Browsing by Author "Sousa, A."
Now showing 1 - 10 of 17
Results Per Page
Sort Options
- Biomechanical performance of hybrid electrospun structures for skin regenerationPublication . Dias, J.R.; Baptista-Silva, S.; Sousa, A.; Oliveira, A. L.; Bártolo, P. J.; Granja, P. L.Wound dressings made by electrospun nanofibers have been demonstrating great potential to regenerate skin tissue as compared to the conventional membrane products available in the market. Until today most of the developed dressings have only demonstrated the capability to regenerate the dermis or epidermis. In this study we propose new hybrid electrospun meshes combining polycaprolactone and gelatin. Several approaches, multilayer, coating and blend were stablished to investigate the most appropriate hybrid structure with potential to promote skin regeneration in its full thickness. The structures were evaluated in terms of physico-chemical properties (porosity, water vapor permeability, contact angle and swelling degree) and according to its mechanical and biological performance. Multilayer and blend structures demonstrated to fit most of native skin requirements. However, looking to all the performed characterization we considered multilayer as the most promising hybrid structures, due its high porosity which contributed to an ideal water vapor permeability rate and good mechanical and biological properties. Based on this multilayer structure is a promisor wound dressing.
- Composite Central Face Design—An Approach to Achieve Efficient Alginate MicrocarriersPublication . Valente, J.F.A.; Dias, J.R.; Sousa, A.; Alves, N.Microparticulated drug delivery systems have been used as promising encapsulation systems for protecting drugs for in vitro and in vivo applications, enhancing its stability, providing an increased surface to volume ratio, reducing adverse effects, and hence an improvement in bioavailability. Among the studied microparticles, there is a rising interest in the research of alginate microparticles for pharmaceutical and biomedical fields confirming its potential to be used as an effective matrix for drug and cell delivery. Moreover, calcium alginate has been one of the most extensively forming microparticles in the presence of divalent cations providing prolonged drug release and suitable mucoadhesive properties. Regarding the above mentioned, in this research work, we intended to produce Ca-alginate micro-vehicles through electrospraying, presenting high encapsulation efficiency (EE%), reduced protein release across the time, reduced swelling effect, and high sphericity coefficient. To quickly achieve these characteristics and to perform an optimal combination among the percentage of alginate and CaCl2, design of Experiments was applied. The obtained model presented to be statistically significant (p-value < 0.05), with a coefficient of determination of 0.9207, 0.9197, 0.9499, and 0.9637 for each output (EE%, release, swelling, and sphericity, respectively). Moreover, the optimal point (4% of alginate and 6.6% of CaCl2) was successfully validated.
- DoE to improve supercoiled p53-pDNA purification by O-phospho-L-tyrosine chromatographyPublication . Valente, J.F.A.; Sousa, A.; Queiroz, J.A.; Sousa, F.P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O phospho-L-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chro matographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-en coding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was ap plied. The dynamic binding capacity of the O-phospho-L-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/ mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of en dotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies
- Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and BioactivityPublication . Azevedo, G.M.; Valente, J.F.A.; Sousa, A.; Pedro, A.Q.; Pereira, P.; Sousa, F.; Queiroz, J.A.The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.
- Effect of Plasmid DNA Size on Chitosan or Polyethyleneimine Polyplexes FormulationPublication . Valente, J. F. A.; Pereira, P.; Sousa, A.; Queiroz, J. A.; Sousa, F.Gene therapy could be simply defined as a strategy for the introduction of a functional copy of desired genes in patients, to correct some specific mutation and potentially treat the respective disorder. However, this straightforward definition hides very complex processes related to the design and preparation of the therapeutic genes, as well as the development of suitable gene delivery systems. Within non-viral vectors, polymeric nanocarriers have offered an ideal platform to be applied as gene delivery systems. Concerning this, the main goal of the study was to do a systematic evaluation on the formulation of pDNA delivery systems based on the complexation of different sized plasmids with chitosan (CH) or polyethyleneimine (PEI) polymers to search for the best option regarding encapsulation efficiency, surface charge, size, and delivery ability. The cytotoxicity and the transfection efficiency of these systems were accessed and, for the best p53 encoding pDNA nanosystems, the ability to promote protein expression was also evaluated. Overall, it was showed that CH polyplexes are more efficient on transfection when compared with the PEI polyplexes, resulting in higher P53 protein expression. Cells transfected with CH/p53-pDNA polyplexes presented an increase of around 54.2% on P53 expression, while the transfection with the PEI/p53-pDNA polyplexes resulted in a 32% increase.
- Employability and Higher Education: The case study of ESTM master's degreePublication . Viana, A.; Pais, S.; Sousa, A.; Schön, M.The labor market in this globalized world, based on new technologies, is constantly changing. In an increasingly demanding and dynamic world of human resources competences, young people have to focus on their training in order to enter in the labor market better prepared and thus contribute to the success of the entity that receives them and for their own professional and personal fulfillment. The choice of their academic journey is crucial to ensure the achievement of these competences and make that decision is not easy, with a system of higher education such as the Portuguese, where there are so many graduation courses and two systems of teaching, University and Polytechnic. In fact, with the changes introduced in higher education by the so-called Bologna Process, most undergraduate’s courses saw the number of years reduced from five to three years, while others opted for integrated master's degrees. In the case of the School of Tourism and Maritime Technology, the option was to offer three-year degree courses and two-year master's degrees. This study intends to understand where are the students who have bet on their training at the master's level and finish their master's degree at the School of Tourism and Maritime Technology (ESTM) of the Polytechnic of Leiria, if they are working in the area and if the employers recognize these qualifications.
- In situ crosslinked electrospun gelatin nanofibers for skin regenerationPublication . Dias, J. R.; Baptista-Silva, S.; Oliveira, C. M. T. de; Sousa, A.; Oliveira, A. L.; Bártolo, P. J.; Granja, P. L.Due to its intrinsic similarity to the extracellular matrix, gelatin electrospun nanofibrous meshes are promising scaffold structures for wound dressings and tissue engineering applications. However, gelatin is water soluble and presents poor mechanical properties, which generally constitute relevant limitations to its applicability. In this work, gelatin was in situ crosslinked with 1,4-butanediol diglycidyl ether (BDDGE) at different concentrations (2, 4 and 6 wt%) and incubation time-points (24, 48 and 72 h) at 37 °C. The physico-chemical and biological properties of BDDGE-crosslinked electrospun gelatin meshes were investigated. Results show that by changing the BDDGE concentration it is possible to produce nanofibers crosslinked in situ with well-defined morphology and modulate fiber size and mechanical properties. Crosslinked gelatin meshes show no toxicity towards fibroblasts, stimulating their adhesion, proliferation and synthesis of new extracellular matrix, thereby indicating the potential of this strategy for skin tissue engineering.
- Marine recreational fishing in Portugal: current knowledge, challenges, and future perspectivesPublication . Diogo, Hugo; Veiga, Pedro; Pita, Cristina; Sousa, A.; Lima, David; Pereira, João Gil; Gonçalves, Jorge M.S.; Erzini, Karim; Rangel, MafaldaMarine recreational fishing (MRF) in Portugal is a traditional leisure activity with considerable importance for coastal populations. In the absence of available information from the national data collection framework, this article aims to review the existing information on MRF across the country. MRF was an open access fishery until recently, but with rising evidence of overexploitation of coastal resources, a precautionary approach was imposed topdown, without consulting recreational fishers. In Portugal, the MRF participation rate is comparable to the European average (2%). The most important fishing mode (according to the official issued licenses) is shore angling. Mainland Portugal marine recreational catches (0.8% of total commercial landings) are likely underestimated, while the estimate for the Azores (6% of total commercial landings) is probably closer to the national reality. The Portuguese MRF sector faces several challenges, including: (1) the need for a definition of a national data collection framework; (2) the inclusion of MRF harvest estimates in stock assessments for key captured species; (3) management approaches which also take into account the ecosystem approach to fisheries and any potential effects of climate change; (4) additional research on post-release mortalities for the most important MRF species; and (5) a stronger involvement of all stakeholders in the decision-making process of MRF. The latter would be critical to improve the adequacy of regulations to the MRF reality, mitigate conflicts with other sectors (e.g., commercial fishing), and potentially increase fishers compliance.
- Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous supportPublication . Valente, J.F.A.; Sousa, A.; Azevedo, G.A.; Queiroz, J.A.; Sousa, F.p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alter- native to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA pu- rification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macrop- orous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic fea- sibility in case of scale-up.
- Tecnologias de Informação e Comunicação aplicadas em museusPublication . Mateus, Marta; Sousa, A.Information and Communication Technologies (ICT) are present in all our daily lives, but their benefit in tourism is evident. This type of technology accompanies the tourist from the choice of the destination until after their experience. Information and Communication Technologies applied to tourism, and more specifically to museums, are seen as an interactive and educational capital that creates a connection between the object and the tourist, making their experience more immersive and authentic. The Money Museum and the Alviela Science Center are two examples that use this type of technology to transmit knowledge to visitors. In times of economic crisis and the closing of museological spaces due to the Covid-19 pandemic, museums had to adapt to this situation, allowing interaction with the public without leaving home.