Separation of bioactive peptides by electrodialysis with ultrafiltration membranes: membrane characteristics, ex-situ and in-situ digestion and their impact on peptide migration

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Improving silicon probe performance through layer-by-layer coating

Fully comprehending brain function, as the scale of neural networks, will only be possi-ble with the development of tools by micro and nanofabrication. Regarding specifically silicon microelectrodes arrays, a significant improvement in long-term performance of these implants is essential. This project aims to create a silicon microelectrode coating that provides high-quality electrical recordings, while limiting the inflammatory response of chronic implants. To this purpose, a combined chitosan and gold nanoparticles coating was produced allied with electrodes modification by electrodeposition with PEDOT/PSS in order to reduce the im-pedance at 1kHz. Using a dip-coating mechanism, the silicon probe was coated and then charac-terized both morphologically and electrochemically, with focus on the stability of post-surgery performance in anesthetized rodents. Since not only the inflammatory response analysis is vital, the electrodes recording degradation over time was also studied. The produced film presented a thickness of approximately 50 μm that led to an increase of impedance of less than 20 kΩ in average. On a 3 week chronic implant, the impedance in-crease on the coated probe was of 641 kΩ, compared with 2.4 MΩ obtained for the uncoated probe. The inflammatory response was also significantly reduced due to the biocompatible film as proved by histological tests.

Spray coating of oxide and chalcogenide semiconductor layers for TFT application

This work documents the deposition and optimization of semiconductor thin films using chemical spray coating technique (CSC) for application on thin-film transistors (TFTs), with a low-cost, simple method. CSC setup was implemented and explored for industrial application, within Holst Centre, an R&D center in the Netherlands. As zinc oxide had already been studied within the organization, it was used as a standard material in the initial experiments, obtaining typical mobility values of 0.14 cm2/(V.s) for unpatterned TFTs. Then, oxide X layer characteristics were compared for films deposited with CSC at 40°C and spin-coating. The mobility of the spin-coated TFTs was 103 cm2/(V.s) higher, presumably due to the lack of uniformity of spray-coated film at such low temperatures. Lastly, tin sulfide, a relatively unexplored material, was deposited by CSC in order to obtain functional TFTs and explore the device’s potential for working as a phototransistor. Despite the low mobilities of the devices, a sensitive photodetector was made, showing drain current variation of nearly one order of magnitude under yellow light. CSC technique’s simplicity and versatility was confirmed, as three different semiconductors were successfully implemented into functional devices.

Historical reconstructions of raw materials based on a blue smalt coating applied to a seventeenth-century altarpiece

Based on samples cross-sections from the Main Altarpiece of the Coimbra Old Cathedral, where a blue coating performed in 1685 is observed (that was partly covered with a Prussian blue-containing overpaint), the raw materials present in this coating were reproduced and studied. Blue areas were painted with smalt in oil, according to the contract signed by Manoel da Costa Pereira in 1684 and the analysis by Le Gac in 2009. Based on these, three batches of cobalt-based glasses (S1, S2 and S3) were heated and melted in alumina crucibles in the kiln. S1 contained 6.03 % of cobalt oxide, S2 contained 2.10 %, with the addition of 1.49 % of magnesium oxide, and S3 contained 6.82 % of cobalt oxide, with the addition of 4.63% of antimony trioxide. These batches were ground mechanically with water and manually with different vehicles stated in recipes. The results were studied by means of OM, SEM-EDS, X-Ray CT, Colorimetry and Vickers HT. Different binders were also produced and analyzed by means of μ-FTIR, in order to perform their characterization and obtain Standard Spectra. Since anhydrite was identified in the ground layers, gypsum from Óbidos was also characterized by XRD. The main goal of this thesis was to study all the raw materials present in the 1685-blue coating, in order to allow the historically accurate reconstruction of the layers build-up in the next future.

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Bioactive nanocomposite spheres with proved shape memory capability in bone defects

Bioactive glass nanoparticles (BGNPs) promote an apatite surface layer in physiologic conditions that lead to a good interfacial bonding with bone.1 A strategy to induce bioactivity in non-bioactive polymeric biomaterials is to incorporate BGNPs in the polymer matrix. This combination creates a nanocomposite material with increased osteoconductive properties. Chitosan (CHT) is a polymer obtained by deacetylation of chitin and is biodegradable, non-toxic and biocompatible. The combination of CHT and the BGNPs aims at designing biocompatible spheres promoting the formation of a calcium phosphate layer at the nanocomposite surface, thus enhancing the osteoconductivity behaviour of the biomaterial. Shape memory polymers (SMP) are stimuli-responsive materials that offer mechanical and geometrical action triggered by an external stimulus.2 They can be deformed and fixed into a temporary shape which remains stable unless exposed to a proper stimulus that triggers recovery of their original shape. This advanced functionality makes such SMPs suitable to be implanted using minimally invasive surgery procedures. Regarding that, the inclusion of therapeutic molecules becomes attractive.  We propose the synthesis of shape memory bioactive nanocomposite spheres with drug release capability.3   1.  L. L. Hench, Am. Ceram. Soc. Bull., 1993, 72, 93-98. 2.  A. Lendlein and S. Kelch, Angew Chem Int Edit, 2002, 41, 2034-2057. 3.  Ã . J. Leite, S. G. Caridade and J. F. Mano, Journal of Non-Crystalline Solids (in Press)

Performance of a fly ash geopolymeric mortar for coating of ordinary portland cement concrete exposed to harsh chemical environments

Premature degradation of ordinary Portland cement (OPC) concrete infrastructures is a current and serious problem with overwhelming costs amounting to several trillion dollars. The use of concrete surface treatments with waterproofing materials to prevent the access of aggressive substances is an important way of enhancing concrete durability. The most common surface treatments use polymeric resins based on epoxy, silicone (siloxane), acrylics, polyurethanes or polymethacrylate. However, epoxy resins have low resistance to ultraviolet radiation while polyurethanes are sensitive to high alkalinity environments. Geopolymers constitute a group of materials with high resistance to chemical attack that could also be used for coating of concrete infrastructures exposed to harsh chemical environments. This article presents results of an experimental investigation on the resistance to chemical attack (by sulfuric and nitric acid) of several materials: OPC concrete, high performance concrete (HPC), epoxy resin, acrylic painting and a fly ash based geopolymeric mortar. Three types of acids, each with high concentrations of 10%, 20% and 30%, were used to simulate long term degradation by chemical attack. The results show that the epoxy resin had the best resistance to chemical attack, irrespective of the acid type and acid concentration.

Effect of variables on the thickness of an edible coating applied on frozen fish establishment of the concept of safe dipping time

Glazing is a technique used to retard fish deterioration during storage. This work focuses on the study of distinct variables (fish temperature, coating temperature, dipping time) that affect the thickness of edible coatings (water glazing and 1.5% chitosan) applied on frozen fish. Samples of frozen Atlantic salmon (Salmo salar) at -15, -20, and -25 °C were either glazed with water at 0.5, 1.5 or 2.5 °C or coated with 1.5% chitosan solution at 2.5, 5 or 8 °C, by dipping during 10 to 60 s. For both water and chitosan coatings, lowering the salmon and coating solution temperatures resulted in an increase of coating thickness. At the same conditions, higher thickness values were obtained when using chitosan (max. thickness of 1.41±0.05 mm) compared to water (max. thickness of 0.84±0.03 mm). Freezing temperature and crystallization heat were found to be lower for 1.5% chitosan solution than for water, thus favoring phase change. Salmon temperature profiles allowed determining, for different dipping conditions, whether the salmon temperature was within food safety standards to prevent the growth of pathogenic microorganisms. The concept of safe dipping time is proposed to define how long a frozen product can be dipped into a solution without the temperature raising to a point where it can constitute a hazard.

Bioactive dihydroxyfuranonaphthoquinones from the bark of Tabebuia incana A.H. Gentry (Bignoniaceae) and HPLC analysis of commercial pau d'arco and certified T.incana bark infusions

Tabebuia incana A.H. Gentry (Bignoniaceae) is a tree from the Brazilian Amazon having medicinal uses and is one several Tabebuia spp. known as pau d'arco or palo de arco in this region. Fractionation of the bark ethanolic extract afforded a mixture of 5 and 8-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-diones (1 and 2, respectively) identified on the basis of nuclear magnetic resonance (NMR), infrared (IR) and mass (MS) spectra, whose in vitro antimalarial and antitumor activity have been shown previously. This is the first study on T. incana bark, and 2 are described in this species for the first time. Also, high performance liquid chromatography (HPLC) analysis of T. incana bark tea revealed the presence of the 1 + 2 mixture peak corresponding to a concentration in the range 10-6-10-5 M. The chromatograms of teas prepared from commercial pau d' arco and T. incana bark were also studied and the presence of the 1 + 2 peak has potential for quality control of commercial plant materials.

Postharvest shelf life extension of blueberries using a chitosan-based edible coating containing aloe vera juice

Poster

Hot-dip galvanized steel dip-coatedwith ureasilicate hybrid in simulated concrete pore solution: Assessment of coating morphology and corrosion protection efficiency

The barrier effect and the performance of an organic–inorganic hybrid (OIH) sol–gel coating are highlydependent on the coating deposition method as well as processing conditions. In this work, studies onthe influence of experimental parameters using the dip coating method were performed. Factors suchas residence time (Rt), a curing step between each dip step and the number of layers of sol–gel OIHfilms deposited on HDGS to prevent corrosion in highly alkaline environments were studied. These OIHcoatings were obtained using a functionalized siloxane, 3-isociantepropyltriethoxysilane that reactedwith a diamino-functionalized oligopolymer (Jeffamine®D-230). The barrier efficiency of OIH coatings insimulated concrete pore solutions (SCPS) was assessed in the first moments of contact, by electrochemicalimpedance spectroscopy and potentiodynamic methods. The durability and stability of the OIH coatings inSCPS was monitored during eight days by macrocell current density. The morphological characterizationof the surface was performed by scanning electronic microscopy before and after exposure to SCPS.Glow discharge optical emission spectroscopy was used to obtain quantitative composition profiles toinvestigate the thickness of the OIH coatings as a function of the number of layers deposited and theinfluence of the Rt in the coating thickness.

Chemical characterization and bioactive properties of Prunus avium L.: The widely studied fruits and the unexplored stems

The aim of this study was to characterize sweet cherry regarding nutritional composition of the fruits, and individual phytochemicals and bioactive properties of fruits and stems. The chromatographic profiles in sugars, organic acids, fatty acids, tocopherols and phenolic compounds were established. All the preparations (extracts, infusions and decoctions) obtained using stems revealed higher antioxidant potential than the fruits extract, which is certainly related with its higher phenolic compounds (phenolic acids and flavonoids) concentration. The fruits extract was the only one showing antitumor potential, revealing selectivity against HCT-15 (colon carcinoma) (GI50~74 μg/mL). This could be related with anthocyanins that were only found in fruits and not in stems. None of the preparations have shown hepatotoxicity against normal primary cells. Overall, this study reports innovative results regarding chemical and bioactive properties of sweet cherry stems, and confirmed the nutritional and antioxidant characteristics of their fruits.

Nutritional value, bioactive compounds and antioxidant properties of three edible mushrooms from Poland

Mushrooms contain a multitude of biomolecules with nutritional and/or biological activity. Among the bioactive molecules, phenolic compounds and tocopherols are the most responsible for their antioxidant activity. In the present work, Boletus edulis, Lentinus edodes and Xerocomus badius, three edible mushroom species originated from Poland, were analyzed for their chemical composition and antioxidant activity. Carbohydrates were the most abundant macronutrients, followed by proteins and ash. Fructose, mannitol and trehalose were the prevalent sugars, but glucose was only found in B. edulis. Polyunsaturated fatty acids predominated over mono and saturated fatty acids. Palmitic, oleic and linoleic acids were abundant in the three samples. α- and β- Tocopherols were quantified in all the samples, but γ-tocopherol was only identified in X. badius. Oxalic and fumaric acids were quantified in the three samples; quinic acid was only present in L. edodes, and malic and citric acids were only found in X. badius. p-Hydroxybenzoic, protocatechuic and cinnamic acids were quantified in all the species, while p-coumaric acid was only found in B. edulis. This species and X. badius revealed the highest antioxidant properties, being B. edulis more effective in radicals scavenging activity and reducing power, and X. badius in lipid peroxidation inhibition, which is related with the highest amounts in phenolic compounds and tocopherols, respectively.

Biomimetic strategies to engineer mineralised human tissues

In the last few years, many reports have been describing promising biocompatible and biodegradable materials that can mimic in a certain extent the multidimensional hierarchical structure of bone, while are also capable of releasing bioactive agents or drugs in a controlled manner. Despite these great advances, new developments in the design and fabrication technologies are required to address the need to engineer suitable biomimetic materials in order tune cells functions, i.e. enhance cell-biomaterial interactions, and promote cell adhesion, proliferation, and differentiation ability. Scaffolds, hydrogels, fibres and composite materials are the most commonly used as biomimetics for bone tissue engineering. Dynamic systems such as bioreactors have also been attracting great deal of attention as it allows developing a wide range of novel in vitro strategies for the homogeneous coating of scaffolds and prosthesis with ceramics, and production of biomimetic constructs, prior its implantation in the body. Herein, it is overviewed the biomimetic strategies for bone tissue engineering, recent developments and future trends. Conventional and more recent processing methodologies are also described.

Effect of chitosan-Aloe vera coating on postharvest quality of blueberry (Vaccinium corymbosum) fruit

The present study was carried out to evaluate the effect of chitosan-based edible coatings with Aloe vera extract on the postharvest blueberry fruit quality during storage at 5 °C. Firstly, A. vera fractions (pulp and liquid) were extracted from leaves and evaluated in terms of antifungal and antioxidant capacities. The choice of the most adequate chitosan and A. vera fraction concentrations to be incorporated in coating formulation was made based on the wettability of the corresponding coating solutions. Coatings with 0.5% (w/v) chitosan + 0.5% (w/v) glycerol + 0.1% (w/v) Tween 80 + 0.5% (v/v) A. vera liquid fraction presented the best characteristics to uniformly coat blueberry surface. Physico-chemical (i.e., titratable acidity, pH, weight loss) and microbiological analyses of coated blueberries (non-inoculated or artificially inoculated with Botrytis cinerea) were performed during 25 d. Microbiological growth and water loss levels were approximately reduced by 50% and 42%, respectively, in coated blueberries after 25 d compared to uncoated blueberries. After 15 d, weight loss values were 6.2% and 3.7% for uncoated and chitosanA. vera coated blueberries, respectively. Uncoated fruits presented mold contamination after 2 d of storage (2.0 ± 0.32 log CFU g1), whilst fruits with chitosan-based coatings with A. vera presented mold contamination only after 9 d of storage (1.3 ± 0.35 log CFU g1). Overall, coatings developed in this study extend blueberries shelf-life for about 5 d, demonstrating for the first time that the combination of chitosan and A. vera liquid fraction as edible coating materials has great potential in expanding the shelf-life of fruits.