o efeito da fotoinativação nas proteínas do bacteriófago; e (v) o possível desenvolvimento de resistência e recuperação da viabilidade após vários tratamentos fotodinâmicos consecutivos. Para avaliar o efeito dos diferentes parâmetros de luz, suspensões fágicas com 107 UFP mL-1 foram irradiadas com diferentes fontes e doses de luz, intensidades luminosas e tempos de irradiação (30,90 e 270 min) na presença de 0,5; 1,0 e 5,0 μM dos derivados porfirínicos catiónicos Tri- Py+-Me-PF e Tetra-Py+-Me. A eficiência da fotoinativação de diferentes fagos do tipo DNA e RNA, foi avaliada através da irradiação da suspensão fágica com luz branca (40 W m-2) durante 270 min na presença de 0,5 e 5,0 μM do derivado porfirínico Tri-Py+-Me-PF, respetivamente para os fagos do tipo RNA e DNA. O mecanismo através do qual a fotoinativação de fagos de DNA (fago do tipo T4) e de RNA (fago Qb) tem lugar foi avaliado por exposição da suspensão fágica à luz branca com uma potência de 40 W m-2, na presença de fotossensibilizador (Tri-Py+-Me-PF e Tetra-Py+-Me) e inibidores, quer do oxigénio singuleto (azida de sódio e L-histidina) quer de radicais livres (Dmanitol e L-cisteína). Os danos nas proteínas do fago do tipo T4, induzidos pelas espécies reativas de oxigénio geradas por 5,0 μM Tri-Py+-Me-PF, foram avaliados pelo método convencional de SDS-PAGE e por espectroscopia de infravermelho. O possível desenvolvimento de resistência e recuperação da viabilidade após a inativação fotodinâmica dos bacteriófagos foi avaliado após dez ciclos consecutivos de tratamento fotodinâmico incompletos (120 min sob irradiação de luz branca a uma potência de 40 W m-2) na presença de 5,0 μM do derivado porfirínico Tri-Py+-Me-PF. Os resultados deste trabalho mostraram que (i) quando uma quantidade de energia (dose de luz) determinada foi aplicada numa suspensão fágica, a partir de uma mesma fonte irradiação, a fotoinactivação do fago foi tanto mais eficiente quanto mais baixa foi a potência luminosa aplicada; (ii) os bacteriófagos foram eficientemente inativados até ao limite de deteção (redução de 6-7 log); (ii) os fagos do tipo RNA foram inativados mais facilmente do que os fagos do tipo DNA (tempos de exposição mais curtos e com concentração de fotossensibilizador dez vezes menor do que a usada para inativar os fagos do tipo DNA); (iii) o mecanismo do tipo II (via produção de oxigénio singuleto) foi o principal mecanismo através do qual a fotoinativação dos bacteriófagos teve lugar; (iv) foi possível detectar danos no perfil proteico após tratamento fotodinâmico e a espectroscopia de infravermelho apresentou-se como uma metodologia promissora de screening para avaliação dos danos induzidos pela inativação fotodinâmica em proteínas; e (v) após dez ciclos consecutivos de tratamento fotodinâmico, o fago do tipo T4 não revelou nenhum tipo de resistência ao tratamento fotodinâmico nem recuperou a sua viabilidade. Como conclusão, a inativação fotodinâmica microbiana é uma tecnologia bastante eficaz para a fotoinativação de bacteriófagos do tipo DNA e RNA sem invólucro, a qual pode ser considerada como uma alternativa ao tratamento convencional com agentes antivíricos, mesmo com intensidades luminosas baixas, sem o risco associado de desenvolvimento de mecanismos de resistência.

New oxomolybdenum(VI) hybrid materials

In this thesis, 2,2’-bipyridine (bipy), di-tert-butyl-2,2’-bipyridine (di-t-Bubipy), 2,2’-bipyridine-5,5’-dicarboxylic acid (H2bpdc), 2-[3(5)-pyrazolyl]pyridine (pzpy) and 2-(1-pentyl-3-pyrazolyl)pyridine (pent-pp) ligands were used as the N,N-chelate ligands in the formation of discrete [MoO2Cl2L]-type complexes. These complexes were employed as precursors for the preparation in aqueous media of oxomolybdenum(VI) products with a wide range of structural diversity. Three distinct heating methods were studied: hydrothermal, reflux or microwave-assisted synthesis. An alternative reaction with the inorganic molybdenum(VI) trioxide (MoO3) and the ligands di-t-Bu-bipy, H2bpdc and pzpy was also investigated under hydrothermal conditions. The distinct nature of the N,N-chelate ligands and/or the heating method employed promoted the isolation of a series of new oxomolybdenum(VI) hybrid materials that clearly reflected the strong structure-directing influence of these ligands. Thus, this thesis describes the synthesis and characterization of the discrete mononuclear [MoO2Cl2(pent-pp)], the dinuclear [Mo2O6(di-t-Bu-bipy)2] and the octanuclear [Mo8O22(OH)4(di-t-Bu-bipy)4] complexes as well as the highly unique polymeric materials {[MoO3(bipy)][MoO3(H2O)]}n, (DMA)[MoO3(Hbpdc)]·nH2O, [Mo3O9(pzpy)]n and [Mo2O6(pent-pp)]n (fine structural details of compound [Mo2O6(pent-pp)]n are presently unknown; however, characterization data strongly pointed toward a polymeric oxide hybrid compound). The catalytic behaviour of the discrete complexes and the polymeric compounds was tested in olefin epoxidation reactions. Compounds [Mo3O9(pzpy)]n and [Mo2O6(pent-pp)]n acted as sources of soluble active species that where identified as the oxodiperoxido complexes [MoO(O2)2(pzpy)] and [MoO(O2)2(pent-pp)], respectively. The majority of the compounds here presented were fully characterized by using solid-state techniques, namely elemental analyses, thermogravimetry, FT-IR, solid-state NMR, electron microscopy and powder X-ray diffraction (both from laboratory and/or synchrotron sources).

Photodynamic inactivation of bacteria by cationic porphyrins : their cellular targets and potential environmental applications

Photodynamic inactivation (PDI) is defined as the process of cell destruction by oxidative stress resulting from the interaction between light and a photosensitizer (PS), in the presence of molecular oxygen. PDI of bacteria has been extensively studied in recent years, proving to be a promising alternative to conventional antimicrobial agents for the treatment of superficial and localized infections. Moreover, the applicability of PDI goes far beyond the clinical field, as its potential use in water disinfection, using PS immobilized on solid supports, is currently under study. The aim of the first part of this work was to study the oxidative modifications in phospholipids, nucleic acids and proteins of Escherichia coli and Staphylococcus warneri, subjected to photodynamic treatment with cationic porphyrins. The aims of the second part of the work were to study the efficiency of PDI in aquaculture water and the influence of different physicalchemical parameters in this process, using the Gram-negative bioluminescent bacterium Vibrio fischeri, and to evaluate the possibility of recycling cationic PS immobilized on magnetic nanoparticles. To study the oxidative changes in membrane phospholipids, a lipidomic approach has been used, combining chromatographic techniques and mass spectrometry. The FOX2 assay was used to determine the concentration of lipid hydroperoxides generated after treatment. The oxidative modifications in the proteins were analyzed by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Changes in the intracellular nucleic acids were analyzed by agarose gel electrophoresis and the concentration of doublestranded DNA was determined by fluorimetry. The oxidative changes of bacterial PDI at the molecular level were analyzed by infrared spectroscopy. In laboratory tests, bacteria (108 CFU mL-1) were irradiated with white light (4.0 mW cm-2) after incubation with the PS (Tri-Py+-Me-PF or Tetra-Py+-Me) at concentrations of 0.5 and 5.0 μM for S. warneri and E. coli, respectively. Bacteria were irradiated with different light doses (up to 9.6 J cm-2 for S. warneri and up to 64.8 J cm-2 for E. coli) and the changes were evaluated throughout the irradiation time. In the study of phospholipids, only the porphyrin Tri-Py+-Me-PF and a light dose of 64.8 J cm-2 were tested. The efficiency of PDI in aquaculture has been evaluated in two different conditions: in buffer solution, varying temperature, pH, salinity and oxygen concentration, and in aquaculture water samples, to reproduce the conditions of PDI in situ. The kinetics of the process was determined in realtime during the experiments by measuring the bioluminescence of V. fischeri (107 CFU mL-1, corresponding to a level of bioluminescence of 105 relative light units). A concentration of 5.0 μM of Tri-Py+-Me-PF was used in the experiments with buffer solution, and 10 to 50 μM in the experiments with aquaculture water. Artificial white light (4.0 mW cm-2) and solar irradiation (40 mW cm-2) were used as light sources.

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.

DPSIR framework application supported by integrated watershed modeling for irrigation from Alqueva dam (Portugal) on Guadiana river estuary

Dissertação de mest., Gestão da Água e da Costa, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

Application of DPSIR framework integrated with multiple regression model to simulate Guadiana catchment - estuary continuum, south eastern Iberia

Dissertação de Mestrado, Gestão da Água e da Costa, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2010

Role of the different actin genes during extrusion of capping protein mutant cells in the Drosophila wing blade epithelium

Dissertação mest., Biotecnologia, Universidade do Algarve, 2008

Estudo do efeito de contaminantes na espécie sentinela, a amêijoa Ruditapes decussatus, por intermédio da análise da expressão proteica: aplicação à monitorização do meio marinho

Tese dout., Ciências e Tecnologias do Ambiente, 2009, Universidade do Algarve

Avaliação da inclusão de EVOCOL em dietas formuladas para juvenis de Linguado (Solea senegalensis, Kaup 1858)

Dissertação de mest., Biologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2010

Dissection of the early steps in the assembly of the 20S proteasome in Saccharomyces cerevisiae

Tese de dout., Bioquímica (Biologia Celular e Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Tolerância à amónia e grau de ureotelia no xarroco, Halobatrachus didactylus (Bloch & Schneider, 1801)

Tese mest., Biologia Marinha, Universidade do Algarve, Faculdade de Ciências do Mar e do Ambiente, 2008

Estudo da reversão das alteracções funcionais da actina após processo de oxidação/redução

Relatório de estágio da licenciatura, Bioquímica, Faculdade de Ciência e Tecnologia da Universidade do Algarve, 2007

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.