Systematics and evolution of coastal and deepwater Hydrozoa from the NE Atlantic

The study of the Portuguese Hydrozoa fauna has been abandoned for more than half a century, except for the Azores archipelago. One of the main aims of this Ph.D. project was to contribute new hydrozoan records leading to a more accurate perception of the actual hydrozoan diversity found in Portuguese waters, including the archipelagos of Azores and Madeira, and neighbouring geographical areas, for habitats ranging from the deep sea to the intertidal. Shallow water hydroids from several Portuguese marine regions (including the Gorringe Bank) were sampled by scuba-diving. Deep-water hydroids, from the Azores, Madeira, Gulf of Cadiz and Alboran Sea, were collected by researchers of different institutions during several oceanographic campaigns. Occasional hydroid sampling by scuba-diving was performed in the UK, Malta and Spain. Over 300 hydroid species were identified and about 600 sequences of the hydrozoan ‘DNA barcode’ 16S mRNA were generated. The families Sertulariidae, Plumulariidae, Lafoeidae, Hebellidae, Aglaopheniidae, Campanulinidae, Halopterididae, Kirchenpaueriidae, Haleciidae and Eudendriidae, were studied in greater detail. About 350 16S sequences were generated for these taxa, allowing phylogenetic, phylogeographic and evolutionary inferences, and also more accurate taxonomic identifications. Phylogenetic analyses integrated molecular and morphological characters. Subsequent results revealed: particularly high levels of cryptic biodiversity, polyphyly in many taxonomic groups, pairs of species that were synonymous, the identity of several varieties as valid species, and highlighted phylogeographic associations of hydroids in deep and shallow-water areas of the NE Atlantic and W Mediterranean. It was proved that many (but not all) marine hydroid species with supposedly widespread vertical and/or horizontal geographical distributions, correspond in fact to complexes of cryptic taxa. This study further revealed that, in the NE Atlantic, shallow environments sustain higher hydrozoan diversity and abundance, but the importance of bathyal habitats as a source of phylogenetic diversity was also revealed. The Azorean seamounts were shown to be particularly important in the segregation of populations of hydroids with reduced dispersive potential. The bathyal habitats of the Gulf of Cadiz proved to harbour a considerably high number of cryptic species, which may mainly be a consequence of habitat heterogeneity and convergence of various water masses in the Gulf. The main causes proposed for speciation and population divergence of hydroids were: species population size, dispersal mechanisms and plasticity to inhabit different environmental conditions, but also the influence of oceanic currents (and its properties), habitat heterogeneity, climate change and continental drift. Higher phylogenetic resolution obtained for the family Plumulariidae revealed particularly that glacial cycles likely facilitated population divergence, ultimately speciation, and also faunal evolutionary transitions from deep to shallow waters.

Phylogenetic and functional diversity of estuarine bacterioneuston

A micro-camada superficial da água (SML) é caracterizada pela ocorrência de grandes quantidades de compostos orgânicos, pela acumulação de contaminantes antropogênicos e é submetida a uma intensa radiação solar, extrema mudança de temperatura e, no caso dos estuários, flutuação de salinidade. Estas propriedades físico-químicas estão, provavelmente, a modular a comunidade bacteriana (bacterioneuston) com propriedades filogenéticas e funcionais específicas. Neste estudo, as abordagens dependentes e independentes do cultivo foram aplicadas para avaliar a estrutura e dinâmica das comunidades bacterioneuston e bacterioplâncton em três localizações geográficas ao longo do estuário da Ria de Aveiro. Além disso, comparámos a diversidade filogenética de grupos específicos (Aeromonas, Pseudomonas e Psychrobacter) presentes em bacterioneuston e bacterioplâncton. Finalmente, as duas comunidades foram comparadas em termos de prevalência e diversidade de bactérias resistentes aos antibióticos e respetivos genes de resistência. Bactérias heterotróficas cultiváveis foram enriquecidas em SML. Eletroforese em gel de gradiente desnaturante (DGGE) permitiu a identificação de filotipos específicos em SML. Além disso, a análise de agrupamento dos perfis de DGGE de ambas as comunidades revelou uma ligeira tendência de agrupamento de acordo com a camada amostrada. As diferenças entre as duas comunidades variaram de acordo com factores espaciais e temporais. Em termos de diversidade filogenética de grupos específicos, não foram identificadas diferenças consistentes entre SML e UW com relação às comunidades de Aeromonas. Com relação ao género Pseudomonas, uma unidade operacional taxonómica cultivável foi consistentemente hiper-representada nas amostras de SML. Metodologias dependentes e independentes do cultivo revelaram a presença de populações de Psychrobacter complexas e muito estáveis em todos os sítios e datas de amostragens, com diferenças significativas entre as comunidades de Psychrobacter presentes em SML e UW. Estirpes representativas de prováveis novas espécies também foram cultivadas. Em termos de resistência aos antibióticos, a prevalência de bactérias resistentes em SML foi alta sugerindo selecção pelas condições presentes em SML. É preciso enfatizar que a resistência aos antibióticos foi incomum entre as bactérias estuarinas e os mecanismos de resistência foram, predominantemente, intrínsecos. Pela combinação de abordagens inovadoras dependentes e independentes do cultivo, este estudo forneceu novas e consistentes informações com relação às diferenças em ambas as comunidades bacterianas e em relação a alguns dos fatores que contribuem para a sua formação.

Resistance to last-resort antibiotics in natural environments

Last-resort antibiotics are the final line of action for treating serious infections caused by multiresistant strains. Over the years the prevalence of resistant bacteria has been increasing. Natural environments are reservoirs of antibiotic resistance, highly influenced by human-driven activities. The importance of aquatic systems on the evolution of antibiotic resistance is highlighted from the assumption that clinically-relevant resistance genes have originated in strains ubiquitous in these environments. We hypothesize that: a) rivers are reservoirs and disseminators of antibiotic resistance; b) anthropogenic activities potentiate dissemination of resistance to last-resort antibiotics. Hence, the main goal of the work is to compare the last-resort antibiotics resistome, in polluted and unpolluted water. Rivers from the Vouga basin, exposed to different anthropogenic impacts, were sampled. Water quality parameters were determined to classify rivers as unpolluted or polluted. Two bacterial collections were established enclosing bacteria resistant to cefotaxime (3rd generation cephalosporin) and to imipenem (carbapenem). Each collection was characterized regarding: phylogenetic diversity, antibiotic susceptibility, resistance mechanisms and mobile genetic elements. The prevalence of cefotaxime- and imipenem-resistant bacteria was higher in polluted water. Results suggested an important role in the dissemination of antibiotic resistance for Enterobacteriaceae, Pseudomonas and Aeromonas. The occurrence of bacteria resistant to non-beta-lactams was higher among isolates from polluted water as also the number of multiresistant strains. Among strains resistant to cefotaxime, extended-spectrum beta-lactamase (ESBL) genes were detected (predominantly blaCTX-M-like) associated to mobile genetic elements previously described in clinical strains. ESBL-producers were often multiresistant as a result of co-selection mechanisms. Culture-independent methods showed clear differences between blaCTX-M-like sequences found in unpolluted water (similar to ancestral genes) and polluted water (sequences identical to those reported in clinical settings). Carbapenem resistance was mostly related to the presence of intrinsically resistant bacteria. Yet, relevant carbapenemase genes were detected as blaOXA-48-like in Shewanella spp. (the putative origin of these genes), and blaVIM-2 in Pseudomonas spp. isolated from polluted rivers. Culture-independent methods showed an higher than the previously reported diversity of blaOXA-48-like genes in rivers. Overall, clear differences between polluted and unpolluted systems were observed, regarding prevalence, phylogenetic diversity and susceptibility profiles of resistant bacteria and occurrence of clinically relevant antibiotic resistance genes, thus validating our hypotheses. In this way, rivers act as disseminators of resistance genes, and anthropogenic activities potentiate horizontal gene transfer and promote the constitution of genetic platforms that combine several resistance determinants, leading to multiresistance phenotypes that may persist even in the absence of antibiotics.

Perfil de ácidos gordos de larvas recém eclodidas produzidas em cativeiro de camarões marinhos ornamentais candidatos a aquacultura

Dissertação de Mestrado, Biologia Marinha, especialização em Pescas e Aquacultura, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2009

Identificação e caracterização de duas isoformas da Ferritina da amêijoa R. decussatus e avaliação de algumas das suas propriedades biológicas

Dissertação de mest., Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Ecological niche modeling, cytogenetics and phylogeography of the genera Geomalacus and Letourneuxia (Gastropoda, Pulmonata) from the Iberian-Moroccan region

First described more that 150 years ago, the systematics of the genera Geomalacus and Letourneuxia (Arionidae, Gastropoda, Pulmonata) is still challenging. The taxonomic classification of arionid species is based on extremely labile characters such as body size or color that depends both on diet and environment, as well as age. Moreover, there is little information on the genetic diversity and population structure of the Iberian slugs that could provide extra clues to disentangle their problematic classification. The present work uses different analytical tools such as habitat suitability (Ecological Niche Modeling - ENM), cytogenetic analysis and phylogeography to establish the geographical distribution and evolutionary history of these pulmonate slugs. The potential distribution of the four Geomalacus species was modeled using ENM, which allowed the identification of new locations for G. malagensis, including a first report in Portugal. Also, it was predicted a much wider distribution for G. malagensis and G. oliveirae than previously known. Classical cytogenetic analyses were assayed with reproductive and a novel use of somatic tissues (mouth and tentacles) returning the number of chromosomes for the four Geomalacus species and L. numidica (n = 31, 2n = 62) and the respective karyotypes. G. malagensis and L. numidica present similar chromosome morphologies and karyotypic formulae, being more similar to each other than the Geomalacus among themselves. We further reconstructed the phylogeny of the genera Geomalacus and Letourneuxia using partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) and the nuclear ribosomal small subunit (18S rRNA), and applied an independent evolutionary rate method, the indicator vectors correlation, to evaluate the existence of cryptic diversity within species. The five nominal species of Geomalacus and Letourneuxia comprise 14 well-supported cryptic lineages. Letourneuxia numidica was retrieved as a sister group of G. malagensis. G. oliveirae is paraphyletic with respect to G. anguiformis. According to our dating estimates, the most recent common ancestor of Geomalacus dates back to the Middle Miocene (end of the Serravallian stage). The major lineage splitting events within Geomalacus occurred during the dry periods of the Zanclean stage (5.3-3.6 million years) and some lineages were confined to more humid mountain areas of the Iberian Peninsula, which lead to a highly geographically structured mitochondrial genetic diversity. The major findings of this are the following: (1) provides updated species distribution maps for the Iberian Geomalacus expanding the known geographic distribution of the concerned species, (2) unravels the cryptic diversity within the genera Geomalacus and Letourneuxia, (3) Geomalacus oliveirae is paraphyletic with G. anguiformis and (4) Letourneuxia numidica is sister group of G. malagensis.

Bio-synthesis of nanosized semiconductors using mine wastes as material sources

This work describes the synthesis of nanosized metal sulfides and respective SiO2 and/or TiO2 composites in high yield via a straightforward process, under ambient conditions (temperature and pressure), by adding to aqueous metals a nutrient solution containing biologically generated sulfide from sulfate-reducing bacteria (SRB). The nanoparticles‘ (NPs) morphological properties were shown not to be markedly altered by the SRB growth media composition neither by the presence of bacterial cells. We further extended the work carried out, using the effluent of a bioremediation system previously established. The process results in the synthesis of added value products obtained from metal rich effluents, such as Acid Mine Drainage (AMD), when associated with the bioremediation process. Precipitation of metals using sulfide allows for the possibility of selective recovery, as different metal sulfides possess different solubilities. We have evaluated the selective precipitation of CuS, ZnS and FeS as nanosized metal sulfides. Again, we have also tested the precipitation of these metal sulfides in the presence of support structures, such as SiO2. Studies were carried out using both artificial and real solutions in a continuous bioremediation system. We found that this method allowed for a highly selective precipitation of copper and a lower selectivity in the precipitation of zinc and iron, though all metals were efficiently removed (>93% removal). This research has also demonstrated the potential of ZnS-TiO2 nanocomposites as catalysts in the photodegradation of organic pollutants using the cationic dye, Safranin-T, as a model contaminant. The influence of the catalyst amount, initial pH and dye concentration were also evaluated. Finally, the efficiency of the precipitates as catalysts in sunlight mediated photodegradation was investigated, using different volumes of dye-contaminated water (150 mL and 10 L). This work demonstrates that all tested composites have the potential to be used as photocatalysts for the degradation of Safranin-T.

Identification of novel molecular determinants of tissue mineralization in fish

The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.

Vitivirus presentes em castas portuguesas de Vitis vinifera: caraterização molecular e diagnóstico

Dissertação de mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Lancefield group C and G streptococci in human infection : molecular typing, virulence and antimicrobial susceptibility

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2014

The dynamics of fibroblasts/ECM in neonatal cardiac injury

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2014

Phylogenetic insights of Avena genus based on genomic analysis of 45S rDNA molecular organization

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014

Micromorphology of the labellum and structure of the osmophore in a group of closely related species of the sexually deceptive Orchid genus Ophrys (orchidaceae)

Tese de doutoramento, Biologia (Biologia Celular), Universidade de Lisboa, Faculdade de Ciências, 2016

Isolamento e caracterização de uma proteína tóxica produzida por um isolado açoriano de Bacillus weihenstephanensis

Dissertação de Mestrado, Biotecnologia em Controlo Biológico, 27 de Junho de 2013, Universidade dos Açores.

Caracterização molecular da resistência a antimicrobianos em Escherichia coli produtoras de β-lactamases de espectro alargado isoladas em cães e estudo da partilha de clones bacterianos entre animais de companhia e coabitantes humanos

A pressão seletiva originada pelo uso excessivo de antimicrobianos na medicina humana e veterinária tem contribuído para a emergência de estirpes bacterianas multirresistentes, sendo os estudos mais escassos relativamente à sua presença nos animais de companhia. Porque os animais e os seus proprietários partilham o mesmo espaço habitacional, apresentando comportamentos de contacto próximo, existe uma hipótese elevada de transferência microbiana inter-espécie. Ante esta possibilidade é importante escrutinar o papel dos animais de companhia enquanto reservatórios de estirpes e de genes de resistência, bem como a sua envolvência na disseminação de estirpes bacterianas multirresistentes. Importa também, investigar o papel das superfícies e objetos domésticos partilhados por ambos, como potenciadores deste fenómeno. O objetivo deste trabalho foi, identificar o filogrupo e fazer a caracterização molecular dos genes que conferem resistência aos β-lactâmicos e às quinolonas, em quarenta isolados de Escherichia coli produtoras de β-lactamases de espectro alargado (ESBL), obtidas em zaragatoas fecais de cães consultados no Hospital Veterinário do ICBAS-UP. Complementarmente pretendeu-se inferir sobre a partilha de clones de Escherichia coli e Enterococcus spp. com elevadas resistências, em cinco agregados familiares (humanos e seus animais de companhia) assim como avaliar a potencial disseminação de estirpes multirresistentes no ambiente doméstico. Previamente foram recolhidas zaragatoas de fezes, pelo e mucosa oral dos animais e em alguns casos, dos seus proprietários, e ainda do ambiente doméstico. As zaragatoas foram processadas e as estirpes isoladas com base em meios seletivos. Foram realizados testes de suscetibilidade antimicrobiana de modo a estabelecer o fenótipo de resistência de cada isolado. O DNA foi extraído por varias metodologias e técnicas de PCR foram utilizadas para caracterização de filogrupos (Escherichia coli) e identificação da espécie (Enterococcus spp.). A avaliação da proximidade filogenética entre isolados foi efetuada por ERIC PCR e PFGE. No conjunto de quarenta isolados produtores de ESBL e/ou resistentes a quinolonas verificou-se que 47,5% pertenciam ao filogrupo A, havendo uma menor prevalência do filogrupo D (25,0%), B1 (17,5%), e B2 (10,0%).A frequência de resistência nestes isolados é factualmente elevada, sendo reveladora de uma elevada pressão seletiva. Com exceção de dois isolados, os fenótipos foram justificados pela presença de β-lactamases. A frequência da presença de genes foi: 47% blaTEM, 34% blaSHV, 24% blaOXA , 18% blaCTX-M-15, 8% blaCTX-M-2, 3% blaCTX-M-9. Nos isolados resistentes às quinolonas verificou-se maioritariamente a presença de mutações nos genes cromossomais gyrA e parC, e em alguns casos a presença de um determinante de resistência mediado por plasmídeo – qnrS. Nos cinco “agregados familiares” (humanos e animais) estudados foi observada uma partilha frequente de clones de E. coli e Enterococcus faecalis com múltiplas resistências, isolados em fezes e mucosa oral de cães e gatos e fezes e mãos dos respetivos proprietários, evidenciando-se assim uma possível transferência direta entre coabitantes (agregados A, C, D, E). Ficou também comprovado com percentagens de similaridade genotípica superiores a 94% que essa disseminação também ocorre para o ambiente doméstico, envolvendo objetos dos animais e de uso comum (agregados A, E). Os resultados obtidos reforçam a necessidade de um uso prudente dos antimicrobianos, pois elevados padrões de resistências terão um impacto não só na qualidade de vida dos animais mas também na saúde humana. Adicionalmente importa sensibilizar os proprietários para a necessidade de uma maior vigilância relativamente às formas de interação com os animais, bem como para a adoção de medidas higiénicas cautelares após essa mesma interação.