Publications by authors named "Diana Ribeiro"

25 Publications

  • Page 1 of 1

Process Development for Flexible Films of Industrial Cellulose Pulp Using Superbase Ionic Liquids.

Polymers (Basel) 2021 May 28;13(11). Epub 2021 May 28.

Centre for Mechanical Engineering, Materials and Processes (CEMMPRE), Department of Chemical Engineering, University of Coimbra, Rua Sílvio Lima-Polo II, 3030-790 Coimbra, Portugal.

Due to environmental concerns, more attention has been given to the development of bio-based materials for substitution of fossil-based ones. Moreover, paper use is essential in daily routine and several applications of industrial pulp can be developed. In this study, transparent films were produced by industrial cellulose pulp solubilization in tetramethylguanidine based ionic liquids followed by its regeneration. Films were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), UV/Vis spectroscopy, proton nuclear magnetic resonance (H-NMR), dynamic scanning calorimetry (DSC), thermal analysis (TG), and X-ray diffraction (XRD). Mechanical tests showed that films have a good elongation property, up to 50%, depending on ionic liquid incorporation. The influence of the conjugated acid and dissolution temperature on mechanical properties were evaluated. These results revealed the potential of this methodology for the preparation of new biobased films.
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http://dx.doi.org/10.3390/polym13111767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199285PMC
May 2021

mU54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs.

Int J Mol Sci 2021 Mar 14;22(6). Epub 2021 Mar 14.

Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, 3810 Aveiro, Portugal.

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (mU) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces mU54 tRNA hypomodification and tsRNA formation. More specifically, mU54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5'tRNA-derived stress-induced RNAs (5'tiRNAs), namely 5'tiRNA-Gly and 5'tiRNA-Glu, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently mU54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel mU54 as a tRNA cleavage protective mark.
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http://dx.doi.org/10.3390/ijms22062941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001983PMC
March 2021

Emerging Roles of tRNAs in RNA Virus Infections.

Trends Biochem Sci 2020 09 3;45(9):794-805. Epub 2020 Jun 3.

iBiMED, Institute of Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, Portugal. Electronic address:

Viruses rely on the host cell translation machinery for efficient synthesis of their own proteins. Emerging evidence highlights different roles for host transfer RNAs (tRNAs) in the process of virus replication. For instance, different RNA viruses manipulate host tRNA pools to favor viral protein translation. Interestingly, specific host tRNAs are used as reverse transcription primers and are packaged into retroviral virions. Recent data also demonstrate the formation of tRNA-derived fragments (tRFs) upon infection to facilitate viral replication. Here, we comprehensively discuss how RNA viruses exploit distinct aspects of the host tRNA biology for their benefit. In light of the recent advances in the field, we propose that host tRNA-related pathways and mechanisms represent promising cellular targets for the development of novel antiviral strategies.
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http://dx.doi.org/10.1016/j.tibs.2020.05.007DOI Listing
September 2020

Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum.

FEBS J 2020 07 19;287(13):2723-2743. Epub 2019 Dec 19.

UCIBIO, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Caparica, Portugal.

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.
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http://dx.doi.org/10.1111/febs.15162DOI Listing
July 2020

Effect of Sleeve Gastrectomy on Angiogenesis and Adipose Tissue Health in an Obese Animal Model of Type 2 Diabetes.

Obes Surg 2019 09;29(9):2942-2951

Institute of Physiology and Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Polo III, Azinhaga de Santa Comba, Celas, 3000-548, Coimbra, Portugal.

Introduction: Metabolic surgery has become an accepted option for the treatment of obesity and associated metabolic diseases like hypertension and type 2 diabetes. Adipose tissue dysfunction and ectopic storage of excess lipids are thought to be involved in the underlying pathophysiological process.

Objectives: The present study aims to clarify the effect of sleeve gastrectomy (SG) on adipose tissue microvasculature and health in an animal model of adipose type 2 diabetes.

Methods: After weaning, diabetic Goto-Kakizaki rats were either fed on standard rat chow or high-calorie diet. At 4 months, animals on high-calorie diet were randomized to SG, sham surgery, or control group. Non-diabetic Wistar rats served as further controls. At 6 months, glucose and lipid metabolisms were studied in vivo. After sacrifice, periepididymal adipose tissue was collected for histology and analysis of parameters of adipose tissue metabolism and insulin sensitivity.

Results: SG decreased body and adipose tissue weight and improved glycemic and lipid profiles. Fasting glycemia, area under the curve after intraperitoneal insulin tolerance test, and insulin resistance were decreased in operated animals. SG also reduced circulating triglycerides and cholesterol while increasing serum adiponectin and adipose tissue peroxisome proliferator-activated receptor γ (PPAR-γ) and perilipin A. Additionally, surgery improved adipose tissue vascular function and markedly increased vascular endothelial growth factor, cluster of differentiation 31, and endothelial nitric oxide synthase.

Conclusions: In our obese animal model of type 2 diabetes, SG significantly improved adipose tissue health and angiogenesis while reducing insulin resistance, involving PPAR-γ and markers of sprouting angiogenesis and endothelial function.
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http://dx.doi.org/10.1007/s11695-019-03935-zDOI Listing
September 2019

Spindly and Bub3 expression in oral cancer: Prognostic and therapeutic implications.

Oral Dis 2019 Jul 4;25(5):1291-1301. Epub 2019 Apr 4.

Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Instituto Universitário de Ciências da Saúde, CESPU, Gandra, Portugal.

Objectives: Bub3 and Spindly are essential proteins required for the activation and inactivation of the spindle assembly checkpoint, respectively. Here, we explored the clinicopathological significance and the therapeutic potential of the opposing roles of the two proteins in oral squamous cell carcinoma (OSCC).

Materials And Methods: Bub3 and Spindly expression was evaluated by immunohistochemistry in 62 tissue microarrays from OSCC and by real-time PCR in OSCC cell lines and in normal human oral keratinocytes. The results were analyzed as to their clinicopathological significance. RNA interference-mediated Spindly or Bub3 inhibition was combined with cisplatin treatment, and the effect on the viability of OSCC cells was assessed.

Results: Overexpression of Bub3 and Spindly was detected in OSCC patients. High expression of Spindly, Bub3, or both was an independent prognostic indicator for cancer-specific survival and was associated with increased cellular proliferation. Accordingly, Bub3 and Spindly were upregulated in OSCC cells comparatively to their normal counterpart. Inhibition of Bub3 or Spindly was cytotoxic to OSCC cells and enhanced their chemosensitivity to cisplatin.

Conclusions: The data point out Bub3 and Spindly as potential markers of proliferation and prognosis, and highlight the potential therapeutic benefit of combining their inhibition with cisplatin.
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http://dx.doi.org/10.1111/odi.13089DOI Listing
July 2019

New Alkoxy Flavone Derivatives Targeting Caspases: Synthesis and Antitumor Activity Evaluation.

Molecules 2018 Dec 31;24(1). Epub 2018 Dec 31.

Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.

The antitumor activity of natural flavonoids has been exhaustively reported. Previously it has been demonstrated that prenylation of flavonoids allows the discovery of new compounds with improved antitumor activity through the activation of caspase-7 activity. The synthesis of twenty-five flavonoids (⁻) with one or more alkyl side chains was carried out. The synthetic approach was based on the reaction with alkyl halide in alkaline medium by microwave (MW) irradiation. The in vitro cell growth inhibitory activity of synthesized compounds was investigated in three human tumor cell lines. Among the tested compounds, derivatives , , , , , , and revealed potent growth inhibitory activity (GI < 10 μM), being the growth inhibitory effect of compound related with a pronounced caspase-7 activation on MCF-7 breast cancer cells and yeasts expressing human caspase-7. A quantitative structure-activity relationship (QSAR) model predicted that hydrophilicity, pattern of ring substitution/shape, and presence of partial negative charged atoms were the descriptors implied in the growth inhibitory effect of synthesized compounds. Docking studies on procaspase-7 allowed predicting the binding of compound to the allosteric site of procaspase-7.
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http://dx.doi.org/10.3390/molecules24010129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337158PMC
December 2018

Mitosis inhibitors in anticancer therapy: When blocking the exit becomes a solution.

Cancer Lett 2019 01 9;440-441:64-81. Epub 2018 Oct 9.

CESPU, Instituto de Investigação e Formação Avançada Em Ciências e Tecnologias da Saúde, Instituto Universitário de Ciências da Saúde, Gandra PRD, Portugal; Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade Do Porto, Porto, Portugal. Electronic address:

Current microtubule-targeting agents (MTAs) remain amongst the most important antimitotic drugs used against a broad range of malignancies. By perturbing spindle assembly, MTAs activate the spindle assembly checkpoint (SAC), which induces mitotic arrest and subsequent apoptosis. However, besides toxic side effects and resistance, mitotic slippage and failure in triggering apoptosis in various cancer cells are limiting factors of MTAs efficacy. Alternative strategies to target mitosis without affecting microtubules have, thus, led to the identification of small molecules, such as those that target spindle Kinesins, Aurora and Polo-like kinases. Unfortunately, these so-called second-generation of antimitotics, encompassing mitotic blockers and mitotic drivers, have failed in clinical trials. Our recent understanding regarding the mechanisms of cell death during a mitotic arrest pointed out apoptosis as the main variable, providing an opportunity to control the cell fates and influence the effectiveness of antimitotics. Here, we provide an overview on the second-generation of antimitotics, and discuss possible strategies that exploit SAC activity, mitotic slippage/exit and apoptosis induction, in order to improve the efficacy of anticancer strategies that target mitosis.
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http://dx.doi.org/10.1016/j.canlet.2018.10.005DOI Listing
January 2019

Synthesis of New Glycosylated Flavonoids with Inhibitory Activity on Cell Growth.

Molecules 2018 05 5;23(5). Epub 2018 May 5.

Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.

Natural flavonoids and xanthone glycosides display several biological activities, with the glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein, to give further insights into the inhibitory activity on cell growth of these classes of compounds, the synthesis of four flavonoids (, , , and ) and one xanthone () containing one or more acetoglycoside moieties was carried out. Acetyl groups were introduced using acetic anhydride and microwave irradiation. The introduction of one or two acetoglycoside moieties in the framework of 3,7-dihydroxyflavone () was performed using two synthetic methods: the Michael reaction and the Koenigs-Knorr reaction. The in vitro cell growth inhibitory activity of compounds , , , , and was investigated in six human tumor cell lines: A375-C5 (malignant melanoma IL-1 insensitive), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). The new flavonoid 3-hydroxy-7-(2,3,4,6-tetra--acetyl-β-glucopyranosyl) flavone () was the most potent compound in all tumor cell lines tested, with GI values < 8 μM and a notable degree of selectivity for cancer cells.
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http://dx.doi.org/10.3390/molecules23051093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102538PMC
May 2018

3D-Models of Insulin-Producing β-Cells: from Primary Islet Cells to Stem Cell-Derived Islets.

Stem Cell Rev Rep 2018 Apr;14(2):177-188

Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Gothenburg, Sweden.

There is a need for physiologically relevant assay platforms to provide functionally relevant models of diabetes, to accelerate the discovery of new treatment options and boost developments in drug discovery. In this review, we compare several 3D-strategies that have been used to increase the functional relevance of ex vivo human primary pancreatic islets and developments into the generation of stem cell derived pancreatic beta-cells (β-cells). Special attention will be given to recent approaches combining the use of extracellular matrix (ECM) scaffolds with pancreatic molecular memory, which can be used to improve yield and functionality of in vitro stem cell-derived pancreatic models. The ultimate goal is to develop scalable cell-based platforms for diabetes research and drug screening. This article will critically assess key aspects related to in vitro pancreatic 3D-ECM models and highlight the most promising approaches for future research.
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http://dx.doi.org/10.1007/s12015-017-9783-8DOI Listing
April 2018

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters.

PLoS One 2017 8;12(11):e0187665. Epub 2017 Nov 8.

Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Gothenburg, Sweden.

It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187665PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678888PMC
November 2017

Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation.

Proc Natl Acad Sci U S A 2017 10 2;114(42):11127-11132. Epub 2017 Oct 2.

Department of Biology and Biological Engineering, Chalmers University of Technology, 41258 Gothenburg, Sweden;

Extracellular vesicles (EVs) are small vesicles released by cells to aid cell-cell communication and tissue homeostasis. Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in pancreatic islets of patients with type 2 diabetes (T2D). IAPP is secreted in conjunction with insulin from pancreatic β cells to regulate glucose metabolism. Here, using a combination of analytical and biophysical methods in vitro, we tested whether EVs isolated from pancreatic islets of healthy patients and patients with T2D modulate IAPP amyloid formation. We discovered that pancreatic EVs from healthy patients reduce IAPP amyloid formation by peptide scavenging, but T2D pancreatic and human serum EVs have no effect. In accordance with these differential effects, the insulin:C-peptide ratio and lipid composition differ between EVs from healthy pancreas and EVs from T2D pancreas and serum. It appears that healthy pancreatic EVs limit IAPP amyloid formation via direct binding as a tissue-specific control mechanism.
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http://dx.doi.org/10.1073/pnas.1711389114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651775PMC
October 2017

[Eosinophilic pneumonia, how to differentiate: classification and diagnostic approach].

Rev Med Suisse 2016 Nov;12(539):1958-1965

Service de pneumologie, CHUV, 1011 Lausanne.

Eosinophilic pneumonia is characterized by pulmonary eosinophilia associated with and/or alveolar eosinophilia. Furthermore, blood eosinophilia may be absent particularly in patients who have benefited beforehand of corticosteroids. The most common causes are parasites and drugs. According to the classification, we distinguish idiopathic pneumonia with eosinophils, those related to vasculitis and those of known etiology. Extrapulmonary involvement is frequently found in the subgroup of vasculitis. It is important to diagnose and start treatment quickly to reduce the risk of complications. The primary treatment is based on corticosteroids and in severe cases of vasculitis immunosuppressants are necessary.
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November 2016

Extracellular Vesicles Derived from Osteogenically Induced Human Bone Marrow Mesenchymal Stem Cells Can Modulate Lineage Commitment.

Stem Cell Reports 2016 Mar 25;6(3):284-91. Epub 2016 Feb 25.

3B's Research Group-Biomaterials, Biodegradable and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark, Barco, 4805-017 Guimarães, Portugal; ICVS/3B's PT Government Associated Laboratory, Braga/Guimarães, Portugal. Electronic address:

The effective osteogenic commitment of human bone marrow mesenchymal stem cells (hBMSCs) is critical for bone regenerative therapies. Extracellular vesicles (EVs) derived from hBMSCs have a regenerative potential that has been increasingly recognized. Herein, the osteoinductive potential of osteogenically induced hBMSC-EVs was examined. hBMSCs secreted negatively charged nanosized vesicles (∼ 35 nm) with EV-related surface markers. The yield of EVs over 7 days was dependent on an osteogenic stimulus (standard chemical cocktail or RUNX2 cationic-lipid transfection). These EVs were used to sequentially stimulate homotypic uncommitted cells during 7 days, matching the seeding density of EV parent cells, culture time, and stimuli. Osteogenically committed hBMSC-EVs induced an osteogenic phenotype characterized by marked early induction of BMP2, SP7, SPP1, BGLAP/IBSP, and alkaline phosphatase. Both EV groups outperformed the currently used osteoinductive strategies. These data show that naturally secreted EVs can guide the osteogenic commitment of hBMSCs in the absence of other chemical or genetic osteoinductors.
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http://dx.doi.org/10.1016/j.stemcr.2016.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788762PMC
March 2016

Protoplast Transformation as a Plant-Transferable Transient Expression System.

Methods Mol Biol 2016 ;1405:137-48

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.
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http://dx.doi.org/10.1007/978-1-4939-3393-8_13DOI Listing
November 2016

Effect of glucocorticoids on growth and bone mineral density in children with nephrotic syndrome.

Eur J Pediatr 2015 Jul 10;174(7):911-7. Epub 2015 Jan 10.

Division of General Pediatrics, Department of Pediatrics, Children's Hospital, University Hospitals of Geneva, 6 Rue Willy-Donzé, 1211, Geneva, Switzerland,

Unlabelled: Glucocorticosteroids (GCs) are the first-line treatment for idiopathic nephrotic syndrome (NS), but prolonged administration interferes with growth and bone mineralization. We conducted a retrospective study to analyze the long-term impact of prednisone on growth and bone mineral density (BMD) in children with NS. Data from children with NS followed during almost 10 years were analyzed. Height and spine BMD values were converted to Z-scores (standard deviation [SD]). The mean cumulative dose of GCs received was calculated and correlated to patient's growth and spine BMD using linear regression and subgroup analysis. We included 30 patients diagnosed at 3.7 years old (interquartile range (IQR) 2.6-4.8) and followed over 9.8 years (IQR 6.6-11.7). The one half of NS patients was steroid sensitive and one half dependent or resistant. The median cumulative dose of GCs received was 0.27 mg/kg/day (IQR 0.18-0.35). Growth and spine BMD were both negatively associated with the cumulative dose of GCs (P=0.001 and P=0.037, respectively). Final height Z-scores were significantly lower in patients receiving >0.2 mg/kg/day GCs (P=0.001). No difference was observed in spine BMD between subgroups.

Conclusion: Increasing doses of GCs were significantly associated with lower height and BMD Z-scores. A significant effect on growth was observed with cutoff doses above 0.2 mg/kg/day.
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http://dx.doi.org/10.1007/s00431-014-2479-zDOI Listing
July 2015

Increased adipogenesis of human adipose-derived stem cells on polycaprolactone fiber matrices.

PLoS One 2014 24;9(11):e113620. Epub 2014 Nov 24.

Reagent and Assay Development Discovery Sciences R&D, Astra Zeneca, Mölndal, Sweden.

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells--differentiated to adipocytes in vitro--are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics--studied by coherent anti-Stokes Raman scattering microscopy--to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113620PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242727PMC
January 2016

Isolation of microsatellite markers for the red mangrove, Rhizophora mangle (Rhizophoraceae).

Appl Plant Sci 2013 Sep 2;1(9). Epub 2013 Sep 2.

Institute for Coastal Research, Universidade Federal do Pará (UFPA), Alameda Leandro Ribeiro s/n, Aldeia, 68600-000 Bragança, Pará, Brazil.

Unlabelled:

Premise Of The Study: Three species of the mangrove tree genus Rhizophora are found in the New World and along the west coast of Africa. Of these, R. mangle is the most abundant and has a complex interbreeding relationship with the sympatric R. racemosa and R. harrisonii. The development of additional microsatellite markers would permit paternity analyses and investigation of the hybrid origin of these species. •

Methods And Results: Using an enriched library method, via hybridization with biotinylated oligonucleotides complementary to repetitive poly AG/TC, primers for 11 microsatellite markers of R. mangle were developed and characterized in populations in Pará and São Paulo (Brazil) and Florida (USA). Ten of these markers were transferable to R. racemosa and R. harrisonii. •

Conclusions: The microsatellite markers presented here will be useful in studies of contemporary and historical gene flow between American and West African Rhizophora species.
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http://dx.doi.org/10.3732/apps.1300003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103143PMC
September 2013

Instructive nanofibrous scaffold comprising runt-related transcription factor 2 gene delivery for bone tissue engineering.

ACS Nano 2014 Aug;8(8):8082-94

Inducer molecules capable of regulating mesenchymal stem cell differentiation into specific lineages have proven effective in basic science and in preclinical studies. Runt-related transcription factor 2 (RUNX2) is considered to be the central gene involved in the osteoblast phenotype induction, which may be advantageous for inducing bone tissue regeneration. This work envisions the development of a platform for gene delivery, combining liposomes as gene delivery devices, with electrospun nanofiber mesh (NFM) as a tissue engineering scaffold. pDNA-loaded liposomes were immobilized at the surface of functionalized polycaprolactone (PCL) NFM. Human bone-marrow-derived mesenchymal stem cells (hBMSCs) cultured on RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFM showed enhanced levels of metabolic activity and total protein synthesis. RUNX2-loaded liposomes immobilized at the surface of electrospun PCL NFMs induce a long-term gene expression of eGFP and RUNX2 by cultured hBMSCs. Furthermore, osteogenic differentiation of hBMSCs was also achieved by the overexpression of other osteogenic markers in medium free of osteogenic supplementation. These findings demonstrate that surface immobilization of RUNX2 plasmid onto elestrospun PCL NFM can produce long-term gene expression in vitro, which may be employed to enhance the osteoinductive properties of scaffolds used for bone tissue engineering strategies.
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http://dx.doi.org/10.1021/nn5021049DOI Listing
August 2014

On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells.

J Tissue Eng Regen Med 2015 Sep 7;9(9):1056-66. Epub 2013 Oct 7.

3Bs Research Group - Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra S. Cláudio do Barco, 4806-909, Caldas das Taipas, Guimarães, Portugal.

Stem cells have received considerable attention by the scientific community because of their potential for tissue engineering and regenerative medicine. The most frequently used method to promote their differentiation is supplementation of the in vitro culture medium with growth/differentiation factors (GDFs). The limitations of that strategy caused by the short half-life of GDFs limit its efficacy in vivo and consequently its clinical use. Thus, the development of new concepts that enable the bioactivity and bioavailability of GDFs to be protected, both in vitro and in vivo, is very relevant. Nanoparticle-based drug delivery systems can be injected, protect the GDFs and enable spatiotemporal release kinetics to be controlled. Liposomes are well-established nanodelivery devices presenting significant advantages, viz. a high load-carrying capacity, relative safety and easy production, and a versatile nature in terms of possible formulations and surface functionalization. The main objective of the present study was to optimize the formulation of liposomes to encapsulate dexamethasone (Dex). Our results showed that the optimized Dex-loaded liposomes do not have any cytotoxic effect on human bone marrow-derived mesenchymal stem cells (hBMSCs). More importantly, they were able to promote an earlier induction of differentiation of hBMSCs into the osteogenic lineage, as demonstrated by the expression of osteoblastic markers, both phenotypically and genotypically. We concluded that Dex-loaded liposomes represent a viable nanoparticle strategy with enhanced safety and efficacy for tissue engineering and regenerative medicine.
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http://dx.doi.org/10.1002/term.1817DOI Listing
September 2015

Superficial siderosis of the central nervous system: an usual cause of sensorineural hearing loss.

Braz J Otorhinolaryngol 2013 Mar-Apr;79(2):257

Serviço de Otorrinolaringologia do Hospital Geral, University and Hospital Center of Coimbra, Coimbra, Portugal.

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http://dx.doi.org/10.5935/1808-8694.20130044DOI Listing
September 2013

Superficial siderosis of the central nervous system: an usual cause of sensorineural hearing loss.

Braz J Otorhinolaryngol 2013 Mar-Apr;79(2):257

Serviço de Otorrinolaringologia do Hospital Geral, University and Hospital Center of Coimbra, Coimbra, Portugal.

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http://dx.doi.org/10.5935/1808-8694.20130044DOI Listing
September 2013

Odontomas: a clinicopathologic study in a Portuguese population.

Quintessence Int 2009 Jan;40(1):61-72

Objective: Odontoma is a tumorlike malformation (hamartoma) that contains odontogenic epithelium with odontogenicectomesenchyme. Frequency and distribution of odontogenic tumor among a Portuguese population were analyzed and compared with previous reports.

Method And Materials: A total of 65 odontogenic tumor cases were collected from the files of the Department of Pathology of Hospital Sao Joao,Porto,Portugal, and the Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), from January 1993 to December 2006. Of these cases, 48 were retrieved and analyzed. The final diagnosis of each case was based on the 2005 WHO histopathologic classification of odontogenictumors, and to the authors' best knowledge, the present series represents the first study on odontomas in a northern Portuguese population.

Results: Of the 65 odontogenic tumors cases, 64 (98.5%) were benign and 1 (1.5%), an ameloblasticcarcinoma, was malignant. Odontoma was the most frequent odontogenic tumor (73.9%), followed by unicysticameloblastoma(7.7%) and calcifying cystic odontogenic tumor (7.7%). Of the 48 odontomas(26 males and 22 females), 34 (70.8%) were compound and 14 (29.2%) were complex. Most odontomas(72.9%) occurred in patients under the age of 30, with a peak incidence in the second decade of life. Twenty-eight (58.3%)odontomas were in the maxilla and 20 (41.7%) in the mandible (P<.05). Twenty-eight (58.3%) of the 48 odontomas were associated with 33 impacted teeth, including 31 permanent teeth, 1 primary tooth, and 1 supernumerary tooth. The maxillary central incisor (n= 6; 19.4%) and the maxillary canine (n= 6; 19.4%) were most commonly associated with odontoma, followed by the mandibular canine (n= 5; 16.0%) and maxillary third molar (n= 4; 12.9%).

Conclusion: This study provides clinical and pathological information on odotogenic tumors in a nothern Portuguese population
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January 2009

Contribution of spoligotyping to the characterization of the population structure of Mycobacterium tuberculosis isolates in Portugal.

Infect Genet Evol 2007 Sep 21;7(5):609-17. Epub 2007 May 21.

Unidade de Ensino e Investigação (UEI) de Micobactérias, Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Lisbon, and Serviço de Patologia Clínica, Hospital Fernando Fonseca, Amadora, Portugal.

Tuberculosis is a major health problem in Portugal. To begin characterizing the population structure of Mycobacterium tuberculosis, spoligotyping was used for the systematic typing, through consecutive sampling, of patient isolates from the Amadora-Sintra area of Greater Lisbon. Distribution amongst major spoligotype families, including the Latin American Mediterranean (LAM), T, Haarlem and Beijing, was compared to that of the international spoligotype database SpolDB4 and to the European countries of traditional Portuguese immigration represented in SpolDB4. Spoligotypes from 665 isolates were analyzed and 97 shared international types (SITs) identified. In SpolDB4 Portugal is represented by part of the spoligotypes from this study explaining the reduced number of unidentified patterns. The importance of the LAM family, and especially of LAM1 and LAM9 sub-families that alone represented 38% of all the isolates in this study as compared to 8% relative to the European sub group, led us to believe that at least in this respect the population structure was closer to that of Africa and South America than to Europe. Spoligotypes characteristic of Portugal or Portuguese related settings were identified. These included SIT244 a T1 sub-family predominant in Portugal and Bangladesh, SIT64 a LAM 6 sub-family common to Portugal and Brazil, and SIT1106 a LAM 9 sub-family. These studies were the first in Portugal stressing the importance of monitoring the population structure of M. tuberculosis isolates, an important step towards gaining an understanding of tuberculosis and the dynamics of this disease.
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http://dx.doi.org/10.1016/j.meegid.2007.05.007DOI Listing
September 2007
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