João P. Silva

Wound healing activity of the human antimicrobial peptide LL37

Antimicrobial peptides (AMPs) are part of the innate immune system and are generally defined as cationic, amphipathic peptides, with less than 50 amino acids, including multiple arginine and lysine residues. The human cathelicidin antimicrobial peptide LL37 can be found at different concentrations in many different cells, tissues and body fluids and has a broad spectrum of antimicrobial and immunomodulatory activities. The healing of wound is a complex process that involves different steps: hemostasis, inflammation, remodeling/granulation tissue formation and re-epithelialization. Inflammation and angiogenesis are two fundamental physiological conditions implicated in this process. We have recently developed a new method for the expression and purification of recombinant LL37. In this work, we show that the recombinant peptide P-LL37 with a N-terminus proline preserves its immunophysiological properties in vitro and in vivo. P-LL37 neutralized the activation of macrophages by lipopolysaccharide (LPS). Besides, the peptide induced proliferation, migration and tubule-like structures formation by endothelial cells. Wound healing experiments were performed in dexamethasone-treated mice to study the effect of LL37 on angiogenesis and wound regeneration. The topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization. Taken together, these results clearly demonstrate that LL37 may have a key role in wound regeneration through vascularization.

Oxidative DNA damage protection and repair by polyphenolic compounds in PC12 cells

Biological systems are frequently exposed to excessive reactive oxygen species, causing a disturbance in the cells natural antioxidant defence systems and resulting in damage to all biomolecules, including nucleic acids. In fact, oxidative DNA damage is described as the type of damage most likely to occur in neuronal cells. In this study, three polyphenolic compounds, luteolin, quercetin and rosmarinic acid, were investigated for their protective effects against oxidative DNA damage induced in PC12 cells, a neuronal cell model. Although luteolin and quercetin prevented the formation of strand breaks to a greater extent than rosmarinic acid, this last one presented the highest capacity to repair strand breaks formation. In addition, rosmarinic acid was the only compound tested that increased the repair of oxidized nucleotidic bases induced with the photosensitizer compound [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl) carbonyl]-2-pyrrolidine-methanol (Ro 19-8022). The activity of repair enzymes was indicated by the in vitro base excision repair assay, using a cell-free extract obtained from cells previously treated with the compounds to incise DNA. The protective effect of rosmarinic acid was further confirmed by the increased expression of OGG1 repair gene, observed through real time RT-PCR. The data obtained is indicative that rosmarinic acid seems to act on the intracellular mechanisms responsible for DNA repair, rather than by a direct effect on reactive oxygen species scavenging, as deducted from the effects observed for luteolin and quercetin. Therefore, these results suggest the importance of these polyphenols, and in particular rosmarinic acid, as protectors of oxidative stress-induced DNA damage that commonly occurs in several pathological conditions, such as neurodegenerative diseases.

Studies on the hemocompatibility of bacterial cellulose

Among the strategies to improve a materials hemocompatibility, pre-coating with the tripeptide Arg-Gly-Asp (RGD) is used to favor endothelialization thus lowering thrombogenicity. The blood compatibility of native and RGD-modified bacterial cellulose (BC) was studied in this work for the first time. The plasma recalcification time and whole blood clotting results demonstrate the hemocompatibility of BC. A significant amount of plasma protein adsorb to BC fibres, however, according to analysis by intrinsic tryptophan fluorescence techniques when albumin, γ-globulin, and fibrinogen from pure protein solutions adsorb to BC do not undergo detectable conformational modifications. Human microvascular endothelial cells cultured on RGD-modified BC readily form a confluent cell layer, inhibiting the adhesion of platelets. As a general conclusion, both native and RGD-modified BCs may be classified as hemocompatible materials.

Hemocompatibility study of a bacterial cellulose/polyvinyl alcohol nanocomposite

Bacterial cellulose (BC) has been suggested to be a suitable biomaterial for the development of cardiovascular grafts. The combination of BC with polyvinyl alcohol (PVA) results in nanocomposites with improved properties. Surprisingly, there are very few studies on the BC-blood interaction. This is the focus of this paper. We present the first thorough assessment of the hemocompatibility of the BC/PVA nanocomposite. Whole blood clotting time, plasma recalcification, Factor XII activation, platelet adhesion and activation, hemolytic index and complement activation are all determined. The platelet activation profiles on BC and BC/PVA surfaces are comprehensively characterized. BC and BC/PVA outperformed ePTFE--used as a point of comparison--thus evidencing their suitability for cardiovascular applications.

MENTOR — A methodology for enterprise reference ontology development

Ontologies facilitate the computational understanding, communication and seamless interoperability between people and organizations. They allow key concepts and terms relevant to a given domain to be identified and defined in an open and unambiguous way. Therefore, ontologies facilitate the use and exchange of data, information, and knowledge among people and organizations, towards intelligent systems interoperability. Tasks on distributed and heterogeneous systems demand support from more than one ontology. Multiple ontologies need to be accessed by the same, but also different systems. The distributed nature of ontology development has led to dissimilar ontologies for the same or overlapping domains. Thus, various parties with different ontologies often do not understand each other. To solve these problems, it is necessary to use ontology mapping geared for interoperability. This paper proposes a methodology to support the development of a common reference ontology for a group of enterprises sharing a business domain. This methodology is based on the concept of Mediator Ontology (MO), which assists the semantic transformations among each enterprisepsilas ontology and the referential one. The MO enables each organization to keep its own terminology, glossary and ontological structures, providing seamless communication and interaction with the others. A use case is described and the proposed methodology demonstrated.

The effect of nanotube surface oxidation on the electrical properties of multiwall carbon nanotube/poly(vinylidene fluoride) composites

Carbon nanotube/poly(vinylidene fluoride) (CNT/PVDF) composites were prepared using CNT with different oxidation and thermal treatments. The oxidation procedure leads to CNT with the most acidic characteristics that lower the degree of crystallinity of the polymer and contribute to a large increase of the dielectric constant. The surface treatments, in general, increase the percolation threshold and decrease conductivity. The surface treatments do not seem to affect CNT interactions and similar degrees of dispersion are achieved in all cases, as shown by the SEM results. The maximum value of the dielectric constant is ~630. It is demonstrated that the composite conductivity can be attributed to a hopping mechanism that is strongly affected by the surface treatment of the CNT.

The Role of Solvent Evaporation in the Microstructure of Electroactive β-Poly(Vinylidene Fluoride) Membranes Obtained by Isothermal Crystallization

Electroactiveβ-poly(vinylidene fluoride) (PVDF) membranes were obtained by isothermal crystallization from the solution. Different morphologies and microstructures were obtained by crystallizing at different temperatures. The mechanism and kinetics of solvent evaporation from the polymeric solution were investigated using isothermal thermogravimetric analysis. The kinetic parameters and the activation energy were also calculated. The solvent evaporation is ruled by two steps, related with a metastable– unstable–metastable transition in the solution phase diagram. Scanning electron microscopy revealed the porous structure and the variations of the morphology with the variation of the isothermal evaporation temperature. Finally, the infrared spectroscopy measurements confirm that the polymer crystallizes in the electroactiveβ-phase of PVDF.

Neuronal cells’ behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds

In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations.

Protective role of new nitrogen compounds on ROS/RNS-mediated damage to PC12 cells

Reactive oxygen (ROS) and nitrogen (RNS) species are known to be involved in many degenerative diseases. This study reports four new nitrogen compounds from organic synthesis, identified as FMA4, FMA7, FMA762 and FMA796, which differ mainly by the number of hydroxyl groups within their phenolic unit. Their potential role as antioxidants was evaluated in PC12 cells by assessing their protection against oxidative and nitrosative insults. The four compounds, and particularly FMA762 and FMA796, were able to protect cells against lipid peroxidation and intracellular ROS/RNS formation to a great extent. Their protective effects were likely mediated by their free radicals scavenging ability, as they appeared to be involved neither in the induction of natural antioxidant enzymes like GSH-PX and SOD, nor in the inhibition of NOS. Nevertheless, these results suggest a promising potential for these compounds as ROS/RNS scavengers in pathologies where oxidative/nitrosative stress are involved.

Production and Characterization of a New Bacterial Cellulose/Poly(Vinyl Alcohol) Nanocomposite

Bacterial cellulose (BC) is characterized for its high water holding capacity, high crystallinity, an ultrafine fiber network and high tensile strength. This work demonstrates the production of a new interpenetrated polymer network nanocomposite obtained through the incorporation of poly(vinyl alcohol) (PVA) on the BC matrix and evaluates the effect of oven drying on the morphological, mechanical and mass transfer properties of the composite membranes. Both the addition of PVA and oven drying induce the appearance of larger pores (circa 1–3 µm in average diameter) in dried BC/PVA membranes. Both types of treatments also affect the permeability of the composite, as assessed by the diffusion coefficients of polyethylene glycol (PEG) molecules (900, 8,000, 35,000 and 100,000 Da) across the membranes. Finally, the Young’s modulus of dry pristine BC decreases following PVA incorporation, resulting in a change from 3.5 GPa to 1 GPa and a five-fold loss in tensile strength.