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RNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.

The progress made on the use of new biopharmaceuticals has a high influence on the global health, and since the approval of humulin (recombinant insulin) by FDA, new protein-based pharmaceuticals have been introduced in the market such as the interferon alfa 2b (IFNα2b), which has been used in in the treatment of oncological diseases for more than 30 years. Nowadays, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavoring a reduction of its production costs. Currently, the downstream processing of biopharmaceuticals is dominated by chromatography and, despite being highly effective, is responsible for their current high-cost [1]. Accordingly, there is a crucial need to develop effective alternative purification platforms. In this way, aqueous two-phase systems (ATPS) emerge. Due to their high-water content, ATPS may be considered a biocompatible technique for the purification of (bio)molecules. Additionally, the use of ionic liquids (ILs) in low concentrations as adjuvants in ATPS affords an enhanced performance in what concerns to (bio)molecules selectivity. In this work it is shown the use of polymer-polymer ATPS comprising ILs as adjuvants to purify recombinant proteins. Several variables were studied in the extraction of the target proteins, namely pH, polymer molecular weight, and the effect of different families of ILs and their concentration. Overall, imidazolium-based ILs seem responsible for an improved ATPS performance towards a higher purity, and is additionally shown that electrostatic interactions between the negatively charged protein and ILs cations may account for this behavior. published

The development of new drugs has a high impact on the global health, and since the approval of humulin (recombinant insulin) by FDA, several protein-based biopharmaceuticals have been introduced in the market. These products are usually attained through biotechnological processes using engineered biological sources and have led to remarkable advances in the treatment of distinct human diseases, as for example the case of interferon alfa 2b (IFNα2b) which has been used in in the treatment of oncological diseases for more than 30 years [1]. Currently, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavouring a reduction of its production costs, which is intimately related to the necessity of improving their downstream processes, since this field is mainly dominated by chromatographic techniques [2] and are responsible for their current high-cost. Therefore, envisaging to develop new, cost-effective, efficient and sustainable techniques for proteins purification, polymer-polymer aqueous two-phase systems (ATPS) with ionic liquids (ILs) as adjuvants are herein proposed for the extraction and purification of IFNα2b from Escherichia coli BL21 cultures. The recombinant protein was produced in the form of inclusion bodies, followed by a dialysis step in order to solubilize the target biomolecule in a specific buffer, with the goal of maintaining the biomolecule stability. Afterwards, several polymers where studied as phase-forming components of ATPS, namely polyethylene glycol (PEG), polypropylene glycol (PPG), as well as Dextran. Furthermore, several ILs belonging to the cholinium, imidazolium, tetralkylammonium and tetralkylphosphonium families, were tested as co-adjuvants for ATPS formation in order to extract and purify of IFNα2b. published

Mestrado em Engenharia Química O processo de metalização em vácuo permite a obtenção de revestimentos semelhantes ao efeito cromado, sendo utilizado essencialmente na indústria automóvel e de decoração. Neste processo, um substrato é revestido com uma camada fina de um metal através de vaporização. O processo apresenta elevadas taxas de revestimento e é ambientalmente inofensivo, no entanto, o revestimento de peças com área elevada é limitado às dimensões da câmara de vácuo e, o de peças irregulares resulta em revestimentos deficientes e irregulares. Na presente dissertação, como alternativa à metalização em vácuo, desenvolveu-se um processo de pintura, para obtenção de um efeito semelhante, por aplicação à pistola. Neste sentido, foi concebido um sistema de pintura constituído por um fundo de poliéster, um revestimento acrílico base de alto brilho, um revestimento de alumínio com efeito cromado e por um revestimento de poliuretano de topo de dois componentes de alto brilho e transparente. Este sistema de pintura foi aplicado em amostras de MDF (painel de aglomerado de fibras de densidade média) e ABS (acrilonitrilo butadieno estireno), excluindo-se a aplicação do fundo poliéster neste último. O MDF sofreu previamente um processo de lixagem e aplicação de um fundo branco ou de poliéster transparente. Numa primeira fase, foram realizados estudos sobre a influência da cor do fundo (no caso do MDF) e do revestimento acrílico base no efeito desejado. Posteriormente, desenvolveram-se formulações com diferentes pigmentos de alumínio por forma a selecionar o que permitisse a maximização do efeito cromado e, por último, foi formulado um verniz de alto brilho de topo transparente de dois componentes para proteção do revestimento de alumínio, que não sofresse amarelecimento nem afetasse em demasia o efeito cromado, e que garantisse dureza e resistência ao risco. Por fim, algumas amostras de MDF revestidas com este esquema de pintura foram estudadas por espectroscopia de ultravioleta-visível, obtendo-se resultados a nível da refletância total e da refletância difusa. A realização deste trabalho permitiu desenvolver um sistema de pintura e um revestimento de alumínio que maximizasse o efeito cromado. O sistema de pintura final é constituído por um fundo de poliéster branco ou transparente, um revestimento acrílico negro de alto brilho, um revestimento de alumínio com o pigmento metalizado em vácuo LPG046 e um revestimento de poliuretano de topo de dois componentes de alto brilho e transparente, com valor de brilho de 157 GU, dureza suficiente e baixa resistência ao risco. Através do estudo de espectroscopia verificou-se que o revestimento de topo aumenta a refletância difusa em cerca de 18%, o que se traduz numa diminuição do efeito cromado. Vacuum metallizing process allows obtaining coatings similar to chrome effect, being mainly used in the automotive and decoration industries. In this process, a substrate is coated with a thin layer of a metal through vaporization. This process has high coating rates and is environmentally harmless, however, the coating of pieces with high area is limited to the dimensions of the vacuum chamber and, the coating of irregular pieces leads to an uneven and irregular coating. In this work, as an alternative to vacuum metalizing, it was developed a painting system to achieve a similar effect, through spray gun application. As such, it was developed a painting system that consists in a polyester background coat, a high gloss acrylic base coat, an aluminum chrome effect coating and two component high gloss and clear polyurethane top coat. The referred paint system was applied to MDF (medium density fiberboard) and ABS (acrylonitrile butadiene styrene) samples, with exclusion of the application of the polyester background coat is the latter case. MDF was previously submitted to a sanding process and coated with a white or clear polyester coating. In a first stage, the influence of the background coat (for MDF) and acrylic base coat color, on the desired effect, were studied. Afterwards, formulations with different aluminum pigments were tested in order to select the one that allowed the maximization of the chrome effect and, finally, it was formulated a high gloss two component clear top coat for protection that would not suffer yellowing nor affect, in a great extent, the chrome effect and ensure hardness and scratch resistance. Finally, some MDF samples coated with this paint system were studied by ultraviolet-visible spectroscopy, aiming to obtain results of total reflectance and diffuse reflectance. This work allowed us to develop a paint system and an aluminum coating that would maximize chrome effect. The final paint system is constituted by a white or clear polyester background coat, a high gloss acrylic black coating, an aluminum coating with the LPG046 vacuum metallized pigment and a high gloss two component clear top coat, with a gloss value of 157 GU, sufficient hardness and low scratch resistance. Based on the spectroscopy study it was found that the top coat increases the diffuse reflectance by about 18%, which leads to a reduction of the chrome effect.

Exame público realizado em 16 Março 2015. Relatório de estágio realizado no âmbito do Mestrado em Psicologia Clínica.

The relevance of RNA in many biological functions has been recognized, broadening the scope of RNA research activities, from basic to applied sciences, also aiming the translation to clinical fields. The preparation and purification of RNA is a critical step for further application, since the quality of the template is crucial to ensure reproducibility and biological relevance. Therefore, the establishment of new tools that allows the isolation of pure RNA with high quality is of particular importance. New chromatographic strategies for RNA purification were considered, exploiting affinity interactions between amino acids and nucleic acids. In the present study, a single arginine-affinity chromatography step was employed for the purification of RNA from a total eukaryotic nucleic acid extract, thus eliminating several steps compared with current RNA isolation procedures. The application of this process resulted in a high RNA recovery yield of 96 ± 17% and the quality control analysis revealed a high integrity (28S:18S ratio = 1.96) in RNA preparations as well as a good purity, demonstrated by the scarce detection of proteins and the reduction on genomic DNA contamination to residual concentrations. Furthermore, the performance of the new RNA isolation method was tested regarding the applicability of the isolated RNA in modern molecular biology techniques. Hence, this new affinity approach will simplify the isolation and purification of RNA, which can bring great improvements in biomedical investigation.