Estudos de susceptibilidade à artemisinina e derivados em Plasmodium falciparum

A resistência do Plasmodium falciparum à maioria dos antimaláricos em uso é um dos maiores obstáculos ao controlo eficaz da doença. Excepcionalmente, a resistência in vivo à artemisinina e seus derivados não foi ainda detectada. Contudo, é amplamente aceite que existe um risco real de aparecimento de resistência a esta classe de antimaláricos. Deste modo, este trabalho teve como finalidade investigar diversos aspectos do fenótipo e genótipo do perfil de fármaco-susceptibilidade à artemisinina e seus derivados no parasita Plasmodium falciparum, recorrendo a populações naturais de parasitas e a um modelo de laboratório. Neste contexto, foi efectuado um estudo multicêntrico, onde se avaliou a susceptibilidade in vitro a derivados da artemisinina em populações naturais de P. falciparum provenientes do Ruanda, da República Democrática de São Tomé e Príncipe (RDSTP) e do Brasil e se investigou o perfil genotípico de determinados genes candidatos nas mesmas amostras. No laboratório, utilizou-se o clone P. falciparum Dd2 para seleccionar clones resistentes à artemisinina in vitro. Em todos os parasitas efectuaram-se estudos de associação entre os níveis de susceptibilidade à artemisinina e derivados e mutações ou alteração no número de cópias de genes previamente apontados como potenciais moduladores da resposta a esta classe de antimaláricos tais como os genes pfcrt, pfmdr1, pfATPase6, pftctp e pfubp-1, bem como genes que codificam proteínas envolvidas nos mecanismos de defesa ao stress oxidativo, propostos no âmbito deste trabalho: pfsod1, pfgst, pfγgcs, pfgr, pfgpx, pftrx1, pftrx2 e pfprx. Os resultados obtidos para as populações naturais de P. falciparum Africanas e do Brasil em termos de susceptibilidade in vitro às artemisininas, parecem estar em conformidade com o descrito em outras áreas endémicas onde se observa que apesar de existir uma variação significativa na sua susceptibilidade, não existe evidência de resistência. No entanto, verificou-se existir uma correlação positiva entre as respostas aos derivados da artemisinina e a amodiaquina, antimalárico de diferente classe, o que indicia a existência de um fenótipo de tolerância cruzada entre compostos de classes diferentes. Apesar de não ter sido detectada uma associação estatisticamente significativa entre a resposta dos isolados às artemisininas e os genes analisados, esta componente dotrabalho permitiu que se avaliasse a estrutura populacional do parasita P. falciparum em termos de fenótipo bem como do perfil de genes apontados como moduladores das respostas a estes fármacos antes da aplicação em larga escala dos mesmos. Estes dados constituem assim uma ferramenta singular de fármaco-vigilância, dado poderem ser utilizados em futuros ensaios como termo de comparação que permite avaliar a evolução das populações parasitárias em termos fenotípicos e genotípicos. A selecção in vitro de clones resistentes à artemisinina partindo de progenitores sensíveis, P. falciparum Dd2, permitiu obter uma população de parasitas com susceptibilidade 100 vezes reduzida em relação ao seu progenitor, a qual se designou Dd2-ARTmut. No entanto, a resistência obtida demonstrou ser instável, tendo desaparecido na ausência de pressão de fármaco, dando origem a uma estirpe que se passou a denominar Dd2-ARTmutREV. A comparação entre os clones Dd2-ARTmut e o progenitor sensível Dd2 em relação a potenciais genes moduladores da susceptibilidade às artemisininas, revelou ter ocorrido amplificação do gene pfmdr1, bem como um aumento da sua expressão. Curiosamente, a amplificação do gene pfmdr1 foi mantida após a reversão da resistência, na estirpe Dd2-ARTmutREV, levantando novas questões sobre o efeito causal desta amplificação na modulação das respostas à artemisinina e seus derivados.Resistance of Plasmodium falciparum to most antimalarials in use is one of the major obstacles to gaining effective control of the disease. Exceptionally, in vivo resistance to artemisinin and its derivatives has not been detected as yet. However, it is widely accepted that there is a real risk that resistance emerges also to this class of drugs. To this purpose, the objective of this work was to investigate several aspects of the phenotype and genotype of responses to artemisinin and its derivatives in Plasmodium falciparum, making use of natural parasite populations and of an in vitro culture system. In this context, a multicentric study was conducted, where the in vitro susceptibility to artemisinin derivatives in natural populations of P. falciparum was inspected in samples from Rwanda, the Democratic Republic of Sao Tome and Principe (RDSTP) and Brazil, and the genotypic profile of certain candidate genes was also assessed in the same samples. In the laboratory, the P. falciparum clone Dd2 was used to select in vitro resistance to artemisinin. In all parasites, the association between the levels of susceptibility to artemisinins and mutations or changes in the number of copies of genes identified as potential modulators of the response to this class of antimalarials was investigated. The panel of genes included those previously reported by others, such as pfcrt, pfmdr1, pfATPase6, pftctp and pfubp-1, as well as genes that encode proteins involved in the mechanism of oxidative stress defense, proposed within the context of this work pfsod1, pfgst, pfγgcs, pfgr, pfgpx, pftrx1, pftrx2 and pfprx. The results obtained for the natural populations of P. falciparum from Africa and Brazil in terms of in vitro susceptibility to artemisinins, appear to be within the trends of other endemic areas and are consistent with the notion that, although natural parasite populations of P. falciparum vary significantly with respect to their susceptibility to this class of drugs, there is no evidence of resistance. However, a positive correlation was found between the responses to artemisinins and amodiaquine, a different class of antimalarial, which indicates the existence of a cross-resistance phenotype between different classes of compounds. Although a statistically significant association between the response to the artemisinins and the genes analyzed was not detected, this component of the work allowed to gain insights into the population structure of P. falciparum parasite in terms of phenotype and genetic profile of potential modulators of the responses to these drugs before their large scale deployment. These data are therefore a unique tool for drug-surveillance since they can be used in future trials as a benchmark that can help evaluate evolution in terms genotypic and phenotypic of parasitic populations. The in vitro selection of artemisinin-resistant parasites from sensitive progenitors, P. falciparum Dd2, gave rise to a parasite strain with 100-fold reduced susceptibility in relation to its progenitor, which was named Dd2-ARTmut. However, resistance proved to be unstable, disappearing in the absence of drug pressure, resulting in a strain that was renamed Dd2-ARTmutREV. Comparison between Dd2-ARTmut and sensitive progenitor Dd2 in relation to putative modulators of susceptibility to artemisinin proved that amplification of the gene pfmdr1 has occurred, as well as an increase in its expression. Interestingly, this amplification of the pfmdr1 gene was maintained after resistance reversion in the strain Dd2-ARTmutREV, raising new questions about the causal effect of this amplification in the modulation of responses to the artemisinins

Socioeconomic impact of waste over the lifetime of project LIFEPAYT

Project LIFE PAYT – Tool to reduce waste in South Europe (LIFE 15 ENV/PT/000609). Co-funded by the LIFE programme of the European CommissionN/

Socioeconomic baseline on waste for households in Aveiro, Portugal

Project LIFE PAYT – Tool to reduce waste in South Europe (LIFE 15 ENV/PT/000609) Co-funded by the LIFE programme of the European Commissioninfo:eu-repo/semantics/publishedVersio

Socioeconomic indicators to monitor the impacts of the transition to PAYT tariffs

info:eu-repo/semantics/publishedVersio

Boron nutrition affects membrane leakage and the chemical composition of leaves and fruits of Olea europaea

Boron (B) is an essential microelement for plants, being its deficiency the most frequent micronutrient disorder in olive tree. The main functions of boron are related to cell wall strength and development, membrane function, cell division, fruit and seed development, water relations, sugar transport and hormone development. The study conducted in Bragança (Northeast Portugal), under rainfed conditions, shows that application of Bfertilizer decreased the symptoms of oxidative stress on leaves, both in summer and, with higher extent, in winter. In fact, B-treated trees presented lower electrolyte leakage, in a closely association with higher concentration of total thiols. Moreover, boron increased the concentration of soluble sugars, while decreased the accumulation of starch in both seasons. Meanwhile, total soluble proteins and total phenols levels were higher in Bfertilized plants during the summer period, whereas in winter, after frost events, the concentration of phenols was higher in B-starved trees. The chemical composition of fruits at final harvest revealed that B-supply increases the soluble sugars and the pulp ash content, decreases organic matter and dietary fiber, whilst the crude protein concentration was similar between treatments. Thus, the present study showed that addition of Bfertilizer affects the metabolism of olive tree and give new insights about the effect of boron on plant physiology and biochemistry that will help to refine the improvement in Bfertilizer recommendations for olive growing areas.info:eu-repo/semantics/publishedVersio

Trophic ecology of common bottlenose dolphins in a pelagic insular environment inferred by stable isotopes

The common bottlenose dolphin (Tursiops truncatus) is a top marine predator widely dispersed in coastal and pelagic habitats and with a generalist feeding behavior. Yet, information on the trophic ecology of animals inhabiting pelagic environments is still scarce. Using carbon (& delta;C-13: C-13/C-12) and nitrogen (& delta;N-15: N-15/N-14) stable isotope ratios, we identified and quantified the main groups of prey assimilated by bottlenose dolphins inhabiting an oceanic habitat (Madeira Island, East Atlantic). Bottlenose dolphins assimilated pelagic, schooling fish (such as blue jack mackerel, Trachurus picturatus) and mesopelagic and demersal squids, which reinforces the pelagic dietary composition of insular/oceanic dolphins. Also, intra-seasonal differences were found in their stable isotope ratios, which suggest intraspecific variability in the feeding behavior among individuals living in the same area. Sex was not the main factor contributing to these differences, suggesting the lack of trophic niche segregation between adult males and females in this offshore environment. Nonetheless, further studies including different life stages and information on the ecophysiological requirements are necessary to disclose the factors responsible for the observed variability. This study showed that insular dolphins fed primarily on economically important pelagic prey, highlighting the need of developing management strategies that integrate conservation in fisheries plans.NORTE01-0145-FEDER-000031; M1420-01-0145-FEDER-000001-OOM; LA/P/0069/2020; M1420-09-5369-FSE-000002info:eu-repo/semantics/publishedVersio

Geographic Structuring of the Plasmodium falciparum Sarco(endo)plasmic Reticulum Ca2+ ATPase (PfSERCA) Gene Diversity

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte