Study of transcripts and reactive oxygen species involved in the defence responses of Quercus suber against Phytophthora cinnamomi

The present work has the merit of exploring an insight into the activation of defence genes of Quercus suber during response to infection by Phytophthora cinnamomi. Thus, cDNA-AFLP methodology was used to identify gene fragments differentially present in the mRNA profiles of host cells of micropropagated Q. suber plantlets roots infected with zoospores of P. cinnamomi at different post challenge time points. Six candidate genes were selected based on their interesting cDNA-AFLP expression patterns and homology to genes known to play a role in defence. These six genes encode a cinnamyl alcohol dehydrogenase 2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), thaumatin-like protein (QsTLP), chitinase (QsCHI) and a 1,3-beta glucanase (QsGLU). The current work has been successful in evaluation of the expression of these genes by qRT-PCR. Data analysis revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the early hours of inoculation, while transcript profiles of thaumatin-like protein showed decreasing. No expression was detected for 1,3-beta-glucanase (QsGLU). Furthermore, the choice of suitable reference genes in any new experimental system is absolutely crucial in qRT-PCR; for this reason in this study and for the first time a set of potential reference genes were analyzed and validated for qRT-PCR normalization in the patho-system Phytophthora-Q. suber. Four candidate reference genes polimerase II (QsRPII), eukaryotic translation initiation factor 5A(QsEIF-5A), b-tubulin (QsTUB) and a medium subunit family protein of Clathrin adaptor complexes (QsCACs) were evaluated to determine the most stable internal references in Q. suber. Analysis of stability of genes was carried out using Genex software. Results indicated all these four potential reference genes assumed stable expression. Data analysis revealed that QsRPII and QsCACs were the two most stable genes, while genes QsTUB and QsEIF-5A were the third and the fourth most stable gene, respectively. In this study, a plasmid-based quantitative PCR method was developed to measure P. cinnamomi colonization during infection process of Q. suber. Plasmid-based detection of P. cinnamomi showed a gradual accumulation of the pathogen DNA in cork oak root tips up to 24 h post infection. The higher increase in P. cinnamomi/plasmid DNA ratio occurred between 18 and 24 h. One of the primary objectives of this research was to study the effect of cinnamomins (elicitins secreted by P. cinnamomin) on inducing defence mechanism against the pathogen, as recent histological and ultra-structural studies showed that P. cinnamomi was restricted to the outer cortex root fragments pre-treated with capsicien and cryptogein, suggesting that elicitins can stimulate plant defence reactions against P. cinnamomi. To complement these studies and to have a clear view of the nature of the interaction, the role of cinnamomins in the production of the oxidative burst [ROS and ROS scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)] and in the defence responses was evaluated. Cork oak seedlings were pretreated with alpha-cinnamomin and then inoculated with P. cinnamomi mycelia. Results showed a significant higher production of reactive oxygen species (ROS) (H2O2 and O2•-) in elicitin and non-elicitin treated roots in interaction with P. cinnamomi in comparison to the corresponding control. The plant group inoculated with the pathogen after cinnamomin treatment showed an earlier increase in H2O2 production but this was lower as compared with that group inoculated with P. cinnamomi alone. Also, in elicitin pre-treated group generally, a lower level of O2•− production during infection was observed as compared with inoculated roots with P. cinnamomi alone without elicitin treatment. Furthermore, in this study, we evaluated activities of antioxidant enzymes upon challenge with P. cinnamomi, with and without pretreatment with alpha cinnamomin. Results indicated that the activities of defense enzymes POD, SOD and CAT increased after P. cinnamomi inoculation when compared with those in the control group. Also, in the group treated with alpha-cinnamomin followed by P. cinnamomi inoculation, a higher level of enzymatic activities was detected as compared with elicitin non-treated group, which suggest the protective effect of alpha-cinnamomin against the pathogen due to higher elevated levels of defense enzymes POD, SOD and CAT during the infection period. Furthermore, a sensitive qPCR method was applied to measure the pathogen biomass in elicited and non-elicited Q. suber roots challenged with P. cinnamomi to elucidate the effect of cinnamomins on the colonization of P. cinnamomi. Plasmid-based quantification of P. cinnamomi showed a significant decrease in accumulation of the pathogen DNA in cork oak roots after treatment with alpha and beta-cinnamomins which attest the role of cinnamomins in promoting defense responses in cork oak against P. cinnamomi invasion.

Instrumentação e controlo para regulação de pH em sistemas de cultivo de organismos aquáticos

A Ria Formosa é um tesouro ambiental sem paralelo, preservando uma fauna e flora únicas no mundo. A riqueza deste habitat é de enorme importância para a região, e extremamente apetecível para cientistas oriundos de todas as partes do globo, que aqui frequentemente se deslocam para conduzirem estudos científicos e experiências. O Centro de Ciências do Mar (CCMAR) da Universidade do Algarve (que inclui o Centro Experimental do Ramalhete) conduz estudos e experiências neste palco, estudos que são de inquestionável valor para o conhecimento e desenvolvimento científico. Um assunto que está a merecer a atenção da comunidade científica mundial nos últimos anos é a questão da acidificação dos oceanos. A diminuição gradual do pH das águas pode vir a ter graves repercussões nos ecossistemas marinhos, e o Centro Experimental do Ramalhete tem vindo a conduzir experiências com fauna e flora provenientes da Ria Formosa em águas com níveis de pH mais reduzido, condições que se prevê que os oceanos venham a ter no futuro. Os equipamentos de instrumentação e controlo a que o Centro tem acesso condicionam as experiências que ali são levadas a cabo pelos investigadores, pelo que o desenvolvimento de equipamentos adequados incorporando tecnologias apropriadas permitiria a realização de novas e melhores experiências no campo da biologia marinha. Ao nível do controlo existe uma lacuna no mercado, entre controladores para aquariofilia demasiado simples e controladores industriais demasiado dispendiosos e complexos. Esta dissertação pretende colmatar essa lacuna através do desenvolvimento de um protótipo de um sistema distribuído microcontrolado para aquisição de dados e controlo de pH que vá ao encontro das necessidades dos investigadores do Centro e que se pretende simples, modular, flexível, económico e expansível no futuro. O foco centra-se no desenvolvimento da instrumentação necessária para as medições de temperatura e pH, e depois no estudo de uma malha de controlo PID utilizando como base um modelo do sistema obtido através de resultados experimentais, para o controlo automático do pH.

An integrated and comparative approach to genetic selection of the aquaculture species Dicentrarchus labrax

The European sea bass, Dicentrarchus labrax, is one of the most important marine species cultivated in Southern Europe and has not benefited from selective breeding. One of the major goals in the sea bass (D. labrax) aquaculture industry is to understand and control the complexity of growth associated traits. The aim of the methodology developed for the studies reported in the thesis was not only to establish genetic and genomic resources for sea bass, but to also develop a conceptual strategy to efficiently create knowledge in a research environment that can easily be transferred to the aquaculture industry. The strategy involved; i) establishing an annotated sea bass transcriptome and then using it to, ii) identify new genetic markers for target QTL regions so that, iii) new QTL analysis could be performed and marker based resolution of the DNA regions of interest increased, and then iv) to merge the linkage map and the physical map in order to map the QTL confidence intervals to the sea bass genome and identify genes underlying the targeted traits. Finally to test if genes in the QTL regions that are candidates for divergent growth phenotypes have modified patterns of transcription that reflects the modified whole organism physiology SuperSAGE-SOLiD4 gene expression was used with sea bass with high growth heterogeneity. The SuperSAGE contributed to significantly increase the transcriptome information for sea bass muscle, brain and liver and also led to the identification of putative candidate genes lying in the genomic region of growth related QTL. Lastly all differentially expressed transcripts in brain, liver and muscle of the European sea bass with divergent specific growth rates were mapped to gene pathways and networks and the regulatory pathways most affected identified and established the tissue specific changes underlying the divergent SGR. Owing to the importance of European sea bass to Mediterranean aquaculture and the developed genomics resources from the present thesis and from other studies it should be possible to implement genetic selection programs using marker assisted selection.

Eco ventilação: desenvolvimento de uma ventilação mais ecológica

Neste trabalho é desenvolvido um estudo comparativo de vários sistemas de ventilação, baseados em jactos localizados. Foram avaliados, para cada um deles, o nível da qualidade do ar interior, conforto térmico, desconforto térmico local (risco de resfriamento) a que os ocupantes estão sujeitos e o índice ADI (Air Distribution Index). O estudo foi efectuado numericamente, a partir de dois softwares acoplados, e experimentalmente, a partir de uma sala de aula desenvolvida a escala real. Nesta sala de aulas, equipada com 6 ou 12 ocupantes e com 6 secretárias, foram analisados sistemas de ventilação com jactos verticais descendentes, localizados em cima (mais afastado e mais próximo do nível da cabeça dos ocupantes) e à frente dos ocupantes, e com jactos horizontais, localizados em frente dos ocupantes, em cima e em baixo da secretária. O estudo numérico, da interacção do escoamento com os ocupantes, foi efectuado a partir do acoplamento de um software que simula a resposta térmica dos ocupantes (Human Thermal Comfort) e um que simula o escoamento tridimensional em espaços ocupados (Virtual Air Flow - 3D), utilizando o modelo de turbulência RNG. Foi utilizada uma malha não uniforme, com um maior refinamento junto dos obstáculos e nas entradas e saídas de ar. No estudo do projecto do sistema de ventilação baseado em jactos verticais descendentes localizados em cima e mais afastado do nível da cabeça dos ocupantes, foram efectuadas medições experimentais de forma a validar os softwares acoplados e um modelo desenvolvido para calcular o escoamento unidireccional no interior de condutas. Neste estudo foi ainda utilizado um modelo de cálculo das temperaturas das superfícies. Por fim foi realizada uma simulação num contexto mais real, com 6 ocupantes sentados e um ocupante em pé, simulando 6 alunos e um professor, onde foi utilizado o acoplamento dos modelos Human Thermal Comfort e Virtual Air Flow - 3D, o modelo numérico de escoamentos unidireccionais no interior de condutas e o modelo de cálculo das temperaturas das superfícies. São ainda feitas sugestões para a partir deste trabalho, se efectuarem estudos em geometrias mais complexas.

Study of influence of regulatory polymorphisms of expression in development of breast cancer

The human genome has millions of genetics variants that can affect gene expression. These variants are known as cis-regulatory variants and are responsible for intra-species phenotypic differences and individual susceptibility to disease. One of the diseases affected by cis-regulatory variants is breast cancer. Breast cancer is one of the most common cancers, with approximately 4500 new cases each year in Portugal. Breast cancer has many genes mutated and TP53 has been shown to be relevant for this disease. TP53 is one of the most commonly mutated genes in human cancer and it is involved in cell cycle regulation and apoptosis. Previous work by Maia et al has shown that TP53 has differential allelic expression (DAE), which suggests that this gene may be under the influence of cis-regulatory variants. Also, its DAE pattern is totally altered in breast tumours with normal copy number. We hypothesized that cis-regulatory variants affecting TP53 may have a role in breast cancer development and treatment. The present work aims to identify the cis-regulatory variants playing a role in TP53 expression, using in silico, in vitro and in vivo approaches. By bioinformatic tools we have identified candidate cis-regulatory variants and predicted the possible transcription factor binding sites that they affect. By EMSA we studied DNA-protein interactions in this region of TP53. The in silico analysis allowed us to identified three candidate cis-regulatory SNPs which may affect the binding of seven transcription factors. However, the EMSA experiments have not been conclusive and we have not yet confirmed whether any of the identified SNPs are associated with gene expression control of TP53. We will carry out further experiments to validate our findings.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

On the solvation in lipid bilayers measured by pyrene fluorescence: thermal and molecular composition influence on equivalent polarity in lipidic mixtures

Phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol (CHOL) are major constituents of mammalian cell membranes. DPPC/CHOL and DPPC/DMPC are well-known binary mixtures. POPC/CHOL, DOPC/CHOL, egg-SM/CHOL, egg-SM/POPC and egg-SM/DOPC are less studied, but also important for the comprehension of the POPC/egg-SM/CHOL mixtures. These provide complex media for which polarity is hard to access. It is mainly determined by the water penetrating the bilayer (unevenly distributed creating a polarity gradient), though the influence of the dipoles from phospholipids (e.g. –PO, –CO, –OH) and the double bond in the steroid ring of CHOL cannot be neglected. CHOL derivatives are an interesting tool to verify the influence of the double bonds in the polarization of its surroundings. Pyrene fluorescence was used to access an equivalent polarity (associated to the dielectric constant) near the lipid/water interface of lipid bilayers. POPC/CHOL and DOPC/CHOL have similar thermal behavior and variation with CHOL content, though for lower CHOL content the equivalent polarity is higher for the DOPC/CHOL mixtures. The studies with DPPC and DMPC showed that pyrene does not seem to have a marked preference for either ordered or disordered phases. For DPPC/CHOL and egg-SM/CHOL the highlight goes to the behavior of the mixtures at higher CHOL amounts, where there is a substantial change in the thermal behavior and polarity values especially for the egg-SM/CHOL mixture. Egg-SM/POPC and egg-SM/DOPC show different behavior depending on which phospholipid has a higher molar proportion. The ternary mixtures analyzed do not exhibit significant differences, though there is the indication of the existence of a more ordered environment at lower temperatures and a less ordered environment for higher temperatures. The presence of 7DHC or DCHOL in egg-SM bilayers showed a tendency for the same behavior detected upon mixing higher amounts of CHOL.

Du roman au théâtre: adaptations théâtrales dans la dramaturgie franco-portugaise au XIXe siècle

L’adaptation de romans à la scène au XIXe siècle dans la dramaturgie franco-portugaise passe par l’étude des relations et des contaminations intergénériques et interculturelles qui s’établissent entre les genres romanesque et dramatique dans les deux cultures. Pour ce faire, nous nous sommes consacrés dans une première partie aux questions théoriques qu’un tel exercice soulève. Effectivement, nous ne pourrions pas prétendre étudier et cerner un tel phénomène littéraire et artistique sans un abordage pluridimensionnel qui nous mène vers son approche à la lumière des études de Traduction et de Réception, d’un côté et, de l’autre, des études sociologiques. Questionner les fondements théoriques de l’adaptation théâtrale, sa production et sa circulation dans les deux pays supposait, pour nous, dès le départ, délimiter non seulement l’horizon d’attente d’une telle pratique artistique mais aussi sa place et son impact dans le champ littéraire et le polysystème culturel de l’époque. En contrepoint, nous nous sommes ensuite intéressés à la question de l’adaptation théâtrale et des contact zones, cette fois en passant par la problématique de la transculturation qu’engendre le contact de deux littératures différentes, s’affrontant dans le champ culturel et littéraire d’arrivée. Dans une deuxième partie, nous nous sommes concentrés sur la contextualisation historico-générique de l’adaptation théâtrale. Le recours à la transposition scénique de romans révèle un désir de gloire au niveau financier et artistique. La pratique est courante, voire généralisée dans le champ littéraire français du XIXe siècle. Parmi les adeptes de l’exercice de transposition, Dumas père est certainement l’auteur le plus prolifique. En outre, la transmodalisation générique s’insère dans un mouvement de création collective, puisque la collaboration entre auteurs dramatiques est très répandue. Par la suite, nous nous sommes intéressés à la genèse des textes à travers la transmodalisation du roman au théâtre : du roman-feuilleton au roman naturaliste. Nous avons constaté que le genre romanesque comme le genre dramatique sont empreints de contaminations intergénériques qui passent par la théâtralisation du roman puis par la romanisation du théâtre. Ainsi, l’adaptation apparaît comme la concrétisation de la fusion des genres romanesque et dramatique qui donnent lieu à un genre hybride. L’adaptation est alors le résultat d’une série de procédés et de techniques : la transmodalisation du roman s’adapte aux nécessités génériques du théâtre. De la sorte, nous avons distingué cinq types d’adaptations : l’adaptation fidèle, l’adaptation partielle, l’adaptation libre, l’adaptation pastichée et l’adaptation de l’adaptation. Dans une troisième partie, nous avons abordé le phénomène de l’adaptation dans le contexte littéraire portugais au XIXe siècle. À travers la présence accrue du théâtre français sur les scènes lisboètes, nous avons observé les rouages de la transposition d’un modèle étranger qui peut passer par différents processus, comme la traduction ou l’appropriation par l’adaptation théâtrale. Cela nous a amené à présenter cinq modèles d’adaptations théâtrales issues d’hypotextes français : l’adaptation française, la traduction fidèle de l’adaptation française, la traduction libre de l’adaptation française, l’adaptation à la couleur locale de l’adaptation française et l’adaptation portugaise d’un roman français. Nous avons également présenté quelques exemples de cas concrets, notamment Le Comte de Monte-Cristo adapté au répertoire lisboète. L’étude de ce phénomène culturel nous a permis de mieux cerner son impact sur le champ théâtral lisboète du XIXe siècle, dans la mesure où il contribue à la transculturation de la dramaturgie portugaise de l’époque.

Farmacogenómica na terapêutica das leucemias agudas

As leucemias agudas são doenças raras, quando comparadas com outros tipos de neoplasias constituindo, no entanto, a doença maligna mais comum na infância. São responsáveis por 30% de todos os tipos de cancros diagnosticados em crianças menores de 15 anos em países industrializados. Enquanto que a leucemia linfoblástica aguda apresenta uma taxa de incidência superior em crianças até aos 15 anos, correspondendo a cerca de 80% das leucemias em crianças e adolescentes, a leucemia mieloide aguda é rara abaixo dos 40 anos, sendo que a sua incidência aumenta progressivamente com a idade. As estratégias atuais de tratamento dividem a terapêutica em duas etapas: a primeira possui como objetivo induzir a remissão da doença e a segunda consiste numa terapêutica de pós-remissão com o objetivo de erradicar a doença residual mínima, evitar recidivas e promover a cura. Após anos de pesquisa na área da farmacogenómica, observa-se que as diferenças genéticas entre indivíduos podem explicar alguma da variabilidade observada na farmacocinética, eficácia e toxicidade de alguns fármacos. Embora muitos estudos relacionem diferentes respostas farmacológicas com a variabilidade genética, a maioria daqueles aplicam-se à população adulta pelo que a atenção dada à população pediátrica tem sido muito menor. Assim, o aperfeiçoamento na terapêutica das leucemias agudas é urgente. Atualmente encontra-se perfeitamente estabelecida a relação entre reações adversas à 6-mercaptopurina e polimorfismos no gene que codifica para a enzima tiopurina s-metiltransferase, sendo um dos poucos exemplos na área da farmacogenética a ser “traduzido” para a prática clínica.

Estudo de casos clínicos à luz do modelo relacional dialógico

Com base nas concepções e trabalho empírico dos técnicos da Psicologia temos vindo a ter conhecimento do papel fundamental dos cuidadores nas etapas de desenvolvimento psicológico do ser humano. Quando não há um relacionamento baseado em trocas afetivas e emocionais contingentes, entre o indivíduo e os seus cuidadores, podem surgir alterações ou até mesmo uma perturbação na sua evolução. Faremos referência ao contributo do Modelo Relacional Dialógico desenvolvido por Maria Rita Mendes Leal, que apresenta uma proposta genética para a construção do “Eu”, segundo a qual, é postulado, que o ser humano nasce biologicamente apto para comunicar e relacionar-se com os parceiros da própria espécie. Neste trabalho apresentamos os passos do desenvolvimento sócio emocional em paralelo com estruturas de personalidade, pretendendo-se contribuir para a compreensão da estruturação de cada entidade, bem como, de comportamentos desadaptativos caraterísticos de diferentes psicopatologias. Para este efeito, são apresentados dois estudos de casos clínicos, de forma a ilustrar este processo de intervenção dinâmico-relacional mutuamente contingente.

Recreational shore fishing in southern Portugal: biological and socio-economic aspects and perspectives for management

Although overfishing is a concern for many fish stocks, it was for a long time only associated with commercial fishing exploitation, with less or no attention being given to the recreational fisheries. Recent research has shown however that the impact of recreational fishing on particular species can be considerable, and that the recreational harvest needs to be taken into account if fisheries are to be accurately assessed and effectively managed. In Portugal, the first recreational fishing regulations were only recently implemented. However, mirroring other European countries, regulations lacked scientific support, and specific knowledge of the activity was limited to a few studies with limited coverage. This thesis aimed to characterize the biological and socioeconomic aspects of the recreational shore angling activity in southern Portugal, to investigate whether the regulations in place were adequate and effective, and to provide recommendations for improved management and conservation of the inshore fisheries resources. A combined aerial-roving survey was conducted to gather data on fishing effort, catch, fishing trips and socioeconomic aspects (including anglers’ perceptions of regulations) of the recreational angling activity. The analysis of anglers’ catches suggested that compliance with daily bag limits was high, with less than 0.5% of creels exceeding the 10 kg angler-1 day-1 bag limit. Overall, 11.5% of the retained fishes were undersized, but non-compliance with minimum size limits was found to be high for some species (e.g. seabass, 73% undersized). In terms of the impact of recreational shore angling, the total estimated catches corresponded to less than 1% of the commercial landings for the same period (shared species). However, shore angling catches for white sea bream (Diplodus sargus) were found to be considerable, corresponding to 65% of the commercial landings (39.4% of total catch). In terms of anglers’ perceptions about the recreational fishing regulations in Portugal, the present study has shown that the majority of anglers accepted the existence of some kind of SRF regulations, but in general there was a partial or total disagreement with the recreational fishing restrictions recently put in place. Most anglers perceived themselves as not being involved in the decision-making process and claimed that some restrictions lacked a meaningful rationale (e.g. prohibition of fishing from piers/jetties). Fishers’ awareness with regard to specific aspects of the restrictions (such as the rationale for minimum size limits) was found to be very limited. During the same period, catches from sport fishing competitions were examined to test for differences with the recreational activity in terms of catches, and evaluate long term trends in catch and mean size of fish. Catches of the sport fishing competitions were found to be different from those observed for recreational fishing, being dominated by different species (e.g. garfish, mullets), and suggesting different fishing strategies of the the two types of anglers. High percentages of undersized fish were observed to be captured (and retained) during the competitions (in particular seabass, with 100% undersized), probably as a result of a single allowable minimum size (AMS) of 15 cm for all species in use in competitions. Lastly, catch and release fishing experiments were carried out to assess post-release mortality of three recreationally important species: two banded sea bream Diplodus vulgaris; black sea bream Spondyliosoma cantharus; and gilthead sea bream Sparus aurata. Post-release mortalities were found to be low (0-12%). The main predictor of mortality for Sparus aurata was anatomical hooking location, with 63% of the fishes that died being deeply hooked. The results support the release of fish, either from mandatory (e.g. minimum landing sizes) or voluntary practices. In summary, this thesis has demonstrated that the impact of recreational fishing for particular species is significant and needs to be taken into account for more effective management and stock assessment purposes. It has also highlighted several management issues that should be addressed in order to promote more adequate regulations in the future and prevent noncompliance issues. A periodic monitoring of the recreational fishing activity including all fishing modes (i.e. spear fishing, boat, and shore angling) would also be beneficial to ensure a timely knowledge on the global recreational fishing activity and support future management actions.

Molecular structure and functional analysis of runx3 in zebrafish

Tese de doutoramento, Ciências Biomédicas, Universidade do Algarve, Departamento de Ciências Biomédicas e Medicina, 2014

Identification and molecular characterization of bone-related micrornas: functional implications

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

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.

Connectivity patterns and early life history of the black-faced blenny Tripterygion delaisi (Cadenat and Blache, 1970)

Tese de doutoramento, Ciências do Mar, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015