Instituto Gulbenkian Ciencia

Oeiras, Portugal

Instituto Gulbenkian Ciencia

Oeiras, Portugal
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Soares P.I.P.,New University of Lisbon | Dias S.J.R.,Instituto Portugues Of Oncologia Of Lisbon | Dias S.J.R.,Instituto Gulbenkian Ciencia | Novo C.M.M.,CEDOC | And 4 more authors.
Mini-Reviews in Medicinal Chemistry | Year: 2012

Osteosarcoma is the most common primary bone tumor in children and adolescents, with a 5-year disease free survival rate of 70%. Current chemotherapy regimens comprise a group of chemotherapeutic agents in which doxorubicin is included. However, tumor resistance to anthracyclines and cardiotoxicity are limiting factors for its usage. Liposomal formulations of doxorubicin improve its anti-cancer effects but are still insufficient. The research in this area has lead to the production of anthracyclines analogues, such as ladirubicin, the leading compound of alkylcyclines. This new anticancer agent has shown promising results in vivo and in vitro, being effective against osteosarcoma cell lines, including those with a multidrug resistant phenotype. In phase I clinical trials, this molecule caused mild side effects and did not induce significant cardiotoxicity at doses ranging from 1 to 16 mg/m2, resulting in a peak plasma concentration (Cmax) ranging from 0.5 to 1.5 μM. The recommended doses for phase II studies were 12 and 14 mg/m2 in heavily and minimally pretreated/non-pretreated patients, respectively. Phase II clinical trials in ovary, breast, colorectal cancer, NSCLC and malignant melanoma are underway. Given the improved molecular targeting efficacy of these new compounds, ongoing approaches have sought to improve drug delivery systems, to improve treatment efficacy while reducing systemic toxicity. The combination of these two approaches may be a good start for the discovery of new treatment for osteosarcoma. © 2012 Bentham Science Publishers.

Braga S.,Instituto Portugues Of Oncologia | Braga S.,Instituto Gulbenkian Ciencia | Cardoso J.,Instituto Gulbenkian Ciencia | Cardoso J.,Precision for Medicine | And 13 more authors.
Current Cancer Drug Targets | Year: 2017

Background: The antiangiogenic drug sunitinib has never been evaluated as single agent in untreated breast cancer patients. Objective: We aimed to characterize the activity of sunitinib, alone and with docetaxel, in untreated locally advanced or operable breast cancer and to uncover the mechanisms of response. Method: Patients were treated with an upfront window of sunitinib followed by four cycles of sunitinib plus docetaxel. Response, resistance and toxicity were evaluated according to standard clinical parameters, magnetic resonance imaging, positron emission tomography, standard pathology characterization, molecular pathology and gene expression profiling. Results: Twelve patients were included. We detected primary resistance to sunitinib in the upfront window in untreated breast cancer, as evidenced by four non-responding patients. At surgery, five patients had viable tumor in the breast and axilla, four had viable tumor cells in the breast alone and three were taken off study and thus not evaluated, due to unacceptable toxicity. Early functional imaging was useful in predicting response. There were no clinical complete responses. Comparison of tumor gene expression profiling data between early responders and non-responders allowed us to identify the up-regulation of VEGF and angiogenic pathways in non-responders. Specifically, in tumors resistant to single-agent sunitinib we detected a transcriptional response to hypoxia characterized by over-expression of several HIF1a target genes. Conclusion: In this report of single-agent sunitinib treatment in untreated localized breast cancer patients, we found evidence of primary resistance to sunitinib, likely mediated by up-regulation of hypoxia responsive genes. © 2017 Bentham Science Publishers.

Mateus R.,Institute Medicina Molecular | Mateus R.,Instituto Gulbenkian Ciencia | Pereira T.,Institute Medicina Molecular | Pereira T.,Instituto Gulbenkian Ciencia | And 8 more authors.
PLoS ONE | Year: 2012

Background: Zebrafish (Danio rerio) has a remarkable capacity to regenerate many organs and tissues. During larval stages the fin fold allows the possibility of performing long time-lapse imaging making this system very appealing to study the relationships between tissue movements, cell migration and proliferation necessary for the regeneration process. Results: Through the combined use of transgenic fluorescently-labeled animals and confocal microscopy imaging, we characterized in vivo the complete fin fold regeneration process. We show, for the first time, that there is an increase in the global rate of epidermal growth as a response to tissue loss. Also enhanced significantly is cell proliferation, which upon amputation happens in a broad area concerning the amputation level and not in a blastema-restricted way. This reveals a striking difference with regard to the adult fin regeneration system. Finally, an accumulation of migratory, shape-changing fibroblasts occurs proximally to the wound area, resembling a blastemal-like structure, which may act as a signaling center for the regeneration process to proceed. Conclusions: These findings provide a novel in vivo description of fundamental mechanisms occurring during the fin fold regeneration process, thereby contributing to a better knowledge of this regenerative system and to reveal variations in the epimorphic regeneration field. © 2012 Mateus et al.

Pires A.E.,Instituto Gulbenkian Ciencia | Pires A.E.,Instituto Portugues Of Oncologia Of Lisbon | Afonso A.F.,Instituto Gulbenkian Ciencia | Afonso A.F.,Instituto Portugues Of Oncologia Of Lisbon | And 9 more authors.
Journal of Immunotherapy | Year: 2010

Natural antibodies are unique self molecules endowed with both suppressive and activating functions on various cells of the immune system and are recognized as a fundamental link between the adaptive and innate immune system. Here, we examine the role of natural antibodies, using polyclonal immunoglobulins (Ig), as a promoter of T-cell reconstitution in a context of lymphopenia. We have established a mouse model to mimic immunologic recovery in adult patients with severe hypothymic function subjected to autologous hematopoietic precursor cell transplantation. Thymectomized mice were transplanted and treated with low doses of Ig or its Fab or Fc fragments. The animals displayed, during early phases of Ig treatment, a significant increase of T-cell reconstitution displaying a naive CD4 +phenotype. In addition, the Igtreated animals exhibited an increase dilution of single-joint T-cell receptor excision circles (sjTRECs) in peripheral blood, suggesting an early increase in proliferation of T cells stimulated by the natural antibodies. These results unveil a novel and considerable effect of intravenous Ig treatment in situations of severe lymphopenia as a stimulator of proliferation of peripheral naive T cells, possibly protecting diverse immune repertoires. Copyright © 2010 by Lippincott Williams & Wilkins.

Mateus R.,New University of Lisbon | Lourenco R.,New University of Lisbon | Fang Y.,National Health Research Institute | Brito G.,University of Lisbon | And 5 more authors.
Development (Cambridge) | Year: 2015

Caudal fin regeneration is characterized by a proliferation boost in the mesenchymal blastema that is controlled precisely in time and space. This allows a gradual and robust restoration of original fin size. However, how this is established and regulated is not well understood. Here, we report that Yap, the Hippo pathway effector, is a chief player in this process: functionally manipulating Yap during regeneration dramatically affects cell proliferation and expression of key signaling pathways, impacting regenerative growth. The intracellular location of Yap is tightly associated with different cell densities along the blastema proximal-distal axis, which correlate with alterations in cell morphology, cytoskeleton and cell-cell contacts in a gradient-like manner. Importantly, Yap inactivation occurs in high cell density areas, conditional to F-actin distribution and polymerization. We propose that Yap is essential for fin regeneration and that its function is dependent on mechanical tension, conferred by a balancing act of cell density and cytoskeleton activity. © 2015. Published by The Company of Biologists Ltd.

PubMed | Francis Crick Institute, Eli Lilly and Company, University of Washington, New York University and 9 more.
Type: Journal Article | Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology | Year: 2016

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. 2016 International Society for Advancement of Cytometry.

Campos C.,University of Geneva | Campos C.,Instituto Gulbenkian Ciencia | Kamiya M.,Ecole Polytechnique Federale de Lausanne | Banala S.,Ecole Polytechnique Federale de Lausanne | And 2 more authors.
Developmental Dynamics | Year: 2011

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP-tag technology and O6-benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long-term imaging and allows, unlike most of the autofluorescent proteins, the use of acid fixatives such as trichloroacetic acid. Furthermore, the use of O6-benzylguanine derivatives bearing caged fluorescein allows cell lineage tracing through photo-deprotection of the fluorophore and its detection either through fluorescence microscopy or through immunohistochemistry after fixation using anti-fluorescein antibodies. © 2011 Wiley-Liss, Inc.

Holt R.,University of Oxford | Sykes N.H.,University of Oxford | Conceicao I.C.,Instituto Nacional Of Saude Dr Ricardo Jorge | Conceicao I.C.,Instituto Gulbenkian Ciencia | And 12 more authors.
European Journal of Human Genetics | Year: 2012

There is strong evidence that rare copy number variants (CNVs) have a role in susceptibility to autism spectrum disorders (ASDs). Much research has focused on how CNVs mediate a phenotypic effect by altering gene expression levels. We investigated an alternative mechanism whereby CNVs combine the 5′ and 3′ ends of two genes, creating a fusion gene. Any resulting mRNA with an open reading frame could potentially alter the phenotype via a gain-of-function mechanism. We examined 2382 and 3096 rare CNVs from 996 individuals with ASD and 1287 controls, respectively, for potential to generate fusion transcripts. There was no increased burden in individuals with ASD; 122/996 cases harbored at least one rare CNV of this type, compared with 179/1287 controls (P<0.89). There was also no difference in the overall frequency distribution between cases and controls. We examined specific examples of such CNVs nominated by case-control analysis and a candidate approach. Accordingly, a duplication involving REEP1-POLR1A (found in 3/996 cases and 0/1287 controls) and a single occurrence CNV involving KIAA0319-TDP2 were tested. However, no fusion transcripts were detected by RT-PCR. Analysis of additional samples based on cell line availability resulted in validation of a MAPKAPK5-ACAD10 fusion transcript in two probands. However, this variant was present in controls at a similar rate and is unlikely to influence ASD susceptibility. In summary, although we find no evidence that fusion-gene generating CNVs lead to ASD susceptibility, discovery of a MAPKAPK5-ACAD10 transcript with an estimated frequency of 1/200 suggests that gain-of-function mechanisms should be considered in future CNVs studies. © 2012 Macmillan Publishers Limited. All rights reserved.

Caiado F.,Instituto Portugues Of Oncologia Francisco Gentil And Epe | Caiado F.,Instituto Gulbenkian Ciencia | Carvalho T.,Instituto Portugues Of Oncologia Francisco Gentil And Epe | Carvalho T.,Instituto Gulbenkian Ciencia | And 8 more authors.
Biomaterials | Year: 2011

Severe skin loss constitutes a major unsolved clinical problem worldwide. For this reason, in the last decades there has been a major push towards the development of novel therapeutic approaches to enhance skin wound healing. Neo-vessel formation through angiogenesis is a critical step during the wound healing process. Besides the contribution of pre-existing endothelial cells (EC), endothelial progenitor cells (EPCs) have also been implicated in wound healing acting either by differentiating into EC that incorporate the neo-vessels, or via the production of paracrine factors that improve angiogenesis. Here we tested the importance of different extracellular matrices (ECM) in regulating the angiogenic and wound healing potential of cord blood-derived EPC (CB-EPC). We compared the properties of several ECM and particularly of fibrin fragment E (FbnE) in regulating EPC adhesion, proliferation, differentiation and healing-promotion in vitro and in vivo. Our results show that CB-EPCs have increased adhesion and endothelial differentiation when plated on FbnE compared to collagens, fibronectin or fibrin. Using integrin neutralizing antibodies, we show that CB-EPC adhesion to FbnE is mediated by integrin α5β1. Gene expression analysis of CB-EPCs plated on different substrates revealed that CB-EPC grown on FbnE shows increased expression of paracrine factors such as VEGF-A, TGF-β1, SDF-1, IL-8 and MIP-1α. Accordingly, conditioned media from CB-EPC grown on FbnE induced EC tube formation and monocyte migration in vitro. To test the wound healing effects of FbnE in vivo we used an FbnE enriched scaffold in a cutaneous wound healing mouse model. In accordance with our in vitro data, co-administration of the FbnE enriched scaffold with CB-EPC significantly accelerated wound closure and wound vascularization, compared FbnE enriched scaffold alone or to using collagen-based scaffolds. Our results show that FbnE modulates several CB-EPC properties in vivo and in vitro, and as such promotes wound healing. We suggest the use of FbnE-based scaffolds represents a promising approach to resolve wound healing complications arising from different pathologies. © 2011 Elsevier Ltd.

Caiado F.,Instituto Portugues Of Oncologia Francisco Gentil | Caiado F.,Instituto Gulbenkian Ciencia | Dias S.,Instituto Portugues Of Oncologia Francisco Gentil | Dias S.,Instituto Gulbenkian Ciencia
Fibrogenesis and Tissue Repair | Year: 2012

In the last decade there have been multiple studies concerning the contribution of endothelial progenitor cells (EPCs) to new vessel formation in different physiological and pathological settings. The process by which EPCs contribute to new vessel formation in adults is termed postnatal vasculogenesis and occurs via four inter-related steps. They must respond to chemoattractant signals and mobilize from the bone marrow to the peripheral blood; home in on sites of new vessel formation; invade and migrate at the same sites; and differentiate into mature endothelial cells (ECs) and/or regulate pre-existing ECs via paracrine or juxtacrine signals. During these four steps, EPCs interact with different physiological compartments, namely bone marrow, peripheral blood, blood vessels and homing tissues. The success of each step depends on the ability of EPCs to interact, adapt and respond to multiple molecular cues. The present review summarizes the interactions between integrins expressed by EPCs and their ligands: extracellular matrix components and cell surface proteins present at sites of postnatal vasculogenesis. The data summarized here indicate that integrins represent a major molecular determinant of EPC function, with different integrin subunits regulating different steps of EPC biology. Specifically, integrin α4β1 is a key regulator of EPC retention and/or mobilization from the bone marrow, while integrins α5β1, α6β1, αvβ3 and αvβ5 are major determinants of EPC homing, invasion, differentiation and paracrine factor production. β2 integrins are the major regulators of EPC transendothelial migration. The relevance of integrins in EPC biology is also demonstrated by many studies that use extracellular matrix-based scaffolds as a clinical tool to improve the vasculogenic functions of EPCs. We propose that targeted and tissue-specific manipulation of EPC integrin-mediated interactions may be crucial to further improve the usage of this cell population as a relevant clinical agent. © 2012 Caiado and Dias; licensee BioMed Central Ltd.

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