Utensilagem óssea do paleolítico superior português

Dissertação mest., Arqueologia - Teoria e Métodos, Universidade do Algarve, 2007

A indústria óssea do Paleolítico Superior do sul da Península Ibérica: a gestão do risco e da incerteza

Tese de Doutoramento, Arqueologia, Faculdade de Ciências Humanas e Sociais, Universidade do Algarve, 2015

Caracterização química e bioquímica dos frutos de ARBUTUS UNEDO L. e de RUBUS FRUTICOSUS AGG.: contribuição para a sua valorização

Dissertação mest., Agricultura sustentável, Universidade do Algarve, 2007

Endocrine regulation of extracellular matrix proteins in calcified tissue in teleost fish

Tese dout., Biologia, Universidade do Algarve, 2005

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

The role of lymphotoxin-B receptor signaling in t-cell acute lymphoblastic leukemia development

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

O fruto de Monstera deliciosa: caracterização físico-química e potencial para produção de aguardente

Dissertação de mest., Tecnologia de Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2012

Estudo do comportamento biológico de biomaterial xenogénico em defeito ósseo crítico

Dentre as várias opções de biomateriais para a regeneração óssea, os de origem xenogénica são considerados como uma das alternativas ao autoenxerto, devido às suas propriedades biológicas e físico-químicas, além da grande disponibilidade, e de seu uso contribuir com a diminuição da morbilidade ao indivíduo. Vários autores consideram o BioOss®, uma hidroxiapatite bovina, como padrão de excelencia entre os biomateriais de substituição óssea independentemente de sua forma ser macro ou microgranular, apesar do tamanho da partícula ser também um fator importante nas reações tissulares envolvidas na neoformação óssea. O presente estudo teve como objetivo verificar o comportamento biológico de BioOss® na forma microgranular, com partículas entre 0,25 a 1,00 mm e 0,4 a 0,6 mm, implantadas em defeito ósseo crítico nos grupos GB e GBS, respectivamente, e analisados comparativamente a um controlo negativo, GC, com defeito preenchido apenas por coágulo sanguíneo, avaliados nos pontos biológicos de 15 e 45 dias. Como resultados, no grupo GC, ocorreu neoformação óssea reacional restrita às bordas e presença de tecido fibroso na área do defeito. Nos grupos GB e GBS, observou-se mínima reação inflamatória, abundante angiogênese e alguma osteogênese, além das bordas, ao longo do defeito, em continuidade à dura-máter. Como conclusão, o BioOss® foi biocompatível, promoveu osteogênese por osteocondução e integrou-se parcialmente ao osso neoformado, com melhores resultados para o grupo GBS.

Identification of genes regulated by mir-363*, a regulator of bone marrow endothelial cell properties

As células estaminais hematopoiéticas residem na medula óssea e possuem capacidade para se auto-renovar e dar origem a todos os tipos de células sanguíneas. O endotélio da medula óssea é constituído por células endoteliais de medula óssea (BMEC) e compreende dois nichos com funções distintas: o nicho osteoblástico e o nicho vascular. O nicho osteoblásctico proporciona condições para a quiescência de células estaminais hematopoiéticas, enquanto no nicho vascular ocorre proliferação e diferenciação das mesmas. Quando ocorre um desequilíbrio na expressão de genes que codificam para proteínas envolvidas na mobilização de células do nicho osteoblástico para o nicho vascular – factores angiócrinos – ocorre uma desestabilização do microambiente medular, que se pode traduzir num processo tumoral. Os microRNAs (miRNAs) são uma classe de RNAs não codificantes, de cadeia simples, que regula a expressão génica. Os miRNAs são sequências endógenas de RNA que possuem entre 19 e 25 nucleótidos de tamanho. Os miRNAs são reguladores da expressão genica, induzindo o silenciamento a nível da pós-transcrição, através da sua ligação com uma sequência específica para a qual possuem afinidade, na região 3’ não traduzida (3’ UTR) dos seus mRNA alvo, conduzindo à inibição da tradução ou à sua degradação. Os miRNAs estão envolvidos na regulação de genes de diversas vias afectando processos fundamentais como hematopoiese, apoptose, proliferação celular e tumorigénese. Os níveis de expressão dos miRNAs estão alterados no cancro, podendo actuar directamente como supressores de tumor ou como oncogenes, sendo neste caso denominados de oncomirs. Os perfis dos níveis de expressão de vários miRNAs foram estudados, tendo-se verificado que se alteram durante o processo de carcinogénese, podendo actuar directamente como supressores de tumor ou como oncogenes, sendo neste caso denominados de oncomirs. Apesar do miR-363* estar envolvido na regulação da expressão de genes que regulam propriedades das células endoteliais e medula óssea, os genes sobre os quais exerce a sua função ainda não foram identificados.O objectivo do presente estudo é a identificação dos genes directamente regulados pelo miR-363* (genes alvo) e a sua relevância para a disfunção medular e a sua caracterização nos síndromes mielodisplásicos. A estratégia usada baseou-se na redução ou aumento forçados dos níveis de miR-363* em células endoteliais e subsequente análise da expressão génica através de microarrays de cDNA do genoma humano. A redução do miR-363* vai implicar o aumento da expressão dos seus genes alvo, assim como o aumento dos níveis do miR-363* vai induzir a degradação e consequente redução dos seus genes alvos. A intersecção dos dados gerados através do estudo da expressão com bases de dados que possuem algoritmos para previsão de genes alvo directos dos miRNAs (miRBase e MicroCosm Targets) permitiu restringir os genes a analisar a sete genes, nomeadamente BST1, ESAM, FCER1G, IKBKG, SELE, THBS3 e TIMP1. A interacção directa destes candidatos a alvos directos do miR-363* foi posteriormente validada. Para tal, as 3’UTR dos genes foram clonadas num vector que contém o gene da luciferase. Uma vez as clonagens realizadas, efectuaram-se ensaios funcionais em células endoteliais, nomeadamente HUVEC, nas quais se co-transfectaram os vectores gerados, anti-miRs ou pre-miRs (para diminuir ou aumentar o nível de miRNA) e o plasmídeo controlo da Renilla para normalização dos ensaios de luciferase. A variação da luminescência obtida em presença do aumento ou redução do miR-363* deu uma forte indicação da regulação directa do miR-363* nesses alvos. No entanto, a confirmação desta interacção directa foi efectuada através de ensaios de mutagénese, nos quais de induziram mutações na 3’UTR nos locais de ligação do miRNA, seguidos dos ensaios funcionais como acima descritos. Esta estratégia sugere que o TIMP1, inibidor da metaloprotease-9 (MMP-9), é regulado directamente pelo miR-363*. Adicionalmente, os níveis de expressão dos alvos directos do miR-363* foram estudados em 17 amostras de aspirados de medula óssea de doentes com síndromes mielodisplásicos. Os síndromes mielodisplásicos são caracterizados como um grupo heterogéneo de condições, que apresentam citopenias (produção deficiente de eritrócitos, leucócitos e/ou megacariócitos) e medula óssea displástica e hipercelular. A escalonagem dos doentes foi feita de acordo com o sistema de prognóstico IPSS elaborado pela Organização Mundial de Saúde, e que consiste numa tabela de risco de progressão de síndromes mielodisplásicos para leucemia mielóide aguda (LMA) e que agrupa os doentes em baixo risco – que compreende os níveis baixo e intermédio 1 – e em alto risco – que compreende os níveis intermédio 2 e alto. Dos genes regulados pelo miR-363*, o destacam-se o TIMP1, estando aumentando em doentes com mau prognóstico, e o THBS3 que apresenta um aumento nos doentes com prognóstico intermédio. Em suma, os estudos realizados permitiram a identificação de genes regulados pelo miR-363* e contribuiram para o conhecimento de como o miR-363* contribui para a disfunção medular, particularmente em síndromes mielodisplásicos, pela desregulação das propriedades endoteliais.

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.

Insights into molecular and cellular events involved in normal and pathological vertebral development and fusion using zebrafish as model organism

The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.

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.

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.

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.

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