Otimização da extração sólido-líquido de antioxidantes de subprodutos florestais pelo método de superfície de resposta

Portugal continental apresenta uma vasta área florestal, que representa cerca de 35,4% da ocupação total do solo, com predominância de espécies como o eucalipto (Eucalyptus globulus) e o pinheiro-bravo (Pinus pinaster). Estas espécies apresentam uma elevada importância a nível económico, designadamente devido à sua ampla utilização, nomeadamente na indústria de celulose e papel, gerando elevadas quantidades de resíduos. Este resíduo de biomassa florestal é utilizado, na sua totalidade, para a geração de energia, na forma de eletricidade ou aquecimento. No entanto, existem outras opções viáveis, a nível económico, tais como a valorização destes subprodutos como fonte de compostos polifenólicos tornando-os, assim, um produto de valor acrescentado. A extração de compostos fenólicos de subprodutos florestais, como folhas de eucalipto e agulhas de pinheiros tem vindo a aumentar devido, principalmente, à substituição de antioxidantes sintéticos, contribuindo para a valorização de subprodutos florestais. Contudo, apesar de todas as potenciais aplicações e vantagens, apenas algumas centenas de espécies aromáticas identificadas são utilizadas à escala comercial. Neste trabalho foi avaliada a capacidade antioxidante de subprodutos da floresta, otimizando as condições de extração através do estudo dos fatores: tempo de extração, temperatura e composição de solvente através do método de superfície de resposta. O planeamento experimental utilizado teve como base um planeamento de compósito central e a avaliação do perfil de antioxidantes das matrizes analisadas foi realizada através de métodos de quantificação total, como o teor fenólico total, a atividade anti-radicalar – método do DPPH (radical 2,2-difenil-1-picrilhidrazilo) e o método de FRAP. Estes métodos analíticos convencionais foram modificados e, devidamente validados, para a análise em leitor de microplacas. Verificou-se que os extratos de pinheiro e de eucalipto, tanto as amostras verdes com as amostras, apresentam uma promissora capacidade antioxidante. O planeamento fatorial aplicado permitiu otimizar as condições de extração em relação às matrizes verdes. Contudo, o mesmo não se verificou em relação às matrizes secas. A composição (% de água) é sem dúvida o fator com mais efeito em todas as amostras (coeficientes de primeira e segunda ordem no modelo). Também a temperatura foi identificada como um fator com efeito significativo sobre os sistemas em análise.

Recovery of acid and base from brackish water by bipolar membrane electrodialysis

Nesta dissertação pretendeu-se estudar a viabilidade do uso de eletrodiálise com membranas bipolares (BM) na recuperação de ácido clorídrico e de hidróxido de sódio a partir de um efluente industrial que contém 1.4 mol/L de cloreto de sódio. Estas membranas mostraram ser uma ferramenta eficiente para a produção de ácidos e bases a partir do respetivo sal. Foi feita uma seleção de diferentes membranas bipolares (Neosepta, Fumatech e PCA) e aniónicas (PC-SA e PC-ACID 60) na tentativa de encontrar a combinação mais adequada para o tratamento do efluente. Dependendo do critério, o melhor arranjo de membranas é o uso de PC-ACID 60 (membrana aniónica), PC-SK (membrana catiónica) e membranas bipolares do tipo Neosepta para maior pureza dos produtos; membranas bipolares Fumatech para maior eficiência de dessalinização e membranas bipolares PCA para um maior grau de dessalinização. Tecnologicamente foi possível obter uma dessalinização de 99.8% em quatro horas de funcionamento em modo batch com recirculação de todas as correntes. Independentemente da combinação usada é recomendável que o processo seja parado quando a densidade de corrente deixa de ser máxima, 781 A/m2. Assim é possível evitar o aumento de impurezas nos produtos, contra difusão, descida instantânea do pH e uma dessalinização pouco eficiente. A nível piloto o principal fornecedor de membranas e unidade de tratamento “stack” é a marca alemã PCA. Sendo assim realizaram-se ensaios de repetibilidade, contra difusão, avaliação económica e upscaling utilizando as membranas bipolares PCA. A nível económico estudou-se o uso de dois tipos de unidades de tratamento; EDQ 380 e EDQ 1600, para diferentes níveis de dessalinização (50, 75 e 80%). Tendo em conta a otimização económica, é recomendável uma dessalinização máxima de 80%, uma vez que a eficiência de processo a este ponto é de 40%. A aplicação do método com a unidade EDQ 1600 para uma dessalinização de 50% é a mais vantajosa economicamente, com custos de 16 €/m3 de efluente tratado ou 0,78 €/kg Cl- removido. O número de unidades necessárias é 4 posicionados em série.

QUESTIONNAIRES IN THE SCREENING FOR Schistosoma mansoni INFECTION: A STUDY OF SOCIO DEMOGRAPHIC AND WATER CONTACT VARIABLES IN FOUR COMMUNITIES IN BRAZIL

The use of questionnaires has been recommended for identifying, at a lower cost, individuals at risk for schistosomiasis. In this study, validity of information obtained by questionnaire in the screening for Schistosoma mansoni infection was assessed in four communities in the State of Minas Gerais, Brazil. Explanatory variables were water contact activities, sociodemographic characteristics and previous treatment for schistosomiasis. From 677, 1474, 766 and 3290 individuals eligible for stool examination in the communities, 89 to 97% participated in the study. The estimated probability of individuals to be infected, if they have all characteristics identified as independently associated with S.mansoni infection, varied from 15% in Canabrava, to 42% in Belo Horizonte, 48% in Comercinho and 80% in São José do Acácio. Our results do not support the hypothesis that a same questionnaire on risk factors could be used in screening for S.mansoni infection in different communities.

Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extracts

The cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL−1) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48–72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.

Biochemistry of grape berries: post-genomics approaches to uncover the effects of water deficits on ripening

Dissertation presented to obtain the Ph.D degree in Biochemistry, Plant Physiology

Occurrence of Cryptosporidium oocysts and Giardia cysts in raw water from the Atibaia river, Campinas, Brazil

Cryptosporidium parvum and Giardia duodenalis are waterborne parasites that have caused several outbreaks of gastrointestinal disease associated with drinking water. Due to the lack of studies about the occurrence of these protozoa in water in the Southeast of Brazil, an investigation was conducted to verify the presence of cysts and oocysts in superficial raw water of the Atibaia River. The water samples were submitted to membrane filtration (3.0 mum) and elution was processed by (1) scraping and rinsing of membrane (RM method) and (2) acetone-dissolution (ADM method). Microbiologic and chemical parameters were analyzed. Aliquots of the pellets were examined by immunofluorescence (Merifluor, Meridian Diagnostics, Cincinnati, Ohio). All water samples were positive for Cryptosporidium and Giardia, in spite of the high turbidity. Higher recovery rates occurred in samples treated by the RM method than by the ADM technique. The goal for future work is the assessment of viability of cysts and oocysts to determine the public health significance of this finding.

Recycling old screen-printed electrodes with newly designed plastic antibodies on the wall of carbon nanotubes as sensory element for in situ detection of bacterial toxins in water

Using low cost portable devices that enable a single analytical step for screening environmental contaminants is today a demanding issue. This concept is here tried out by recycling screen-printed electrodes that were to be disposed of and by choosing as sensory element a low cost material offering specific response for an environmental contaminant. Microcystins (MCs) were used as target analyte, for being dangerous toxins produced by cyanobacteria released into water bodies. The sensory element was a plastic antibody designed by surface imprinting with carefully selected monomers to ensure a specific response. These were designed on the wall of carbon nanotubes, taking advantage of their exceptional electrical properties. The stereochemical ability of the sensory material to detect MCs was checked by preparing blank materials where the imprinting stage was made without the template molecule. The novel sensory material for MCs was introduced in a polymeric matrix and evaluated against potentiometric measurements. Nernstian response was observed from 7.24 × 10−10 to 1.28 × 10−9 M in buffer solution (10 mM HEPES, 150 mM NaCl, pH 6.6), with average slopes of −62 mVdecade−1 and detection capabilities below 1 nM. The blank materials were unable to provide a linear response against log(concentration), showing only a slight potential change towards more positive potentials with increasing concentrations (while that ofthe plastic antibodies moved to more negative values), with a maximum rate of +33 mVdecade−1. The sensors presented good selectivity towards sulphate, iron and ammonium ions, and also chloroform and tetrachloroethylene (TCE) and fast response (<20 s). This concept was successfully tested on the analysis of spiked environmental water samples. The sensors were further applied onto recycled chips, comprehending one site for the reference electrode and two sites for different selective membranes, in a biparametric approach for “in situ” analysis.

Novel optical PVC probes for on-site detection/determination of fluoroquinolones in a solid/liquid interface: Application to the determination of Norfloxacin in aquaculture water

A novel optical disposable probe for screening fluoroquinolones in fish farming waters is presented, having Norfloxacin (NFX) as target compound. The colorimetric reaction takes place in the solid/liquid interface consisting of a plasticized PVC layer carrying the colorimetric reagent and the sample solution. NFX solutions dropped on top of this solid-sensory surface provided a colour change from light yellow to dark orange. Several metals were tested as colorimetric reagents and Fe(III) was selected. The main parameters affecting the obtained colour were assessed and optimised in both liquid and solid phases. The corresponding studies were conducted by visible spectrophotometry and digital image acquisition. The three coordinates of the HSL model system of the collected image (Hue, Saturation and Lightness) were obtained by simple image management (enabled in any computer). The analytical response of the optimised solid-state optical probe against concentration was tested for several mathematical transformations of the colour coordinates. Linear behaviour was observed for logarithm NFX concentration against Hue+Lightness. Under this condition, the sensor exhibited a limit of detection below 50 μM (corresponding to about 16 mg/mL). Visual inspection also enabled semi-quantitative information. The selectivity was ensured against drugs from other chemical groups than fluoroquinolones. Finally, similar procedure was used to prepare an array of sensors for NFX, consisting on different metal species. Cu(II), Mn(II) and aluminon were selected for this purpose. The sensor array was used to detect NFX in aquaculture water, without any prior sample manipulation.

Solid contact PVC membrane electrodes based on neutral or charged carriers for the selective reading of anionic sulfamethoxazole and their application to the analysis of aquaculture water

Sulfamethoxazole (SMX) is among the antibiotics employed in aquaculture for prophylactic and therapeutic reasons. Environmental and food spread may be prevented by controlling its levels in several stages of fish farming. The present work proposes for this purpose new SMX selective electrodes for the potentiometric determination of this sulphonamide in water. The selective membranes were made of polyvinyl chloride (PVC) with tetraphenylporphyrin manganese (III) chloride or cyclodextrin-based acting as ionophores. 2-nitrophenyl octyl ether was employed as plasticizer and tetraoctylammonium, dimethyldioctadecylammonium bromide or potassium tetrakis (4-chlorophenyl) borate was used as anionic or cationic additive. The best analytical performance was reported for ISEs of tetraphenylporphyrin manganese (III) chloride with 50% mol of potassium tetrakis (4-chlorophenyl) borate compared to ionophore. Nersntian behaviour was observed from 4.0 × 10−5 to 1.0 × 10−2 mol/L (10.0 to 2500 µg/mL), and the limit of detection was 1.2 × 10−5 mol/L (3.0 µg/mL). In general, the electrodes displayed steady potentials in the pH range of 6 to 9. Emf equilibrium was reached before 15 s in all concentration levels. The electrodes revealed good discriminating ability in environmental samples. The analytical application to contaminated waters showed recoveries from 96 to 106%.

Microcystin-LR detection in water by the Fabry–Pérot interferometer using an optical fibre coated with a sol–gel imprinted sensing membrane

Cyanobacteria deteriorate the water quality and are responsible for emerging outbreaks and epidemics causing harmful diseases in Humans and animals because of their toxins. Microcystin-LR (MCT) is one of the most relevant cyanotoxin, being the most widely studied hepatotoxin. For safety purposes, the World Health Organization recommends a maximum value of 1 μg L−1 of MCT in drinking water. Therefore, there is a great demand for remote and real-time sensing techniques to detect and quantify MCT. In this work a Fabry–Pérot sensing probe based on an optical fibre tip coated with a MCT selective thin film is presented. The membranes were developed by imprinting MCT in a sol–gel matrix that was applied over the tip of the fibre by dip coating. The imprinting effect was obtained by curing the sol–gel membrane, prepared with (3-aminopropyl) trimethoxysilane (APTMS), diphenyl-dimethoxysilane (DPDMS), tetraethoxysilane (TEOS), in the presence of MCT. The imprinting effect was tested by preparing a similar membrane without template. In general, the fibre Fabry–Pérot with a Molecular Imprinted Polymer (MIP) sensor showed low thermal effect, thus avoiding the need of temperature control in field applications. It presented a linear response to MCT concentration within 0.3–1.4 μg L−1 with a sensitivity of −12.4 ± 0.7 nm L μg−1. The corresponding Non-Imprinted Polymer (NIP) displayed linear behaviour for the same MCT concentration range, but with much less sensitivity, of −5.9 ± 0.2 nm L μg−1. The method shows excellent selectivity for MCT against other species co-existing with the analyte in environmental waters. It was successfully applied to the determination of MCT in contaminated samples. The main advantages of the proposed optical sensor include high sensitivity and specificity, low-cost, robustness, easy preparation and preservation.

Cytotoxicity Induced by Extracts of Pisolithus tinctorius Spores on Human Cancer and Normal Cell Lines—Evaluation of the Anticancer Potential

Fungi have been considered a potential source of natural anticancer drugs. However, studies on these organisms have mainly focused on compounds present in the sporocarp and mycelium. The aim of this study was to assess the anticancer potential of fungal spores using a bioassay-guided fractionation with cancer and normal cell lines. Crude extracts from spores of the basidiomycetous fungus Pisolithus tinctorius were prepared using five solvents/solvent mixtures in order to select the most effective crude extraction procedure. A dichloromethane/methanol (DCM/MeOH) mixture was found to produce the highest extraction yield, and this extract was fractionated into 11 fractions. Crude extracts and fractions were assayed for cytotoxicity in the human osteocarcinoma cell line MG63, the human breast carcinoma cell line T47D, the human colon adenocarcinoma cell line RKO, and the normal human brain capillary endothelial cell line hCMEC/D3. Cytotoxicity was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. The results showed a reduction in cancer cell viability of approximately 95% with 4 of 11 fractions without a significant reduction in viability of hCMEC/D3 cells. Data demonstrated that spores of P. tinctorius might serve as an interesting source of compounds with potential anticancer properties.

The effect of water inorganic matrix in ibuprofen adsorption onto activated carbon for water and wastewater treatment

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Are coffee silverskin extracts safe for topical use? An in vitro and in vivo approach

Recent changes in regulatory requirements and social views on animal testing have incremented the development of reliable alternative tests for predicting skin and ocular irritation potential of products based on new raw materials. In this regard, botanical ingredients used in cosmetic products are among those materials, and should be carefully reviewed concerning the potential presence of irritant constituents. In particular, cosmetic products used on the face, in vicinity of the eyes or that may come in contact with mucous membranes, should avoid botanical ingredients that contain, or are suspected to contain, such ingredients. In this study, we aimed to evaluate the effect of a new cosmetic ingredient, namely, coffee silverskin (CS), with an in vitro skin and ocular irritation assay using reconstructed human epidermis, EpiSkin™, and human corneal epithelial model, SkinEthics™ HCE, and an in vivo assay. Three different extracts of CS were evaluated. The histology of the models after extracts applications was analysed. The in vitro results demonstrated that extracts were not classified as irritant and the histological analyses proved that extracts did not affect both models structure. The content of caffeine, 5-hydroxymethyl furfural and chlorogenic acid was quantified after the epidermal assay. The in vivo test carried out with the most promising extract (hydroalcoholic) showed that, with respect to irritant effects, these extracts can be regarded as safe for topical application.

Water jet based autonomous surface vehicle for coastal waters operations

This paper presents the design of low cost, small autonomous surface vehicle for missions in the coastal waters and specifically for the challenging surf zone. The main objective of the vehicle design described in this paper is to address both the capability of operation at sea in relative challenging conditions and maintain a very low set of operational requirements (ease of deployment). This vehicle provides a first step towards being able to perform general purpose missions (such as data gathering or patrolling) and to at least in a relatively short distances to be able to be used in rescue operations (with very low handling requirements) such as carrying support to humans on the water. The USV is based on a commercially available fiber glass hull, it uses a directional waterjet powered by an electrical brushless motor for propulsion, thus without any protruding propeller reducing danger in rescue operations. Its small dimensions (1.5 m length) and weight allow versatility and ease of deployment. The vehicle design is described in this paper both from a hardware and software point of view. A characterization of the vehicle in terms of energy consumption and performance is provided both from test tank and operational scenario tests. An example application in search and rescue is also presented and discussed with the integration of this vehicle in the European ICARUS (7th framework) research project addressing the development and integration of robotic tools for large scale search and rescue operations.