Nucella lapillus: imposex transcriptome analysis and phenotypic plasticity

O conhecimento de mecanismos de genómica funcional tem sido maioritariamente adquirido pela utilização de organismos modelo que são mantidos em condições laboratoriais. Contudo, estes organismos não reflectem as respostas a alterações ambientais. Por outro lado, várias espécies, ecologicamente bem estudadas, reflectem bem as interacções entre genes e ambiente mas que, das quais não existem recursos genéticos disponíveis. O imposex, caracterizado pela superimposição de caracteres sexuais masculinos em fêmeas, é induzido pelo tributilestanho (TBT) e trifenilestanho (TPT) e representa um dos melhores exemplos de disrupção endócrina com causas antropogénicas no ambiente aquático. Com o intuito de elucidar as bases moleculares deste fenómeno, procedeu-se à combinação das metodologias de pirosequenciação (sequenciação 454 da Roche) e microarrays (Agilent 4*180K) de forma a contribuir para um melhor conhecimento desta interacção gene-ambiente no gastrópode Nucella lapillus, uma espécie sentinela para imposex. O trancriptoma de N. lapillus foi sequenciado, reconstruído e anotado e posteriormente utilizado para a produção de um “array” de nucleótidos. Este array foi então utilizado para explorar níveis de expressão génica em resposta à contaminação por TBT. Os resultados obtidos confirmaram as hipóteses anteriormente propostas (esteróidica, neuroendócrina, retinóica) e adicionalmente revelou a existência de potenciais novos mecanismos envolvidos no fenómeno imposex. Evidência para alvos moleculares de disrupção endócrina não relacionados com funções reprodutoras, tais como, sistema imunitário, apoptose e supressores de tumores, foram identificados. Apesar disso, tendo em conta a forte componente reprodutiva do imposex, esta componente funcional foi a mais explorada. Assim, factores de transcrição e receptores nucleares lipofílicos, funções mitocondriais e actividade de transporte celular envolvidos na diferenciação de géneros estão na base de potenciais novos mecanismos associados ao imposex em N. lapillus. Em particular, foi identificado como estando sobre-expresso, um possível homólogo do receptor nuclear “peroxisome proliferator-activated receptor gamma” (PPARγ), cuja função na indução de imposex foi confirmada experimentalmente in vivo após injecção dos animais com Rosiglitazone, um conhecido ligando de PPARγ em vertebrados. De uma forma geral, os resultados obtidos mostram que o fenómeno imposex é um mecanismo complexo, que possivelmente envolve a cascata de sinalização envolvendo o receptor retinoid X (RXR):PPARγ “heterodimer” que, até à data não foi descrito em invertebrados. Adicionalmente, os resultados obtidos apontam para alguma conservação de mecanismos de acção envolvidos na disrupção endócrina em invertebrados e vertebrados. Finalmente, a informação molecular produzida e as ferramentas moleculares desenvolvidas contribuem de forma significativa para um melhor conhecimento do fenómeno imposex e constituem importantes recursos para a continuação da investigação deste fenómeno e, adicionalmente, poderão vir a ser aplicadas no estudo de outras respostas a alterações ambientais usando N. lapillus como organismo modelo. Neste sentido, N. lapillus foi também utilizada para explorar a adaptação na morfologia da concha em resposta a alterações naturais induzidas por acção das ondas e pelo risco de predação por caranguejos. O contributo da componente genética, plástica e da sua interacção para a expressão fenotípica é crucial para compreender a evolução de caracteres adaptativos a ambientes heterogéneos. A contribuição destes factores na morfologia da concha de N. lapillus foi explorada recorrendo a transplantes recíprocos e experiências laboratoriais em ambiente comum (com e sem influência de predação) e complementada com análises genéticas, utilizando juvenis provenientes de locais representativos de costas expostas e abrigadas da acção das ondas. As populações estudadas são diferentes geneticamente mas possuem o mesmo cariótipo. Adicionalmente, análises morfométricas revelaram plasticidade da morfologia da concha em ambas as direcções dos transplantes recíprocos e também a retenção parcial, em ambiente comum, da forma da concha nos indivíduos da F2, indicando uma correlação positiva (co-gradiente) entre heritabilidade e plasticidade. A presença de estímulos de predação por caranguejos estimulou a produção de conchas com labros mais grossos, de forma mais evidente em animais recolhidos de costas expostas e também provocou alterações na forma da concha em animais desta proveniência. Estes dados sugerem contra-gradiente em alterações provocadas por predação na morfologia da concha, na produção de labros mais grossos e em níveis de crescimento. O estudo das interacções gene-ambiente descritas acima demonstram a actual possibilidade de produzir recursos e conhecimento genómico numa espécie bem caracterizada ecologicamente mas com limitada informação genómica. Estes recursos permitem um maior conhecimento biológico desta espécie e abrirão novas oportunidades de investigação, que até aqui seriam impossíveis de abordar.

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.

Mitochondrial plasticity in pathophysiological conditions

Both skeletal and cardiac muscles daily burn tremendous amounts of ATP to meet the energy requirements for contraction. So, it is not surprising that the maintenance of mitochondrial morphology, number, distribution and functionality in striated muscle are important for muscle homeostasis. In these tissues mitochondria present the added dimension of two populations, the intermyofibrillar (IMF) and the subsarcolemmal (SS) mitochondria, being IMF the most abundant one. In the present thesis, the molecular mechanisms harboured in mitochondria of striated muscles were studied using animal models, to better comprehend the role of mitochondrial plasticity in several pathophysiological conditions such as aging, diabetes mellitus and bladder cancer. The comparative analysis of IMF and SS populations isolated from heart evidenced a higher respiratory chain activity of mitochondria interspersed in the contractile apparatus. The higher susceptible of SS respiratory chain complexes subunits to carbonylation, but not to nitration, seems to justify the lower respiratory chain activity observed in this mitochondrial population. Our results showed that in heart from aged mice there is an accumulation of dysfunctional mitochondria. The age-related decrease of oxidative phosphorylation activity seems to be justified, at least partially, by the increased proneness of mitochondrial proteins as OXPHOS subunits and MnSOD to oxidative modifications. Moreover, a sedentary lifestyle seems to worsen the functional consequences of aging in heart by increasing mitochondrial proteins susceptibility to nitration. In skeletal muscle from rats with type 1 diabetes mellitus induced by streptozotocin administration, we verified the accumulation of dysfunctional mitochondria due, at least in part, to the impairment of PQC system. Indeed, the decreased activity of AAA proteases was accompanied by the accumulation of oxidatively modified mitochondrial proteins with impact in respiratory chain activity. The diminishing of mitochondria activity also underlies cancer-induced muscle wasting. Indeed, using a rat model of chemically induced urothelial carcinoma we verified that the loss of gastrocnemius mass was related to mitochondrial dysfunction due to, at least partially, the down-regulation of PQC system involving the mitochondrial proteases paraplegin and Lon. PQC impairment resulted in the accumulation of oxidatively modified mitochondrial proteins. In overall, regardless the pathophysiological stimuli that promote mitochondrial alterations, there are similarities in the pattern of disease-related mitochondrial plasticity. The diminished capacity for ATP production in striated muscle seems to be due to increased oxidative damage of mitochondrial proteins, namely subunits of respiratory chain complexes, metabolic proteins and MnSOD. Our data highlighted, for the first time, the impact of mitochondrial PQC system impairment in the accumulation of oxidized proteins, exacerbating the dysfunction of this organelle in striated muscle in several pathophysiological conditions.

Trophic plasticity in the cnidarian-dinoflagellate symbiosis

Coral reefs are of utmost ecological and economical importance but are currently in global decline due to climate change and anthropogenic disturbances. Corals, as well as other cnidarian species, live in symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium. This relationship provides the cnidarian host with alternative metabolic pathways, as the symbionts translocate photosynthetic carbon to the animal. Besides this autotrophic nutrition mode, symbiotic cnidarians also take up organic matter from the environment (heterotrophy). The nutritional balance between auto- and heterotrophy is critical for the functioning, fitness and resilience of the cnidariandinoflagellate symbiosis. New methodological approaches were developed to better understand the role of auto- and heterotrophy in the ecophysiology of cnidarians associated with Symbiodinium, and the ecological implications of this trophic plasticity. Specifically, the new approaches were developed to assess photophysiology, biomass production of the model organism Aiptasia sp. and molecular tools to investigate heterotrophy in the cnidarian-dinoflagellate symbiosis. Using these approaches, we were able to non-invasively assess the photophysiological spatial heterogeneity of symbiotic cnidarians and identify spatial patterns between chlorophyll fluorescence and relative content of chlorophyll a and green-fluorescent proteins. Optimal culture conditions to maximize the biomass production of Aiptasia pallida were identified, as well as their implications on the fatty acid composition of the anemones. Molecular trophic markers were used to determine prey digestion times in symbiotic cnidarians, which vary between 1-3 days depending on prey species, predator species and the feeding history of the predator. This method was also used to demonstrate that microalgae is a potential food source for symbiotic corals. By using a stable isotope approach to assess the trophic ecology of the facultative symbiotic Oculina arbuscula in situ, it was possible to demonstrate the importance of pico- and nanoplanktonic organisms, particularly autotrophic, in the nutrition of symbiotic corals. Finally, we showed the effects of functional diversity of Symbiodinium on the nutritional plasticity of the cnidarian-dinoflagellate symbiosis. Symbiont identity defines this plasticity through its individual metabolic requirements, capacity to fix carbon, quantity of translocated carbon and the host’s capacity to feed and digest prey.

Mitochondrial dysfunction in fatty acid β-oxidation disorders

Mitochondria are central organelles for cell survival with particular relevance in energy production and signalling, being mitochondrial fatty acid β–oxidation (FAO) one of the metabolic pathways harboured in this organelle. FAO disorders (FAOD) are among the most well studied inborn errors of metabolism, mainly due to their impact in health. Nevertheless, some questions remain unsolved, as their prevalence in certain European regions and how pathophysiological determinants combine towards the phenotype. Analysis of data from newborn screening programs from Portugal and Spain allowed the estimation of the birth prevalence of FAOD revealing that this group of disorders presents in Iberia (and particularly in Portugal) one of the highest European birth prevalence, mainly due to the high birth prevalence of medium chain acyl-CoA dehydrogenase deficiency. These results highlight the impact of this group of genetic disorders in this European region. The characterization of mitochondrial proteome, from patients fibroblasts with FAOD, namely multiple acyl-CoA dehydrogenase deficiency (MADD) and long chain acyl-CoA dehydrogenase deficiency (LCHADD), provided a global perspective of the mitochondrial proteome plasticity in these disorders and highlights the main molecular pathways involved in their pathogenesis. Severe MADD forms show an overexpression of chaperones, antioxidant enzymes (MnSOD), and apoptotic proteins. An overexpression of glycolytic enzymes, which reflects cellular adaptation to energy deficiency due to FAO blockage, was also observed. When LCHADD fibroblasts were analysed a metabolic switching to glycolysis was also observed with overexpression of apoptotic proteins and modulation of the antioxidant defence system. Severe LCHADD present increased ROS alongside with up regulation of MnSOD while moderate forms have lower ROS and down-regulation of MnSOD. This probably reflects the role of MnSOD in buffering cellular ROS, maintain them at levels that allow cells to avoid damage and start a cellular response towards survival. When ROS levels are very high cells have to overexpress MnSOD for detoxifying proposes. When severe forms of MADD were compared to moderate forms no major differences were noticed, most probably because ROS levels in moderate MADD are high enough to trigger a response similar to that observed in severe forms. Our data highlights, for the first time, the differences in the modulation of antioxidant defence among FAOD spectrum. Overall, the data reveals the main pathways modulated in FAOD and the importance of ROS levels and antioxidant defence system modulation for disease severity. These results highlight the complex interaction between phenotypic determinants in FAOD that include genetic, epigenetic and environmental factors. The development of future better treatment approaches is dependent on the knowledge on how all these determinants interact towards phenotype.!

A Spiking Neural Network Model of the Medial Superior Olive using Spike Timing Dependent Plasticity for Sound Localisation

Sound localization can be defined as the ability to identify the position of an input sound source and is considered a powerful aspect of mammalian perception. For low frequency sounds, i.e., in the range 270 Hz-1.5 KHz, the mammalian auditory pathway achieves this by extracting the Interaural Time Difference between sound signals being received by the left and right ear. This processing is performed in a region of the brain known as the Medial Superior Olive (MSO). This paper presents a Spiking Neural Network (SNN) based model of the MSO. The network model is trained using the Spike Timing Dependent Plasticity learning rule using experimentally observed Head Related Transfer Function data in an adult domestic cat. The results presented demonstrate how the proposed SNN model is able to perform sound localization with an accuracy of 91.82% when an error tolerance of +/-10 degrees is used. For angular resolutions down to 2.5 degrees , it will be demonstrated how software based simulations of the model incur significant computation times. The paper thus also addresses preliminary implementation on a Field Programmable Gate Array based hardware platform to accelerate system performance.

Technology-related strategies in labour-intensive industries from Southern European Regions: consequences for local employment

Tese dout., Economia, Universidade do Algarve, 2010

Development of an operational tool for oil spill forecast: application to oil exposed regions

Tese de doutoramento, Ciências do Mar, da Terra e do Ambiente (Modelação), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Modeling the suitable habitat of a structural red gorgonian species in the Mediterranean/east Atlantic regions predicts a wider distribution than previously known

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Univerdade do Algarve, 2015

How does developmental plasticity differ between diverse types of nutrition regimes?

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

Study the function of the newly discovered TrkB receptor fragment (TrkB-ICD) formed by calpain cleavage

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

The attractive female body weight and female body dissatisfaction in 26 countries across 10 world regions: results of the International Body Project I

This study reports results from the first International Body Project (IBP-I), which surveyed 7,434 individuals in 10 major world regions about body weight ideals and body dissatisfaction. Participants completed the female Contour Drawing Figure Rating Scale (CDFRS) and self-reported their exposure to Western and local media. Results indicated there were significant cross-regional differences in the ideal female figure and body dissatisfaction, but effect sizes were small across high-socioeconomic-status (SES) sites. Within cultures, heavier bodies were preferred in low-SES sites compared to high-SES sites in Malaysia and South Africa (ds = 1.94-2.49) but not in Austria. Participant age, body mass index (BMI), and Western media exposure predicted body weight ideals. BMI and Western media exposure predicted body dissatisfaction among women. Our results show that body dissatisfaction and desire for thinness is commonplace in high-SES settings across world regions, highlighting the need for international attention to this problem.

Transitions In Regional Science – Regions In Transitions: Regional Research In Central Europe

There are no exact boundaries of what constitutes Central Europe, but it is nevertheless very evident as a zone of interloping forces stretching across Europe. The result has been negotiated, conflictual and contested processes defining an essentially virtual, discursive, yet also actually existing, space of ‘Central Europe’. Yet, and this is the particular message of this collection of essays, ‘transition’ has not merely been a process ‘on the ground’ as object of investigation and discussion, but has also affected the observers, especially academic commentators and analysts – both within Central Europe, but also outside. This includes a growing interaction between discussions and analyses ‘inside’ and ‘outside’ Central Europe on the phenomenon of ‘transition’. The result has been mutual learning processes and changes of ways of looking at things and interpreting them. The book is divided into two parts, roughly reflecting the two key research questions.The first part (A) is devoted to transitions in regional science. Part B focuses on changes in variety of territorial structures and developmentissues caused by Central-European transitions and which are resulting in differential pathways of regional development in the region.

Outermost european regions of the atlantic area network

This paper is part of the results from the project "Implementation Strategies and Development of an Open and Distance Education System for the University of the Azores" funded by the European Social Fund. http://hdl.handle.net/10400.3/2327