Faculdade de Medicina e Ciências Biomédicas
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Browsing Faculdade de Medicina e Ciências Biomédicas by Field of Science and Technology (FOS) "Ciências Médicas::Medicina Clínica"
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- Expression of matrix gla protein (MGP) in breast cancer: a molecular and cellular approachPublication . Teixeira, Karine Rosário da Silva; Conceição, Natércia; Braga, SofiaEvery year around 8.2 million people die due to cancer, in Portugal, breast cancer is the leading cause of women death (in cancer patients). Several risk factors could be pointed being age (over 50 years old), gender (females), reproductive and hormonal factors and genetic predisposition (presence of mutations in BRCA 1 and BRCA 2) the most commons. Staging is done regarding several aspects, e.g.: histology, histopathologic localization, hormone receptors, mutations or expression of cluster genes. Research from last decades is focused into targeted therapies. MGP, matrix Gla protein, was pointed as a possible target. MGP primary structure consists in a signal peptide, a phosphorylation domain and a γ-carboxylase recognition site. MGP has also five Gla residues. For a long time, only an isoform with four exons was known, but recently was discovered a second isoform with five exons, with three additional putative binding sites, conferring, possible, a higher binding capability as well as a presumably higher calcification inhibitory power. Although MGP role in cancer is not fully understood, being upregulated in some types of tumors and downregulated others, in breast cancer, was established, as a prognostic factor, being overexpressed in poor diagnose cases. Being MGP role in tumorigenesis to be defined, the aims of this work were: clarify MGP expression in normal vs tumoral tissue (using in silica method – TCGA database); as well as clarify regulatory mechanisms of MGP (TCGA database); analyze expression of each isoform in breast cancer cell lines (results were obtained performing qRT-PCR followed by electrophoresis and sequencing of the extracted bands) and assess the result of overexpression of each isoform (using XTT and wound healing assay). We could conclude that both variants could be expressed in simultaneous. Every cell line presented E4 isoform but just a few presented E5 isoform. Hound healing assay revealed that an overexpression of E4 and E5 variants slow down the rate of the area invaded by migrating cells, specially an overexpression of E4 isoform. After in silica analyze we could conclude that MGP is more expressed in tumoral tissue vs normal tissue, existing a negative correlation between MGP expression and mir155 expression and methylation, that means that: in tumoral tissue mir155 is less expressed than in normal tissue and occurs an hypomethylation in tumoral tissue. Knowing that MGP inhibits calcification and is correlated with angiogenesis, our hypothesis is that overexpression of MGP in tumoral tissue is a mechanism of cancer to facilitate angiogenesis – a hallmark of cancer.
- Mechanisms of de novo bone formation in a fish model of fin regenarationPublication . Silva, João Pedro Gonçalves Cardeira da; Laizé, Vicent Stéphane; Gavaia, Paulo JorgeUnveiling the basis of animal tissue regeneration may contribute to the better understanding of disease mechanisms and the development of novel therapeutic strategies. Regeneration-competent animals are also valuable tools in drug discovery and for studying basic mechanisms of development. Bone is a dynamic tissue, formed and maintained through a balance between matrix deposition by osteoblasts and resorption by osteoclasts. When bone remodelling fails to homeostatic equilibrium, degenerative and dysplastic diseases may develop. Moreover, human bone is unable to completely restore structure and function after trauma. Hence, this work aimed at uncovering novel players in bone formation and patterning in an innovative system, the zebrafish regenerating caudal fin. A comprehensive set of imaging and analytical methodologies were first development and optimized to track bone formation and regeneration in a precise and timely manner throughout fin epimorphic regeneration. To gain insights into cellular and molecular programs underlying bone regeneration, the transcriptome of regenerates at distinct stages (blastema formation and regenerative outgrowth) was analysed. RNA-Seq data pointed toward a role of osteoclasts and calcitonin in ray patterning. Calcitonin-specific inhibition of osteoclast activity impaired ray bifurcation and demonstrated the central role that osteoclasts play in this process. Evidence toward calcitonin-specific regulation of osteoclast differentiation in the context of ray bifurcation, was also collected. To further study the mechanisms underlying bone formation, a swim-training regeneration model was established. In this model, bone formation during regeneration was stimulated and ray bifurcation was accelerated upon increased mechanical loading, further demonstrating the suitability of this system to investigate underlying mechanisms. This work provided valuable technical advancements for the study of bone regeneration in zebrafish and identified novel players in ray formation and patterning (i.e. bifurcation). Altogether, this work also proposes the zebrafish bifurcating fin ray as a potential model for tetrapod digit specification and associated diseases.