Browsing by Author "Menezes, Carina"
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- Actividade antibacteriana de extractos de Planktothrix agardhii contra Staphylococcus aureusPublication . Menezes, Carina; Dias, ElsaA resistência de bactérias patogénicas à antibioterapia é um dos principais problemas de saúde pública, face à crescente ineficácia dos antibióticos no tratamento de doenças infeciosas. Assim, a pesquisa de novos antibióticos é um importante desafio da investigação em saúde. O potencial farmacológico de cianobactérias tem sido avaliado e descrito, mas, no entanto, ainda não há nenhum composto cianobacteriano que tenha sido aprovado pelas autoridades do medicamento como agente antibacteriano. Neste trabalho avaliámos a atividade de extratos de 40 estirpes de Planktothrix agardhii contra duas bactérias patogénicas: Klebsiella pneumonia e Staphylococus aureus. Para cada espécie bacteriana alvo usou-se uma estirpe de referência (ATCC) e uma estirpe clínica, isolada a partir de amostras de pacientes e estudadas no Laboratório de Resistência aos Antibióticos e Infeções Associadas aos Cuidados de Saúde. As estirpes de P. agardhii foram isoladas de albufeiras portuguesas e têm sido mantidas na coleção “Estela Sousa e Silva Algae Culture Collection” do Laboratório de Biologia e Ecotoxicologia. A biomassa de P. agardhii (200mg) foi extraída com metanol (70%, 10mL/100mg) overnight e sujeita a ciclos de sonicação. O metanol dos extratos foi evaporado e os extratos aquosos resultantes foram purificados em cartuchos C18. A atividade dos extratos contra as bactérias K. pneumonia e S.aureus foi avaliada através do método da Difusão em Disco (EUCAST), usando discos impregnados com 2, 4, 6, 8 e 10 mg de cada extrato. De acordo com os resultados preliminares, nenhum dos extratos apresenta atividade contra K. pneumonia, mas o extrato de P. agardhii LMECYA 256 apresenta um ligeiro halo de inibição relativamente às estirpes de S. aureus, de uma forma aparentemente dependente da dose de extrato aplicada. O S. aureus é uma bacteria Gram-positiva, frequentemente associada a uma vasta gama de patologias, desde simples infeções na pele, até infeções graves como pneumonia e meningite. Assim, revela-se do maior interesse explorar as propriedades antibacterianas de cianobactérias, designadamente do género Planktothrix, contribuindo, desta forma, para um dos desafios atuais da ciência: a identificação de novos antibióticos de origem natural.
- Antioxidant and Cytoprotective Properties of Cyanobacteria: Potential for Biotechnological ApplicationsPublication . Guerreiro, Adriana; Andrade, Mariana; Menezes, Carina; Vilarinho, Fernanda; Dias, ElsaAntioxidant compounds from cyanobacteria may constitute a natural alternative to current synthetic antioxidants, which contain preservatives and suspected toxicity. In this work, we evaluate the antioxidant potential of cyanobacterial strains of distinct species/genus isolated from freshwater (n = 6), soil (n = 1) and wastewater (n = 1) environments. Lyophilized biomass obtained from in-vitro cultures of those strains was extracted with ethanol and methanol. The antioxidant potential was evaluated by chemical (DPPH scavenging method, β-carotene bleaching assay, determination of total phenolic and total flavonoid compounds) and biological (H2O2-exposed HEK293T cell line model) approach. Some strains showed high yields of antioxidant activity by the DPPH assay (up to 10.7% IP/20.7 TE µg/mL) and by the β-carotene bleaching assay (up to 828.94 AAC), as well as significant content in phenolic (123.16 mg EAG/g DW) and flavonoid (900.60 mg EQR/g DW) compounds. Normalization of data in a “per cell” or “per cell volume” base might facilitate the comparison between strains. Additionally, most of the cyanobacterial extracts conferred some degree of protection to HEK293T cells against the H2O2-induced cytotoxicity. Freshwater Aphanizomenon gracile (LMECYA 009) and Aphanizomenon flos-aquae (LMECYA 088), terrestrial Nostoc (LMECYA 291) and wastewater Planktothrix mougeotii (LEGE 06224) seem to be promising strains for further investigation on cyanobacteria antioxidant potential.
- Cianobactérias e cianotoxinas em água tratada: conhecer para prevenirPublication . Dias, Elsa; Sarioglou, Konstantina; Ortiz, Miriam; Menezes, CarinaA monitorização da qualidade da água para consumo humano relativamente à presença de cianobactérias é uma das atribuições do Departamento de Saúde Ambiental do Instituto Nacional de Saúde. A proliferação massiva de cianobactérias (bloom) em reservatórios de água doce superficial tem impactos no ecossistema aquático, nos procedimentos operativos das estações de tratamento de água e acarreta riscos para a saúde humana e animal devido à capacidade toxigénica de algumas espécies cianobacterianas. Neste trabalho descreve-se a deteção de uma densidade elevada da espécie Dolichospermum planctonicum numa amostra de água tratada, bem como a presença de anatoxina-a (neurotoxina). A legislação portuguesa apenas contempla um valor paramétrico de referência para microcistinas (hepatotoxinas). Porém, de acordo com os valores-guia da Organização Mundial da Saúde, a anatoxina-a foi detetada numa concentração abaixo do valor recomendado para água de consumo humano. Os resultados alertam para a importância da monitorização regular de cianobactérias em água bruta e tratada e da adequação da monitorização de cianotoxinas em função do potencial tóxico das espécies presentes. Por outro lado, salienta-se a necessidade de integrar os dados de monitorização a nível do país, pois só assim será possível conhecer o cenário real de ocorrência de blooms tóxicos em Portugal, e, em conformidade, adequar a legislação nacional.
- Coleção de culturas de algas Estela Sousa e SilvaPublication . Menezes, Carina; Churro, Catarina; Paulino, Sérgio; Sam-Bento, Filomena; Alverca, Elsa; Dias, Elsa; Pereira, PauloA coleção de culturas de algas Estela Sousa e Silva (ESSACC) foi criada em 1956 e reside atualmente no Laboratório de Biologia e Ecotoxicologia no Instituto Nacional de Saúde Dr. Ricardo Jorge. A ESSACC foi implementada em resposta à necessidade de um repositório de material biológico para investigação na área do fitoplâncton. Este importante recurso biológico contém culturas monoclonais de algas eucarióticas e cianobactérias provenientes de águas costeiras e albufeiras portuguesas. Atualmente, a coleção mantém acima de 176 isolados vivos, dos quais 151 são cianobactérias de água doce e 25 são dinoflagelados marinhos. Adicionalmente são também mantidos alguns isolados pertencentes a outros grupos de fitoflagelados. Esta coleção permitiu até agora a identificação e caracterização de espécies assim como a produção e purificação de toxinas para aplicação em estudos toxicológicos entre outras diversas áreas de investigação. Deste modo, a ESSACC constitui uma ferramenta importante no fornecimento de culturas de algas a investigadores na área do fitoplâncton, particularmente no estudo de espécies nocivas.
- Comparative study of the cytotoxic effects of microcystin-LR in mammalian cell lines: Vero, HepG2, Caco2 and MDCKPublication . Menezes, Carina; Amorim, Ana; Alverca, ElsaMicrocystin-LR (MCLR) is a natural occurring freshwater cyanotoxin, recognized as one of the most toxic microcystin variants. It is thought to be responsible for cases of livestock and human intoxication due to consumption of toxic cyanobacteria-contaminated water. Although considered a hepatotoxin, MCLR also targets other organs such as the kidneys and intestines. In spite the cellular mechanisms associated with the toxicity of MCLR are still unclear, a previous work in a monkey kidney cell line suggested that the endoplasmic reticulum was an early target of MCLR toxicity and that autophagy was triggered as a cell defense mechanism at subcytotoxic concentrations of MCLR. In the present work, cytotoxic, morphological and ultrastructural effects of MCLR were compared in HepG2 (human liver), Vero (monkey kidney), MDCK (dog kidney) and Caco2 (human intestine) cell lines. MCLR induced a concentration-dependent decrease in cell viability by the NR assay in all cell lines, with HepG2 and Vero showing the lowest cytotoxic thresholds of 25 and 50 μM MCLR, respectively. In these cells, MCLR exposure induced lysosomal damages previously to mitochondrial disruption, reinforcing the role of lysosomes in MCLR-induced toxicity. Immunolabelling and ultrastructural visualization of autophagosomes, showed that autophagy was a response transversal to both cell lines, triggered at subcytotoxic MCLR concentrations, confirming its importance as a defense mechanism to early damages inflicted by the toxin. The analysis of GRP94, an ER stress protein, did not undoubtedly demonstrate that MCLR targets the ER. However, together with the ultrastructural data, suggested that in both HepG2 and Vero cells, the ER has a role in autophagy induction. Additionally, in HepG2 cells, GRP94 down-regulation with increasing MCLR concentrations supported the ER role in the triggering of apoptosis. At high toxin concentrations, ultrastructural alterations consistent with apoptosis were observed for all four cell lines, proving that this is a general MCLR-induced mechanism.
- Comparison of saxitoxin-genes expression and production profiles between Aphanizomenon gracile and Cuspidotrix issastchenkoi strains, isolated from freshwater reservoirsPublication . Reis, Marta; Menezes, Carina; Dias, Elsa; Valério, ElisabeteSaxitoxins (STX) are a group of carbamate alkaloids known to inhibit the axons sodium ion channels, thus affecting the nervous system in vertebrates. These neurotoxins cause paralysis and respiratory failure, ultimately ending in death. They are produced by organisms belonging to two different kingdoms: marine eukaryotic dinoflagellates and freshwater prokaryotic cyanobacteria. The unique biosynthetic pathway, which is responsible for encoding proteins that allow synthesizing and exporting STX, is the cluster sxt, already described in several cyanobacterial species. It has been observed that diverse environmental factors affect differently STX production in cyanobacteria. Among those, temperature is the one that aroused greater interest, since it directly affects cyanobacterial growth rates, however controversial results have been reported. Furthermore, studies on the environmental regulation of STX synthesis and transport are quite rare. In this work, we tested the influence of temperature in two STX producers, Aphanizomenon gracile LMECYA40 and Cuspidothrix issatschenkoi LMECYA31, isolated from Portuguese freshwaters and maintained in the ESSACC. Total STX concentration, the expression of genes linked to STX biosynthesis (sxtA) and transport (sxtM and sxtPer) and the percentage of heterocytes per trichome were determined, in four different phases of cyanobacterial growth (lag, early exponential, late exponential and stationary phases). At 20ºC, a higher expression of the sxt genes and higher production of STX in the late exponential and stationary phase was observed in both species. Also, C. issatschenkoi produced more STX throughout the growth cycle than A. gracile. In A. gracile the percentage of heterocytes per trichome was higher in the lag phase, decreasing along the cell cycle. C. issatschenkoi did not form heterocytes throughout the growth cycle. Further experiments are being performed at 10ºC and 30°C in order to understand the effect of temperatures environmentally relevant in STX producers.
- Cytotoxic and morphological effects of microcystin-LR in in vitro models - a comparision between HepG2, Vero-E6, MDCK and CaCo-2 cell linesPublication . Menezes, Carina; Alverca, Elsa; Dias, Elsa; Sam-Bento, Filomena; Paulino, Sérgio; Pereira, PauloMicrocystin-LR (MCLR), a potent hepatotoxin, is transported selectively into the cells throught specific membrane polypeptides mostly present in the liver. These transporters are also expressed in the brain, kidneys and intestine, although the toxicity of MCLR in these cell types is still poorly understood. In this study, morphological, ultrastructural and biochemical analyses were performed in hepatic, renal and intestinal cell lines in order to evaluate the toxicity of MCLR obtained from semi-purified cyanobacterial extracts. Our results show that after 24h of exposure, MCLR induces the viability decrease of all cell lines, in a concentration-dependent manner. HepG2 cells are the most susceptible, followed by Vero, MDCK and CaCo-2. Ultrastructural analyses show that subcytotoxic concentrations of MCLR induce the formation of large cytoplasmic vacuoles, particularly in the HepG2 cell line. Immunofluorescence microscopy reveals that these vacuoles are enriched in LC3B protein, suggesting autophagy as an early cellular response of HepG2 and Vero cells to MCLR. At cytotoxic MCLR concentrations, lysossomal dysfunction in both cell lines occurs prior to mitochondrial disruption, as demonstrated by the specific labeling with Acridine Orange and Rhodamine-123. This suggests that besides mitochondria, lysossomes may also be an MCLR-early target. Immunolocalization and western blot analysis of the endoplasmic reticulum anti-apoptotic protein GRP94 show distinct MCLR-induced effects in Vero and HepG2 cells: re-localization of GRP94 within Vero cells and decrease of GRP94 expression in the HepG2 cell line. These results demonstrate that all the studied cell lines are susceptible to MCLR although with cell type specificity and differential organelle targeting.
- Descrição de um surto de intoxicação por cianobactérias (algas verdes-azuis) em bovinos no AlentejoPublication . Menezes, Carina; Nova, Rita; Vale, Micaela; Azevedo, Joana; Vasconcelos, Vítor; Pinto, CarlosSuspeita de hepatoxicidade provocada por cianotoxinas, num surto de morte súbita em 25 bovinos numa exploração com 54 vacas de carne em Almodôvar, Alentejo.
- Description of an outbreak of cattle intoxication by cyanobacteria (blue-green algae) in the South of PortugalPublication . Menezes, Carina; Nova, Rita; Vale, Micaela; Azevedo, Joana; Vasconcelos, Vítor; Pinto, CarlosIn 2017, 25 cows from a beef herd consisting of 54 healthy animals, with ages between 1.5 to 4 years old, from Aberdeen Angus breed x Charolais were found dead in Almodôvar, Alentejo, in the South of Portugal. They were grazing on a field of stubble for about 3 weeks when they broke the electric fence that bordered the field and had access to stagnant water from a small stream (Ribeira de Oeiras) that run across the herd. All the animals were seen healthy, by the keeper, until 12 a.m. of Sunday August 27th. When they were last observed before the occurrence, they were resting at the shade of trees and bushes surrounding the Oeiras stream. At this time of the year, this is a normal behaviour due to the high temperatures, and it is common for them to engage in grazing activities in the evening, when the temperature begins to decrease. On the next day (the 28th August), at 7 a.m., the keeper encountered 20 cadavers scattered throughout the land and 5 sick cows. The other 29 animals of the group did not show any clinical signs during the following weeks after the occurrence. In the same period, was reported the death of a heron in the Oeiras stream.
- Distribuição e dinâmica de ocorrências de cianobactérias e compostos associados na Albufeira do RoxoPublication . Dias, Elsa; Menezes, CarinaO presente relatório resultou dos trabalhos desenvolvidos no âmbito do Protocolo de Colaboração entre o Instituto Nacional de Saúde Doutor Ricardo Jorge e a AgdA- Águas Públicas do Alentejo, SA. Pretendeu avaliar-se a distribuição e dinâmica de ocorrência de cianobactérias potencialmente produtoras de COVs e de cianotoxinas na albufeira do Roxo. Especificamente, pretendia-se estudar três locais e três profundidades de colheita, de forma a identificar o ponto mais adequado à captação de água da albufeira. Foi também objetivo do trabalho, avaliar a persistência e viabilidade de fitoplâncton na água tratada à saída da ETA e nos reservatórios da Atalaia e Aljustrel, bem como a presença de COVs. O trabalho visava, ainda, a monitorização de parâmetros físico-químicos nas amostras de água bruta, de forma a identificar eventuais factores associados ao desenvolvimento de florescências cianobacterianas.