CIIMAR@E2S
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Os laboratórios CIIMAR@E2S resultam da integração do P.PORTO como membro associado do Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR). Esta integração tem vindo a ser progressivamente consolidada na E2S através do envolvimento de longa data de investigadores integrados no CIIMAR. A criação dos laboratórios CIIMAR@E2S reforçou a colaboração entre as instituições e potenciou o aumento da capacidade de investigação nas áreas da biotecnologia azul, saúde, ambiente e sustentabilidade. A investigação desenvolvida centra-se principalmente no desenvolvimento de soluções funcionais, inovadoras, seguras e sustentáveis, com aplicações nos domínios da saúde e do ambiente. Na área da biotecnologia aplicada à saúde, a investigação incide na descoberta de compostos naturais com potencial de intervenção em doenças neurodegenerativas e no cancro, bem como no desenvolvimento de alternativas naturais para dermocosméticos e nutracêuticos. No domínio da sustentabilidade ambiental, a investigação está centrada no desenvolvimento de pigmentos derivados de microrganismos para aplicações industriais e na valorização de resíduos têxteis e agroalimentares.
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- Analysis of the Use of Cylindrospermopsin and/or Microcystin-Contaminated Water in the Growth, Mineral Content, and Contamination of Spinacia oleracea and Lactuca sativaPublication . Llana-Ruiz-Cabello, Maria; Jos, Angeles; Cameán, Ana; Oliveira, Flavio; Barreiro, Aldo; Machado, Joana; Azevedo, Joana; Pinto, Edgar; Almeida, Agostinho; Campos, Alexandre; Vasconcelos, Vitor; Freitas, MarisaCyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxins.
- Analysis of the use of microcystin-contaminated water in the growth and nutritional quality of the root-vegetable, Daucus carotaPublication . Machado, J.; Azevedo, J.; Freitas, Marisa; Pinto, Edgar; Almeida, A.; Vasconcelos, V.; Campos, A.Toxic cyanobacterial blooms are often observed in freshwaters and may reflect the increased eutrophication of these environments and alterations in climate. Cyanotoxins, such as microcystins (MCs), are an effective threat to many life forms, ranging from plants to humans. Despite the research conducted to date on cyanotoxins, the risks associated to the use of contaminated water in agriculture require further elucidation. To tackle this aim, a research was conducted with the root-vegetable Daucus carota. The specific aims of this work were the following: (i) to evaluate the effects of MC-LR on the plant growth and photosynthesis; (ii) to evaluate the nutritional quality of carrot roots; and (iii) to measure bioaccumulation. To this purpose, young carrots were grown in soil during 1 month in natural conditions and exposed to Mycrocystis aeruginosa aqueous extracts containing environmentally realistic concentrations of MC-LR (10 and 50 MC-LR μg/L). The results showed that MC-LR may decrease root growth after 28 days of exposure to 50 μg/L and increase photosynthetic efficiency. We also observed changes in mineral and vitamin content in carrots as a result of the exposure to contaminated water. Moreover, MC-LR was detected in carrot roots by ELISA at very low concentration 5.23 ± 0.47 ng MC eq./g FW. The soil retained 52.7 % of the toxin potentially available for plants. This result could be attributed to MC-LR adsorption by soil particles or due to microbial degradation of the toxin. We conclude that the prolonged use of MC-LR-contaminated water may affect crop growth, alter the nutritional value of vegetable products, and potentiate contamination.
- Anticancer potential of spores of the soil fungus Pisolithus tinctoriusPublication . Oliveira, Rui S.; Alves, Ricardo; Preto, Marco; Martins, RosárioCancer is one of the major causes of death worldwide being the search for new anticancer drugs essential for the treatment of this disease. The sporocarps of the soil fungus Pisolithus tinctorius contain pisosterol, a triterpene that has been shown to have antitumor activity against some cancer cell lines. Nevertheless, no studies have focused on the anticancer potential of other structures such as spores, and so the anticancer potential of P. tinctorius, remains largely unknown. The main objective of this study was to evaluate the potential of P. tinctorius spores as a source of anticancer compounds.
- Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcusPublication . Martins, Rosario; Ramos, Miguel; Herfindal, Lars; Sousa, José; Skærven, Kaja; Vasconcelos, VítorAqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.
- Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strainsPublication . Leão, Pedro N.; Costa, Margarida; Ramos, Vitor; Pereira, Alban R.; Fernandes, Virgínia C.; Domingues, Valentina F.; Gerwick, William H.; Vasconcelos, Vítor; Martins, RosárioCyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.
- Application of real-time PCR in the assessment of the toxic cyanobacterium cylindrospermopsis raciborskii abundance and toxicological potentialPublication . Moreira, Cristiana; Martins, António; Azevedo, Joana; Freitas, Marisa; Regueiras, Ana; Vale, Micaela; Antunes, Agostinho; Vasconcelos, VítorCyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.
- Are known cyanotoxins involved in the toxicity of picoplanktonic and filamentous north atlantic marine cyanobacteria?Publication . Frazão, Bárbara; Martins, Rosario; Vasconcelos, VítorEight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.
- Assessment of cyanobacterial biomass as sustainable agricultural fertilizer: soil experiment with plants in Pot †Publication . Massa, Anabella; Azevedo, Joana; Azevedo, Rui; Pinto, Edgar; Costa, Anabela; Vasconcelos, Vitor; Campos, Alexandre; Freitas, MarisaProviding food to the growing human population in a sustainable way is one of the greatest challenges of modern society. In this context, cyanobacterial biomass (CB) can function as a source of macronutrients to increase soil productivity. These organisms can be collected from the environment in considerable amounts, since they tend to grow in large blooms. However, some of these cyanobacterial strains produce toxins that need to be carefully monitored to avoid food accumulation. The objective of this work was to evaluate the possible use of toxic and non-toxic strains of CB as fertilizer supplement in the growth of economically relevant vegetables. One-month old Raphanus sativus (radish) and Spinacia oleracea (spinach) plants were grown in pots in indoor controlled conditions. Six experimental conditions were set: (1) a control with no nutrient addition, (2) a recommended dose of a NK commercial fertilizer (CF), 0.6g of lyophilized CB of (3) a non-toxic strain of Cylindrospermopsis raciborskii, (4) a toxin-producing strain of C. raciborskii, (5) Microcystis aeruginosa, and (6) Anabaena sp. Several variables were estimated: in CB, this included the NPK dose addition, and in plants, the height, dry weight (dw) of the shoot and root, and the mineral content of plant edible parts. The mineral content in CB was estimated and compared with the recommended dose of CF, according to the information given by the fabricant label. We found no significative differences in N composition; nevertheless, there was a significative higher content in P and significative lower content in K in the CB. In the plants, we found no significative statistical differences between the treatments for the dw of radish root and spinach height. In spinach, the dw of the shoot in the M. aeruginosa treatment was significantly lower than the control, CF, and both the toxic and non-toxic C. raciborskii biomass. Additionally, in radish, the plant height and dw of the shoot M. aeruginosa treatment were significantly lower than in the toxic strain of C. raciborskii treatment. When analyzing mineral content in edible parts, we found that spinach treated with control and CF showed a higher content of Ca, Mo, N, P, and K, while in radish, the same two treatments plus the C. raciborskii toxic had higher Co and Fe content. M. aeruginosa amendment seems to impair shoot growth in both plant species. On the contrary, the toxic C. raciborskii CB seems to have a beneficial effect on growth and in mineral uptake on radish plants.
- Avaliação de Impacto na Saúde da desinfeção de águas residuais como barreira à disseminação ambiental da resistência antimicrobianaPublication . Almeida, Liliana; Freitas, Marisa; Miraglia, Simone Georges El Khouri; Abe, Karina CamasmieDesde 1938, com a descoberta da penicilina, que os antibióticos vieram revolucionar a medicina com a sua capacidade de tratamento de infeções bacterianas. Contudo, devido à inata capacidade de adaptação das bactérias a agentes antimicrobianos, verifica-se atualmente que os níveis de resistência a antibióticos estão a aumentar, enquanto o progresso da descoberta de novos agentes tem sido lento e não tem acompanhado esta tendência com a introdução de novas substâncias ativas. Desta forma, a Resistência a Antimicrobianos (RAM) é atualmente um dos problemas de saúde pública mais relevantes a nível global, apresentando consequências clínicas e económicas preocupantes (Loureiro et al., 2015).
- Avaliação do impacte na saúde da expansão da linha Amarela do metro do PortoPublication . Amorim, Inês Ferreira; Miraglia, Simone Georges El Khouri; Abe, Karina Camasmie; Freitas, MarisaA Avaliação do Impacte Ambiental (AIA) é uma ferramenta de caráter preventivo que visa analisar e avaliar os potenciais impactes que uma ação/atividade poderá causar não só no ambiente, mas também, na saúde pública (EC, 1999; REA, 2014). Este processo permite assim fazer uma gestão dos possíveis impactes, positivos ou negativos, iniciando-se com a identificação e avaliação dos indicadores relevantes e terminado com a implementação de medidas de mitigação que impeçam ou minimizem que os impactes negativos possam ocorrer (REA, 2014; APA, 2021).