Pine processionary moth (Thaumetopoea pityocampa) and great tit (Parus major) in Portugal

In the present work, we studied a common outbreaking Lepidoptera species in Portuguese pine stands – Thaumetopoea pityocampa (Den. & Schiff.) - and one of its potential predators – Parus major (L.). The population dynamics of the immature stages of the Lepidoptera was studied in several types of Pinus pinaster (Aiton) plantations in three different areas: Setúbal Peninsula, Abrantes and National Pine Forest of Leiria. Location and plantation structure was the most important factors determining population density of T. pityocampa. Setubal and Abrantes was highly susceptible to attacks by the Lepidoptera, whereas Leiria had lower densities. Young and homogeneous pine stands was more susceptible to attacks than older and more heterogeneous pines stands. However, a desynchronized population of T. pityocampa, in which the larvae develops during summer instead of during winter, reached high densities also in Leiria. The impact of several mortality factors and climatic conditions on the immature stages of the insect (eggs and larvae), in normal and desynchronized populations are discussed, as well as possible evolutionary implications of the sudden appearance of the new version of T. pityocampa. The break of the pupa diapause and adult emergence times the annual life cycle of this insect. Adults from the desynchronized population emerged earlier than adults from the normal population, which in turn determined the change in the larvae development period. Different factors, potentially affecting the timing of adult emergence in both normal and abnormal populations are also discussed. To study P. Major, nest-boxes were placed in the areas of Setúbal and Leiria and they were monitored during three seasons. The nest-boxes increased the density of breeding and wintering birds in the studied pine plantations, indicating that a lack of natural holes are in fact a limiting factor for this populations. The earliest breeding start for this species was recorded in my study area, indicating that Portuguese coastal pines provide good breeding conditions earlier than in other areas of Europe and North Africa. This leads to an overlap between the end of the larvae stage of T. pityocampa and the beginning of the breeding season of P. major. Key-words: Thaumetopoea pityocampa, Parus major, Pinus pinaster, population dynamics, Portugal.

Systems biotechnology of baculovirus-producing insect cells

Dissertation presented to obtain the Ph.D degree in Ciências da Engenharia e Tecnologia, especialidade Biotecnologia

Characterization of Pinus spp. needles by gas chromatography and mass spectrometry: Application to plant-insect interactions

Dissertação apresentada para obtenção do Grau de Doutor em Ciências do Ambiente pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecn

Toxicity of carcinogenic and non-carcinogenic Polycyclic Aromatic Hydrocarbons and their mixtures to aquatic organisms under ecologically-relevant scenarios

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous priority pollutants that tend to be trapped in aquatic sediments due to their high hydrophobicity. Nonetheless, the differential toxicological effects and mechanisms between the various classes of PAHs and their mixtures, as they invariably occur in the environment, are scarcely known, especially under ecologically-relevant scenarios. This thesis aimed at establishing a bridge between the study of mechanistic pathways and environmental monitoring of carcinogenic and non-carcinogenic PAHs, by introducing ecological-relevance in the research with model PAHs. A first bioassay conducted in situ with the mussel Mytilus edulis demonstrated that, dredging operations in harbours increase PAH bioavailability, eliciting genotoxicity, and showed that established environmental guidelines underestimate risk. Subsequent ex situ bioassays were performed with the carcinogenic benzo[b]fluoranthene (B[b]F) and non-carcinogenic phenantrene (Phe), selected following preceding results, and revealed that low-moderate concentrations of these PAHs in spiked sediments induce genotoxic effects to the clam Ruditapes decussatus, therefore contradicting the general notion that bivalves are less sensitive to PAHs than vertebrates due to inefficient bioactivation. Also, it was demonstrated that passive samplers permit inferring on PAH bioavailability but not on bioaccumulation or toxic effects. On the other hand, sea basses (Dicentrarchus labrax), yielded a complex pattern of effects and responses, relatively to genotoxicity, oxidative stress and production of specific metabolites, especially when exposed to mixtures of the PAHs which led to additive, if not synergistic, effects. It was shown that Phe may elicit significant genotoxicity especially in presence of B[b]F, even though the low, albeit realistic, exposure concentrations diluted dose- and time-independent relationships. The present work demonstrated that environmental quality guidelines underestimate the effects of PAHs in realistic scenarios and showed that the significant genotoxic and histopathological effects caused by mixed PAHs may not be reflected by oxidative stress- or CYP-related biomarkers. Besides important findings on the metabolism of PAH mixtures, the work calls for the need to re-evaluate the criteria for assessing risk and for the disclosure of more efficient indicators of toxicological hazard.

Establishment and evaluation of flexible insect cell lines for rapid production of recombinant proteins

Fundação para a Ciência e a Tecnologia (FCT) - (PTDC/EBB-EBI/102266/2008 and SFRH/BD/43830/2008, respectively) and by European Community’s FP7/2007-2013 (grant agreement nº 270089)

The role of Hedgehog signaling during zebrafish larvae fin fold regeneration

Several studies have demonstrated that although the structure of the adult and larval zebrafish caudal fin is different, there are similarities at the cellular and molecular level that turn larval zebrafish fin fold a useful model to study the basic principles of regeneration. In this process, while the essential role for Hedgehog (Hh) signaling is well established in the adult zebrafish caudal fin system, its involvement in juvenile tissue regeneration is still unknown. The aim of this Master thesis was therefore to evaluate the contribution of the Hh signaling pathway to the larval zebrafish fin fold regeneration process. Accordingly, we analyzed the expression of several Hh signaling components through in situ hybridization. Here, we showed that several of these genes are effectively expressed in the larval regenerating fin tissue, suggesting a role for Hh signaling also during larval regeneration. However, divergence in the regulation of few Hh signaling components appears to exist between the adult and larval zebrafish fin regeneration processes. Nevertheless, similarly to adult caudal fin regeneration, when Hh signaling was blocked, by using cyclopamine, the larval fin fold regenerative outgrowth is severely impaired. Since larval zebrafish fin fold is ciliated, and primary cilia are closely related to Hh signaling regulation in vertebrate systems, we further addressed the role of primary cilia during larval fin fold regeneration process. To this end, we used the zebrafish iguana mutant, in which primary cilia are not formed, to study the modulation of Hh signaling expression during larval fin fold regeneration in the absence of primary cilia. Here, we found that several genes were expressed with a delay, coincident with the delay in the mutant fin fold regeneration observed in previous work. We show that Hh signaling in the fin fold is crucial to promote cell proliferation. When Hh signaling is blocked using cyclopamine there is a strong blockage of cell proliferation and regeneration is also blocked. Surprisingly, in iguana mutants where Hh signaling is impaired but not totally blocked, cell proliferation is not detected but regeneration still occurs. This raises the question about the requirement of cell proliferation in larvae fin fold regeneration. By blocking the cell cycle using aphidicolin we demonstrate that cell proliferation is not necessary for zebrafish larvae fin fold regeneration.