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- p53 as the Focus of Gene Therapy: Past, Present and FuturePublication . Valente, Joana; Queiroz, João; Sousa, FaniSeveral gene deviations can be responsible for triggering oncogenic processes. However, mutations in tumour suppressor genes are usually more associated to malignant diseases, with p53 being one of the most affected and studied element. p53 is implicated in a number of known cellular functions, including DNA damage repair, cell cycle arrest in G1/S and G2/M and apoptosis, being an interesting target for cancer treatment.
- DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatographyPublication . Valente, Joana; Sousa, A.; Queiroz, João; Sousa, FaniP53 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 chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. 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 endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.
- Microencapsulated chitosan–dextran sulfate nanoparticles for controled delivery of bioactive molecules and cells in bone regenerationPublication . Valente, Joana; Gaspar, Vítor Manuel Abreu; Antunes, Bernardo Paiva; Coutinho, Paula Isabel Teixeira Gonçalves; Correia, Ilídio Joaquim SobreiraThis research work aimed to synthesize and characterize a novel dual delivery system comprised of BSA-loaded in chitosan–dextran sulfate nanoparticulated carriers and mesenchymal stem cells that are encapsulated into alginate microparticles. The physicochemical and biological characteristics of this novel system, such as, morphology, release, swelling, and cytotoxicity were thoroughly characterized. The results obtained from confocal microscopy demonstrate that chitosan–dextran sulfate nanoparticles and cells are fully encapsulated within alginate microparticles, and spatially dispersed in the microparticle matrix. Moreover, scanning electron microscopy images revealed that these micro-sized carriers possess a rough surface, an important parameter that also promoted proper cell migration and adhesion. Notably, the incorporation of BSA in this duplex nano-micro delivery system extended its release profile throughout time, in comparison with microparticles alone, whilst not eliciting any cell damage. Taken together, these findings suggest that this dual carrier is a versatile delivery system with potential for a spatiotemporally controlled release of bioactive molecules and cells.
- Poly(ester amide)s based on (L)-lactic acid oligomers and α-amino acids: influence of the α-amino acid side chain in the poly(ester amide)s propertiesPublication . Fonseca, Ana; Coelho, Jorge; Valente, Joana; Correia, Tiago R.; Correia, Ilídio Joaquim Sobreira; Gil, Maria; Simões, PedroNovel biodegradable and low cytotoxic poly(ester amide)s (PEAs) based on α-amino acids and (L)-lactic acid (L-LA) oligomers were successfully synthesized by interfacial polymerization. The chemical structure of the new polymers was confirmed by spectroscopic analyses. Further characterization suggests that the α-amino acid plays a critical role on the final properties of the PEA. L-phenylalanine provides PEAs with higher glass transition temperature, whereas glycine enhances the crystallinity. The hydrolytic degradation in PBS (pH = 7.4) at 37 °C also depends on the α-amino acid, being faster for glycine-based PEAs. The cytotoxic profiles using fibroblast human cells indicate that the PEAs did not elicit an acute cytotoxic effect. The strategy presented in this work opens the possibility of synthesizing biodegradable PEAs with low citotoxicity by an easy and fast method. It is worth to mention also that the properties of these materials can be fine-tuned only by changing the α-amino acid.
- Alginate based scaffolds for bone tissue engineeringPublication . Valente, Joana; Valente, Tiago António Martins; Alves, Patrícia; Ferreira, Paula; Silva, Abílio; Correia, I.J.The design and production of scaffolds for bone tissue regeneration is yet unable to completely reproduce the native bone properties. In the present study new alginate microparticle and microfiber aggregated scaffolds were produced to be applied in this area of regenerative medicine. The scaffolds' mechanical properties were characterized by thermo mechanical assays. Their morphological characteristics were evaluated by isothermal nitrogen adsorption and scanning electron microscopy. The density of both types of scaffolds was determined by helium pycnometry and mercury intrusion porosimetry. Furthermore, scaffolds' cytotoxic profiles were evaluated in vitro by seeding human osteoblast cells in their presence. The results obtained showed that scaffolds have good mechanical and morphological properties compatible with their application as bone substitutes. Moreover, scaffold's biocompatibility was confirmed by the observation of cell adhesion and proliferation after 5 days of being seeded in their presence and by non-radioactive assays.