Hábitos de leitura de alunos dos 2.º e 3.º ciclos do ensino básico na E.B. 2,3 Poeta Emiliano da Costa

Dissertação de mest., Promoção e Mediação da Leitura, Faculdade de Ciências Humanas e Sociais, Univ. do Algarve, 2012

Contribuição para a elaboração de um plano de gestão integrada dos campos de Z. noltii na Ria Formosa

Este trabalho representa uma contribuição para a elaboração de um Plano de Gestão Integrada dos campos de Zostera noltii na Ria Formosa. Utilizando como ferramenta os Sistemas de Informação Geográfica (SIG) e através de foto-interpretação, foram cartografados os campos de Z. noltii na zona de sedimentos intertidais da Ria Formosa. Foi criado um modelo SIG para a análise da cartografia obtida com outros dados relevantes para gestão deste recurso natural, nomeadamente, as áreas intertidais cedidas para a cultura de moluscos bivalves, o Plano de Ordenamento da Orla Costeira Vilamoura – Vila Real de Santo António e o Plano de Ordenamento do Parque Natural da Ria Formosa (POPNRF). Considerando a importância da vertente social num sistema de apoio à decisão considerou-se relevante a análise da actividade de cultura de bivalves pela perspectiva dos profissionais (viveiristas) quanto à importância da Z. noltii no ecossistema e a gestão da actividade. Realizou-se entrevistas semi – dirigidas aos actores chave, inquéritos à comunidade de produtores de moluscos bivalves e observação não participada. Os dados obtidos foram manipulados com recurso ao modelo de Estratégia de Actores. Verificou-se que área total de Z. noltii na zona intertidal da Ria Formosa é cerca de 1304 ha, o que corresponde a 45% da área total intertidal. Nas classes de espaço do POPNRF 25% da área ocupada por Z. noltii está incluída em classes onde a acessibilidade é condicionada. A maioria dos viveiristas manifestou o seu descontentamento pela actual gestão da actividade com consequências na sua produtividade. Relativamente a Z. noltii a maioria dos profissionais desconhece a sua função no ecossistema, não considerando necessário implementar medidas de conservação. São propostas como linhas orientadoras de gestão: a implementação de um modelo de gestão participada da actividade de cultivo de bivalves, o desenvolvimento de programas de monitorização participada dos campos de Z. noltii e das áreas de cultivo de bivalves.

Effect of polyunsaturated fatty acids (PUFAs) on the proliferation and extracellular matrix mineralization and gene expression of gilthead seabream skeletal cell lines

The aquaculture industry aims at replacing significant amounts of marine fish oil by vegetable oils in fish diet. Dietary lipids have been shown to alter the fatty acid composition of bone compartments, which would impact the local production of factors controlling bone formation. Knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is however scarce in fish. Two in vitro bone-derived cell systems developed from seabream (an important species for aquaculture in the Mediterranean region) vertebra, capable of in vitro mineralization and exhibiting prechondrocyte (VSa13) and pre-osteoblast (VSa16) phenotype, were used to assess the effect of certain polyunsaturated fatty acids (PUFAs; arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) on cell proliferation, extracellular matrix (ECM) mineralization and gene expression. While all PUFAs promoted morphological changes in both cell lines, VSa16 cell proliferation appeared to be stimulated by PUFAs in a dose dependent manner until 100M, whereas proliferation of VSa13 cells was impaired at concentrations above 10M. AA, EPA and DHA inhibited VSa13 ECM mineralization, alone and in combination, while VSa16 ECM mineralization was only inhibited by AA and EPA. DHA had the opposite effect, increasing mineralization almost by 2 fold. When EFAs were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. Expression of marker genes for bone and lipid metabolisms has been investigated by qPCR and shown to be regulated in pre-osteoblasts exposed to individual PUFAs. Our results show that PUFAs are effectors of fish bone cell lines, altering cell morphology, proliferation and mineralization when added to culture medium. This work also demonstrates the suitability of our in vitro cell systems to get insights into mineralization-related effects of PUFAs in vivo and to evaluate the replacement of fish oils by vegetable oil sources in fish feeds.

Impactes socioculturais do turismo residencial no município do Tarrafal de Santiago - Cabo Verde

Esta dissertação é um estudo de caso sobre os impactes socioculturais do turismo residencial no município de Tarrafal, ilha de Santiago em Cabo Verde. Partindo de um esquema teórico herdeiro da Sociologia e Antropologia do turismo, começaremos por analisar alguns temas centrais acerca do Turismo. Pretende-se dar uma visão de Cabo Verde enquanto destino turístico procurando dar conta da evolução do mercado turístico, seus impactes, considerando que o turismo revela-se como principal motor da economia deste arquipélago. Este estudo baseia-se no turismo residencial não institucionalizado na localidade de Tarrafal e torna-se fundamental para o entendimento das relações estabelecidas entre turistas, bem como entre estes com os residentes locais. São analisadas as perceções sobre o estado atual do turismo e, em particular o turismo residencial nesta localidade, contribuindo assim na busca de estratégias para o desenvolvimento sustentável e de melhor qualidade da atividade turística neste local. A aproximação ao “terreno” poderá trazer informações importantes para a definição das linhas de orientação para o planeamento e gestão desta atividade.

O papel do regulador transcricional CITED2 na leucemia linfoblástica aguda de linfócitos T

A leucemia linfoblástica aguda de Linfócitos T (LLA-T) é uma neoplasia agressiva de precursores de células T do timo, que afeta principalmente crianças e adolescentes. A identificação de fatores moleculares que controlam a iniciação e progressão da LLA-T é fundamental para desenvolvimento de terapêuticas mais específicas e eficientes. Devido a atividade de fatores de transcrição estar muitas vezes desregulada na LLA-T e o regulador transcricional CITED2 controla diversos processos no desenvolvimento e oncogénese, levando assim ao objetivo principal, verificar se o CITED2 está envolvido na iniciação e/ou progressão da LLA-T, para se cumprir este iremos verificar se o CITED2está expresso nas linhas celulares leucémicas, quais os seus efeitos e por último verificar se o seu promotor é influenciada por três vias de sinalização: JAK-STAT, NFkB e NOTCH1. Analisou-se a expressão de CITED2 em linhas celulares de leucemia linfoblástica das células T e de células T normais, realizando o PCR quantitativo. Verificou-se que todas as células expressavam o CITED2, algumas com elevada expressão, por exemplo a linha celular EL4.2 expressa sete vezes mais Cited2 que os timócitos normais. Selecionou-se duas linhas celulares, EL4.2 e as DND41, para efetuar a subexpressão de CITED2, utilizando um plasmídeo lentiviral que expressa um RNA de interferência contra o CITED2. Conseguimos obter transdução estável deste plasmídeo para a linha celular DND41, tendo então efetuado análises de proliferação celular em meio de cultura normal ou meio com redução de soro fetal bovino. Verificou-se que a linha celular DND41/shCITED2 apresentava um maior crescimento celular comparando com a linha DND41/pLKO.1, sendo significativo, a partir das 48 horas, mas análise das fases do ciclo celular não demonstrou qualquer diferença. Analisou-se a resistência destas linhas celulares à apoptose, tratando as células com dexametasona, um agente quimioterápico para leucemias LLA-T. Observou-se que existe uma ligeira tendência para a linha celular DND41/shCITED2 ser mais resistente à apoptose. Mas nas linhas celulares sem tratamento, verificou-se diferenças na fase G0/G1 e G2/M, sendo que a linha celular DND41 sh CITED2 apresenta mais células com ciclo celuar ativo. Analisou-se no presente trabalho, se o promotor do CITED2 seria influenciando pela ativação das vias de sinalização JAK-STAT, NF-kB e NOTCH1. Os nossos resultados indicam que a ativação destas vias regulam negativamente o promotor do CITED2. Tendo em conta o conjunto dos nossos resultados, podemos concluir que a inativação do regulador transcricional CITED2 pode contribuir para o desenvolvimento da LLA-T. No entanto, será necessário desenvolver mais estudos para compreender os mecanismos subjacentes.

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.

Percurso técnico e pedagógico em educação ambiental

O presente Relatório Técnico de Atividades Profissionais apresenta a minha experiência profissional na área da Educação Ambiental (EA), após terminar a Licenciatura em Engenharia do Ambiente em 2001. Tem por objetivo documentar diversas formas de abordagem e de planeamento de ações de educação ambiental, com vista a contribuir para o estabelecimento de linhas de ação futuras a partir da identificação de possíveis potencialidades e constrangimentos. Inclui também a evolução da educação ambiental a nível nacional e internacional, a fim de evidenciar a sua atual importância como linha de orientação estratégica e prioridade das Políticas de Ambiente. Encontram-se ainda apresentados no relatório os principais projetos em que estive envolvida, entre eles: o Projeto DIMEAGUA (relativo à aplicação da Diretiva Quadro da Água às massas de água de transição e costeiras do Guadiana, e dinamizado com alunos do 3º ciclo); o Projeto Coastwatch Europe (sob a temática da Gestão e Proteção do Litoral, em que participaram alunos do 1º, 2º e 3º ciclos); o Projeto Interpretação Ambiental da Zona Ribeirinha de Faro (que abordou temáticas relacionadas com a fauna e flora dos sapais, a poluição, o património natural e arqueológico e a degradação do ambiente natural e foi dinamizado com alunos do 2º ciclo); o Projeto H2Ooooh (Projeto UNESCO sobre o tema Água e os principais problemas relacionados com o seu uso, a sua exploração e a sua oferta limitada, para alunos do 1º ciclo); as celebrações do Ano Internacional da Biodiversidade (no âmbito do qual crie um jogo pedagógico intitulado “O meu Planeta e Eu”); e o Projeto Planeta Azul (com uma abordagem transversal aos conceitos da sustentabilidade, prevenção e segurança e em que participaram alunos do 1º e 2º ciclos). Globalmente pode-se concluir que em projetos de EA há necessidade de se efetuar um bom planeamento das ações, construir uma proposta de projeto consistente, otimizar e articular parcerias, aplicar um processo avaliativo e efetuar uma abordagem interdisciplinar e global das temáticas em estudo.

Synthesis and evaluation of polymer nanoparticles for delivery in RPE cells

Ocular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.

Adaptabilidade de carreira e auto-eficácia na transição ao trabalho: estudo longitudinal com estudantes finalistas do ensino profissional

O impacto do contexto de trabalho no desenvolvimento vocacional tem vindo a ser estudado, há vários anos, no domínio da Psicologia Vocacional. Autores como Mark Savickas, Super e Vondraceck, reconhecem-no como um fator importante para o desenvolvimento vocacional do indivíduo, sobretudo pelos desafios que o exercício de determinada atividade profissional pode vir a colocar. Neste contexto, a experiência de estágio pode ter um efeito significativo na adaptabilidade de carreira e em outros contrutos a esta associados, sobretudo por se tratar de um contruto que traduz a competência de cada um para lidar com os desafios, obstáculos e transições inerentes à aprendizagem em contexto real de trabalho. A presente investigação, através de um estudo longitudinal com 119 alunos do ensino profissional, tem como objectivo avaliar os níveis de adaptabilidade ao longo do ano lectivo, bem como o impacto deste construto na auto-eficácia e na empregabilidade percebida, no final do ano letivo. Tem ainda como objectivo avaliar o impacto da qualidade da experiência de estágio nas diferenças eventualmente observadas nos níveis de adaptabilidade dos estudantes. Os resultados obtidos vão ao encontro da literatura, destacando a estabilidade dos níveis de adaptabilidade ao longo do ano, reforçando ainda, o papel deste construto na qualidade de preditor do sentimento de competência na transição para o mundo do trabalho. As limitações do estudo e as futuras linhas de investigação serão também apresentadas.

Golf destinations' brand personality: the case of the Algarve

The purpose of this research is to develop and validate a measurement scale to assess golf destinations’ brand personality and therefore to perceive the destination personality of the Algarve as a golf destination. Based on literature review on human personality, brand personality, destination brand image and marketing scales validation procedures, an initial 36 unrepeated items were the base for a survey instrument. Those items were generated from the literature, from the results of individual interviews with experts in tourism and golf in the Algarve and from promotional texts in golf- related websites. After content validation, the items were allocated into categories of attributes by a panel of expert judges. A survey was then applied to a convenient sample of 600 golf players in the Algarve, and 545 (valid) questionnaires were analysed to refine the scale. Golf players assessed the components of the relational brand personality (functional, symbolic and experiential) as well as the Algarve as a golf destination. A taxonomy of brand personality was developed and tested in the Algarve as it is recognized as one of the world best golf destination. The developed taxonomy of brand personality was assessed in two ways: 1) through the overall perception of the Algarve as a golf destination and 2) through the perception of specific attributes of the destination grouped into three main categories (functional, symbolic and experiential). Therefore, two multi-dimensional brand personality models were estimated by using structural equation modelling. Findings of this study indicate that golf players ascribe personality characteristics to destinations. The brand personality of the Algarve is translated into three main dimensions enjoyableness, distinctiveness and friendliness when tourists/golf players reveal their overall perception of the destination. The brand personality of golf destination Algarve is reflected in the dimensions reliability, hospitality, uniqueness and attractiveness when tourists assess the components of the relational brand personality. Refined scales consisting of 10 and 12 items were finally derived meeting both reliability and validity requirements. This study does not replicate Aaker’s (1997) personality dimensions and very little parallelism can be drawn with Aaker’s (1997) brand personality scale since only three items from her scale were validated in both models: friendly and cheerful, (sincerity), reliable (competence). The same is verified concerning the ‘Big-five’. The human personality traits (HPT) validated to describe golf destinations personality are only four helpful, pleasant (agreeableness), relaxed (emotional stability), and innovative (intellect or openness). As far as destination image descriptors (DID) are concerned, the items appealing, relaxed and safe were validated, while traits suggested by the interviews and website promotional texts such as calm, natural, spectacular, unique, welcoming, and the best (destination-specific traits) appear to be appropriate to describe the personality of a golf destination. The results suggest that the overall perception of the Algarve´s brand personality is described by the dimensions enjoyableness, distinctiveness and friendliness. Moreover, the relational perspective revealed that the functional attributes of the destination are described by the dimension reliablility, while the symbolic attributes are described by the dimensions hospitablility and uniqueness and finally its experiential attributes are described by the dimension attractiveness. These results show that a golf destination´s brand personality should not just be based on good golf practices. Theoretical and practical implications are discussed in the context of destination brand personality.

The activity of mouse Cerberus like 2 during cardiogenesis - genetic and morphogenetic studies

Cardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.

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

Papel das proteínas de choque térmico na apoptose mediada por espécies reativas de oxigénio em culturas celulares

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

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.