Integrating data from heterogeneous DNA microarray platforms

DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus.

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drugs resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

Influence of saliva and mucin on the adhesion of Candida oral clinical isolates

Objectives: This research work intends to clarify the role of artificial saliva, in particularly the role of mucin, a salivary protein, on the surface properties and adhesion ability of Candida spp. oral clinical isolates to abiotic surfaces. Methods: Four oral clinical isolates of Candida spp. were used: two Candida albicans strains (AC; AM) and two Candida parapsilosis strains (AD; AM2). The strains were isolated from patients using oral prosthesis. The microorganisms were cultured in the absence or presence of mucin and artificial saliva, and their adhesion to an abiotic surface (coated with mucin and artificial saliva) was evaluated. Results: The presence of mucin per se onto the abiotic surface decreased the adhesion of all strains, although the combination of mucin with artificial saliva had reduced this effect. No direct correlation between adhesion and the surface free energies of adhesion of the microorganisms was found. Significance: Candida spp. were human commensal microorganisms that became pathogenic when the host immune defenses were compromised. Medical devices were colonized by Candida spp. particularly, oral prostheses, which might lead to the degradation of the prostheses and systemic infections. The salivary secretions that constantly cover the oral cavity influenced Candida spp. adhesion process. Therefore, it was important to understand the interactions between Candida spp., salivary proteins and the characteristic of oral prosthesis when developing materials for oral prostheses.

Enzymatic phosphorylation of hair keratin enhances fast adsorption of cationic moieties

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by 31P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 minutes for cationic moiety like Methylene Blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance.

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of Retinal Pigment Epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.52.0 swelling/mm3), high mechanical strength (2048 MPa) and non-pyrogenicity (2.12 EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.

Layer-by-layer technique to developing functional nanolaminate films with antifungal activity

The layer-by-layer (LbL) deposition method was used to build up alternating layers (five) of different polyelectrolyte solutions (alginate, zein-carvacrol nanocapsules, chitosan and chitosan-carvacrol emulsions) on an aminolysed/charged polyethylene terephthalate (A/C PET) film. These nanolaminated films were characterised by contact angle measurements and through the determination of water vapour (WVTR) and oxygen (O2TR) transmission rates. The effect of active nanolaminated films against the Alternaria sp. and Rhizopus stolonifer was also evaluated. This procedure allowed developing optically transparent nanolaminated films with tuneable water vapour and gas properties and antifungal activity. The water and oxygen transmission rate values for the multilayer films were lower than those previously reported for the neat alginate or chitosan films. The presence of carvacrol and zein nanocapsules significantly decreased the water transmission rate (up to 40 %) of the nanolaminated films. However, the O2TR behaved differently and was only improved (up to 45 %) when carvacrol was encapsulated, i.e. nanolaminated films prepared by alternating alginate with nanocapsules of zein-carvacrol layers showed better oxygen barrier properties than those prepared as an emulsion of chitosan and carvacrol. These films containing zein-carvacrol nanocapsules also showed the highest antifungal activity (30 %), which did not significantly differ from those obtained with the highest amount of carvacrol, probably due to the controlled release of the active agent (carvacrol) from the zein-carvacrol nanocapsules. Thus, this work shows that nanolaminated films prepared with alternating layers of alginate and zein-carvacrol nanocapsules can be considered to improve the shelf-life of foodstuffs.

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Hydrocarbonoclastic bacteria: from bioremediation to bioenergy feedstock

Tese de Doutoramento em Engenharia Química e Biológica.

PDMS encasing system for integrated lab-on-chip Ag/AgCl reference electrodes

Poly(dimethylsiloxane) (PDMS) is an organosilicon polymer widely used in the fabrication of microfluidic systems to integrate biochips. In this study, we propose the use of an adapted PDMS mould for the creation of a miniaturized, reusable, reference electrode for in-chip electrochemical measurements. Through its integrated microfluidic system it is possible to replenish internal buffer solutions, unclog critical junctions and treat the electrode’s surface, assuring a long term reuse of the same device. Planar Ag/AgCl reference electrodes were microfabricated over a passivated p-type Silicon Wafer. The PDMS mould, containing an integrated microfluidic system, was fabricated based on patterned SU-8 mould, which includes a lateral horizontal inlet access point. Surface oxidation was used for irreversible permanent bondage between flat surfaces. The final result was planar Ag/AgCl reference electrode with integrated microfluidic that allows for electrochemical analysis in biochips

PDMS biofunctionalization study for the development of a microfluidic device: Application to salivary cortisol

The authors acknowledge to Sofia Neves from ICVS for her help in the antibodies selection.

Patterned CNT-PDMS nanocomposites for flexible pressure sensors

This paper reports the fabrication process and characterization of a flexible pressure sensor based on polydimethylsiloxane (PDMS) and multi-walled carbon nanotubes (CNT-PDMS). The proposed approach relies on patterned CNT-PDMS nanocomposite strain gauges fabricated with SU-8 microstructures (with the micropatterns) in a low‑cost and simple fabrication process. This nanocomposite polymer is mounted over a PDMS membrane, which, in turn, lies on top of a PDMS diaphragm like structure. This configuration enables the PDMS membrane to bend when pressure is applied, thereby affecting the nanocomposite strain gauges, effectively changing their electrical resistance. Carbon nanotubes have several advantages such as excellent mechanical properties, high electrical conductivity and thermal stability. Furthermore, the measurement range of the proposed sensor can be adapted according to the application by varying the CNTs content and geometry of microstructure. In addition, the sensor’s biocompatibility, low cost and simple fabrication makes it very appealing for biomechanical strain sensing. The sensor’s sensitivity was about 0.073%ΔR/mmHg.

Improved burn wound healing using a bioactive peptide

[Excerpt] Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. We took advantage of a commercially available hydrogel, Carbopol, a vehicle for topical administration that maintains a moist environment within the wound site. We hypothesized that the incorporation of LLKKK18 conjugated to dextrin would improve the healing process in rat burns. Whereas the hydrogel improves healing, LLKKK18 released from the dextrin conjugates further accelerates wound closure, and simultaneously improving the quality of healing. Indeed, the release of LLKKK18 reduces oxidative stress and inflammation (low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels). Importantly, it induced a faster resolution of the inflammatory stage through early M2 macrophage recruitment. In addition, LLKKK18 stimulates angiogenesis (increased VEGF and microvessel development in vivo), potentially contributing to more effective transport of nutrients and cytokines. Moreover, collagen staining evaluated by Masson’s Trichrome was visually much more intense after treatment with LLKKK18, suggesting higher collagen deposition. (...)

Design of bio-based supramolecular structures through self-assembly of α-lactalbumin and lysozyme

Bovine α-lactalbumin (α-La) and lysozyme (Lys), two globular proteins with highly homologous tertiary structures and opposite isoelectric points, were used to produce bio-based supramolecular structures under various pH values (3, 7 and 11), temperatures (25, 50 and 75 °C) and times (15, 25 and 35 min) of heating. Isothermal titration calorimetry experiments showed protein interactions and demonstrated that structures were obtained from the mixture of α-La/Lys in molar ratio of 0.546. Structures were characterized in terms of morphology by transmission electron microscopy (TEM) and dynamic light scattering (DLS), conformational structure by circular dichroism and intrinsic fluorescence spectroscopy and stability by DLS. Results have shown that protein conformational structure and intermolecular interactions are controlled by the physicochemical conditions applied. The increase of heating temperature led to a significant decrease in size and polydispersity (PDI) of α-La–Lys supramolecular structures, while the increase of heating time, particularly at temperatures above 50 °C, promoted a significant increase in size and PDI. At pH 7 supramolecular structures were obtained at microscale – confirmed by optical microscopy – displaying also a high PDI (i.e. > 0.4). The minimum size and PDI (61 ± 2.3 nm and 0.14 ± 0.03, respectively) were produced at pH 11 for a heating treatment of 75 °C for 15 min, thus suggesting that these conditions could be considered as critical for supramolecular structure formation. Its size and morphology were confirmed by TEM showing a well-defined spherical form. Structures at these conditions showed to be stable at least for 30 or 90 days, when stored at 25 or 4 °C, respectively. Hence, α-La–Lys supramolecular structures showed properties that indicate that they are a promising delivery system for food and pharmaceutical applications.

Flexible thin-film lithium battery

Tese de Doutoramento Programa Doutoral em Engenharia Electrónica e Computadores.