Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

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 characterization of Gla-rich protein (GRP) gene expression and function

Gla-rich protein (GRP) is a vitamin K-dependent protein related to bone and cartilage recently described. This protein is characterized by a large number of Gla (γ-carboxyglutamic acid) residues being the protein with the highest Gla content of any known protein. It was found in a widely variety of tissues but highest levels was found in skeletal and cartilaginous tissues. This small secreted protein was also expressed and accumulated in soft tissues and it was clearly associated with calcification pathologies in the same tissues. Although the biological importance of GRP remains to be elucidated, it was suggested a physiological role in cartilage development and calcification process during vertebrate skeleton formation. Using zebrafish, an accepted model to study skeletal development, we have described two grp paralog genes, grp1 and grp2, which exhibited distinct patterns of expression, suggesting different regulatory pathways for each gene. Gene synteny analysis showed that grp2 gene is more closely related to tetrapod grp, although grp1 gene was proposed to be the vertebrate ortholog by sequence comparison. In addition, we identified a functional promoter of grp2 gene and using a functional approach we confirmed the involvement of transcription factors from Sox family (Sox9b and Sox10) in the regulation of grp2 expression. In an effort to provide more information about the function of grp isoforms, we generated two zebrafish transgenic lines capable to overexpress conditionally grp genes and possible roles in the skeleton development were studied. To better understand GRP function a mammalian system was used and the analysis of knockout mice showed that GRP is involved in chondrocyte maturation and the absence of GRP is associated to proteoglycans loss in calcified articular cartilage. In addition, we detected differences in chondrogenesis markers in articular chondrocyte primary culture. Overall, our data suggest a main role for GRP on chondrocyte differentiation.

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

Controle hormonal da diferenciação e inversão sexual na dourata Sparus aurata (L.)

Tese de Doutoramento, Biologia Marinha, Unidade de Ciências e Tecnologias dos Recursos Aquáticos, Universidade do Algarve, 2001

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.

Assemblage structure and secondary production of mesozooplankton in shallow water volcanic CO2 vents of the Azores

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

Temporal distribution of the short-beaked common dolphin (Delphinus delphis) in the south of Madeira Island (Portugal) and relationship with oceanographic variables

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

Technical and economic viability of traps in the portuguese fishery of deepwater rose shrimp (Parapenaeus longirostris)

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Incorporation of selenium on microalgae as supplement to artemia and zebrafish

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Crescimento, ecologia alimentar e reprodução de Trachurus trecae (Cadenat, 1949), na costa sul de Angola-Namibe

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

Impacto fisiológico do parasita dinoflagelado Amyloodinium ocellatum (Brown, 1931) em corvina-legítima (Argyrosomus regius, Asso, 1801) produzida em aquacultura

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

Inclusão da microalga (Phaedactylum tricornutum) em rações de acabamento para dourada: efeito na pigmentação e outros atributos da qualidade

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2015