Institute Investigacao e Inovacao em Saude I3S

Porto, Portugal

Institute Investigacao e Inovacao em Saude I3S

Porto, Portugal

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Penna G.C.,Federal University of Rio de Janeiro | Penna G.C.,Hospital Materials Dei | Vaisman F.,Federal University of Rio de Janeiro | Vaisman F.,Instituto Nacional Do Cancer INCA | And 8 more authors.
Cytogenetic and Genome Research | Year: 2017

Thyroid cancer derived from follicular cells (TCDFC) comprises well-differentiated (papillary and follicular) carcinoma, poorly differentiated carcinoma, and anaplastic carcinoma. Papillary thyroid carcinoma is the most common endocrine cancer, and its incidence is steadily increasing. Lethality and aggressiveness of TCDFC is inversely correlated with differentiation degree. In this review, an emphasis has been put on molecular markers involved in tumorigenesis of thyroid carcinoma with a focus on aggressive histotypes and the role of such biomarkers in predicting thyroid cancer outcome. Genetic rearrangements in TCDFC (RET/PTC, PAX8/PPARG) and mutations in RAS, BRAF, TERT promoter (TERTp), TP53, PIK3CA, and AKT1 are discussed. The majority of the studies to date indicate that TERTp mutations can serve as a marker of more aggressive disease in all the subtypes of thyroid carcinoma, being the best current marker of poor prognosis, due to its independent association with distant metastases and increased disease-specific mortality. Some studies suggested that a more accurate prediction of thyroid cancer outcome may be possible through a more extensive genetic analysis. The same is true concerning the identification of other mutations that are only relatively frequent in advanced tumors (e.g., TP53, PIK3CA, or AKT1). A better understanding of the prognostic role of TERTp mutation (together with additional ones like BRAF, RAS, PIK3CA, AKT1, or TP53) and the clarification of their putative role in fine-needle aspiration biopsies are likely to allow, in the future, an early refinement of the stratification risk in patients with well-differentiated carcinomas. It is worth noting that, as with any categorical staging system, the risk evaluation within each category (low, intermediate, and high) varies depending on the specific clinicopathologic features of individual patients and the specific biological behavior of the tumor. Finally, besides the diagnostic and/or prognostic significance of the above-mentioned mutations, it is crucial to understand that the molecular pathways and epigenetic alterations will likely turn into interesting targets for new therapies. © 2016 S. Karger AG, Basel.

Martins-Marques T.,University of Coimbra | Pinho M.J.,University of Coimbra | Zuzarte M.,University of Coimbra | Oliveira C.,Institute Investigacao e Inovacao em Saude i3S | And 6 more authors.
Journal of Extracellular Vesicles | Year: 2016

Extracellular vesicles (EVs) are major conveyors of biological information, mediating local and systemic cellto- cell communication under physiological and pathological conditions. These endogenous vesicles have been recognized as prominent drug delivery vehicles of several therapeutic cargoes, including doxorubicin (dox), presenting major advantages over the classical approaches. Although dox is one of the most effective antitumour agents in the clinical practice, its use is very often hindered by its consequent dramatic cardiotoxicity. Despite significant advances witnessed in the past few years, more comprehensive studies, supporting the therapeutic efficacy of EVs, with decreased side effects, are still scarce. The main objective of this study was to evaluate the role of the gap junction protein connexin43 (Cx43) in mediating the release of EV content into tumour cells. Moreover, we investigated whether Cx43 improves the efficiency of dox-based anti-tumour treatment, with a concomitant decrease of cardiotoxicity. In the present report, we demonstrate that the presence of Cx43 in EVs increases the release of luciferin from EVs into tumour cells in vitro and in vivo. In addition, using cell-based approaches and a subcutaneous mouse tumour model, we show that the anti-tumour effect of dox incorporated into EVs is similar to the administration of the free drug, regardless the presence of Cx43. Strikingly, we demonstrate that the presence of Cx43 in dox-loaded EVs reduces the cardiotoxicity of the drug. Altogether, these results bring new insights into the concrete potential of EVs as therapeutic vehicles and open new avenues toward the development of strategies that help to reduce unwanted side effects. © 2016 Tania Martins-Marques et al.

Gaspar P.,Institute Investigacao e Inovacao em Saude I3S | Gaspar P.,University of Porto | Gaspar P.,Abel Salazar Biomedical Sciences Institute | Mirzaian M.,Leiden University | And 8 more authors.
Molecular Genetics and Metabolism | Year: 2016

In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC-MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1-nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plfasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed. © 2016 Elsevier Inc.

Rei M.,University of Porto | Rei M.,S Joao Hospital | Rei M.,Institute of Biomedical Engineering INEB | Rei M.,Institute Investigacao e Inovacao em Saude i3s | And 22 more authors.
European Journal of Obstetrics Gynecology and Reproductive Biology | Year: 2016

Background Visual analysis of cardiotocographic (CTG) tracings has been shown to be prone to poor intra- and interobserver agreement when several interpretation guidelines are used, and this may have an important impact on the technology's performance. Objectives The aim of this study was to evaluate agreement in CTG interpretation using the new 2015 FIGO guidelines on intrapartum fetal monitoring. Study design A pre-existing database of intrapartum CTG tracings was used to sequentially select 151 cases acquired with a fetal electrode, with duration exceeding 60 minutes, and signal loss less than 15%. These tracings were presented to six clinicians, three with more than 5 years’ experience in the labor ward, and three with 5 or less years’ experience. Observers were asked to evaluate tracings independently, to assess basic CTG features: baseline, variability, accelerations, decelerations, sinusoidal pattern, tachysystole, and to classify each tracing as normal, suspicious or pathologic, according to the 2015 FIGO guidelines on intrapartum fetal monitoring. Agreement between observers was evaluated using the proportions of agreement (Pa), with 95% confidence intervals (95%CI). Results A good interobserver agreement was found in the evaluation of most CTG features, but not bradycardia, reduced variability, saltatory pattern, absence of accelerations and absence of decelerations. For baseline classification Pa was 0.85 [0.82–0.90], for variability 0.82 [0.78–0.85], for accelerations 0.72 [0.68–0.75], for tachysystole 0.77 [0.74–0.81], for decelerations 0.92 [0.90–0.95], for variable decelerations 0.62 [0.58–0.65], for late decelerations 0.63 [0.59–0.66], for repetitive decelerations 0.73 [0.69–0.78], and for prolonged decelerations 0.81 [0.77–0.85]. For overall CTG classification, Pa were 0.60 [0.56–0.64], for classification as normal 0.67 [0.61–0.72], for suspicious 0.54 [0.48–0.60] and for pathologic 0.59 [0.51–0.66]. No differences in agreement according to the level of expertise were observed, except in the identification of accelerations, where it was better in the more experienced group. Conclusions A good interobserver agreement was found in evaluation of most CTG features and in overall tracing classification. Results were better than those reported in previous studies evaluating agreement in overall tracing classification. Observer experience did not appear to play a role in agreement. © 2016 Elsevier Ireland Ltd

PubMed | Institute Investigacao e Inovacao em Saude i3S and University of Lisbon
Type: | Journal: Scientific reports | Year: 2016

In cancer, defective E-cadherin leads to cell detachment, migration and metastization. Further, alterations mediated by E-cadherin dysfunction affect cell topology and tissue organization. Herein, we propose a novel quantitative approach, based on microscopy images, to analyse abnormal cellular distribution patterns. We generated undirected graphs composed by sets of triangles which accurately reproduce cell positioning and structural organization within each image. Network analysis was developed by exploring triangle geometric features, namely area, edges length and formed angles, as well as their variance, when compared with the respective equilateral triangles. We generated synthetic networks, mimicking the diversity of cell-cell interaction patterns, and evaluated the applicability of the selected metrics to study topological features. Cells expressing wild-type E-cadherin and cancer-related mutants were used to validate our strategy. Specifically, A634V, R749W and P799R cancer-causing mutants present more disorganized spatial distribution when compared with wild-type cells. Moreover, P799R exhibited higher length and angle distortions and abnormal cytoskeletal organization, suggesting the formation of very dynamic and plastic cellular interactions. Hence, topological analysis of cell network diagrams is an effective tool to quantify changes in cell-cell interactions and, importantly, it can be applied to a myriad of processes, namely tissue morphogenesis and cancer.

Sousa-Pinto B.,University of Porto | Goncalves L.,University of Porto | Rodrigues A.R.,University of Porto | Rodrigues A.R.,Institute Investigacao e Inovacao em Saude I3S | And 10 more authors.
Journal of Nutritional Biochemistry | Year: 2016

Transforming growth factor beta (TGF-β) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-β and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-β, TGF-β receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-β and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-β and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-β mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-β receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-β expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots. © 2016 Elsevier Inc.

Caldeira J.,University of Porto | Caldeira J.,Instituto Of Engineering Biomedica Ineb | Figueiredo J.,University of Porto | Bras-Pereira C.,Instituto Gulbenkian Of Ciencia Igc | And 15 more authors.
Human Molecular Genetics | Year: 2015

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is acommon event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecaddependent invasion. To verify whethermatrix deregulationwas triggered by Ecad loss,we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecadcompetent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. © The Author 2015.

Barros R.,University of Porto | Barros R.,Institute Investigacao e Inovacao em Saude i3S | Pereira D.,Portuguese Oncology Institute Francisco Gentil | Pereira D.,University of Beira Interior | And 12 more authors.
Disease Markers | Year: 2016

Barrett's esophagus (BE) is the replacement of the normal esophageal squamous epithelium by a columnar lining epithelium. It is a premalignant condition for the development of adenocarcinoma of the esophagus and esophagogastric junction. BE is associated with gastroesophageal reflux which might change the expression profile of key transcription factors involved in the establishment of tissue differentiation, namely, SOX2 (associated with esophageal and gastric differentiation) and CDX2 (associated with intestinal differentiation). Here, we sought to characterize the expression profile of SOX2 and CDX2 in the sequential alterations of the esophageal mucosa towards adenocarcinoma and compare it with the well-established gastric and intestinal mucin profiles (MUC5AC, MUC6, and MUC2). We observed that SOX2 and CDX2 expression correlates with gastric and intestinal differentiation in BE, defined by morphological parameters and mucin expression. We show the presence of a complete intestinal profile in BE, without gastric mucins and without SOX2, and we observed an evolutionary modulation of the metaplastic phenotype by SOX2 and CDX2. We observed that adenocarcinomas harbor more frequently a mixed gastric and intestinal phenotype. In conclusion, our study establishes a role for transcription factors SOX2 and CDX2 in the progression from gastric to gastrointestinal differentiation in Barrett's metaplasia. © 2016 Rita Barros et al.

Sousa R.G.,Institute Investigacao e Inovacao em Saude i3S | Sousa R.G.,Institute Engineering Biomedica INEB | Esteves T.,Institute Investigacao e Inovacao em Saude i3S | Esteves T.,Institute Engineering Biomedica INEB | And 10 more authors.
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2015

Immunogold particle detection is a time-consuming task where a single image containing almost a thousand particles can take several hours to annotate. In this work we present a framework for the automatic detection of immunogold particles that can leverage significantly the burden of this manual task. Our proposal applies a Laplacian of Gaussian (LoG) filter to provide its detection estimates to a Stacked Denoising Autoencoder (SdA). This learning model endowed with the capability to extract higher order features provides a robust performance to our framework. For the validation of our framework, a new dataset was created. Based on our work, we determined that solely the LoG detector attained more than 74.1% of accuracy and, when combined with a SdA the accuracy is improved by at most 11.4%. © Springer International Publishing Switzerland 2015.

PubMed | Institute Investigacao e Inovacao em Saude i3S
Type: Journal Article | Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society | Year: 2015

Intervertebral disc (IVD) degeneration is responsible for various spine pathologies and present clinical treatments are insufficient. Concurrently, the mechanisms behind IVD degeneration are still not completely understood, so as to allow development of efficient tissue engineering approaches. A model of rat IVD degeneration directly coupled to herniation is here proposed in a pilot study. Disc injury is induced by needle puncture, using two different needles gauges: a low caliber 25-G needle and a high caliber 21-G needle. Histological, biochemical, and radiographic degeneration was evaluated at 2 and 6 weeks post-injury. We show that the larger caliber needle results in a more extended histological and radiographic degeneration within the IVD, compared to the smaller one. TUNEL quantification indicates also increased cell death in the 21-G group. Analyses of collagen type I (Picrosirius red staining), collagen type II (immunofluorescence), and GAG content (Blyscan assay) indicate that degeneration features spontaneously recover from 2 to 6 weeks, for both needle types. Moreover, we show the occurrence of hernia proportional to the needle gauge. The number of CD68+ macrophages present, as well as cell apoptosis within the herniated tissue are both proportional to hernia volume. Moreover, hernias formed after lesion tend to spontaneously diminish in volume after 6 weeks. Finally, MMP3 is increased in the hernia in the 21-G group at 2 weeks. This model, by uniquely combining IVD degeneration and IVD herniation in the same animal, may help to understand mechanisms behind IVD pathophysiology, such as hernia formation and spontaneous regression. 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:258-268, 2017.

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