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  • PolyThymine cryogels as a novel approach to the purification of mRNA encoding anti- ADAM17 antibodies
    Publication . Rodrigues, Bruno Daniel Lopes; Tomaz, Cândida Ascensão Teixeira; Sousa, Fani Pereira de
    Currently, the metalloprotease "A disintegrin and metalloproteinase 17" (ADAM17) has been gaining prominence due to its important action in signalling pathways where the soluble interleukin receptor - 6 (sIL-6R), the epidermal growth factor receptor (EGF-R) and the tumour necrosis factor alfa receptor (TNF-aR) participate, establishing ADAM17 as an attractive target in cancer therapy and infectious diseases. Thus, due to the high specificity of antibodies to recognize a particular epitope, it was possible to develop an anti-ADAM17 antibody, named MEDI3622, which has been shown to have the ability to specifically block the activity of ADAM17. Furthermore, the messenger Ribonucleic Acid (mRNA) has been showing great potential and applicability in several areas, namely in passive immunization, by the in vivo expression of antibodies, making mRNA a highly attractive method. Thus, in the present work, an mRNA sequence was prepared with the coding sequence of MEDI3622 for expression of this anti-ADAM17 antibody. Therefore, given the great therapeutic potential of mRNA, the high demand from the biopharmaceutical industry and the need to obtain it in a stable and pure form, it is important to optimize the production and purification processes of this biomolecule. The cryogels have emerged as a new alternative to conventional particulate chromatographic media for the purification of high molecular weight molecules, and their potential has already been demonstrated in the purification of the supercoiled isoform of plasmidic deoxyribonucleic acid (pDNA). Cryogels also have a supermacroporous structure that allows for facilitated diffusion, high flow rates, and high capacity. Currently, the strategy of choice for mRNA purification is affinity chromatography, using nucleotides as ligands, whose adenine and thymine base pairing specificity promotes highly selective and specific interactions. Thus, the aim of this work was to synthesize thymine-functionalized cryogels to purify the mRNA sequence produced by in vitro transcription, that encodes the anti-ADAM17 antibody MEDI3622. To this end, the ligand 1-(2-propenyl) thymine (ProT) was synthesized and characterized by nuclear magnetic resonance (NMR), the cryogels were subsequently prepared based on the monomer 2-hydroxyethyl methacrylate (HEMA), by copolymerization of the monomers HEMA and ProT, with different mass ratios of ProT and subjected to cryogenic treatment. The cryogels were characterized by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDXA), and rehydration capacity tests. Subsequently, to evaluate the behaviour of poly-thymine cryogels, purification tests were first performed under different conditions, varying the ionic strength, the type of salt (sodium chloride (NaCl) or ammonium sulphate ((NH4)2SO4)) and the pH in the mobile phase, taking advantage of the affinity between ProT ligands and adenines present in low molecular weight RNA (LMW RNA) used as a model sample, aiming to identify the best conditions under which binding and elution occurred. In the second phase, having already optimized the binding and elution conditions, the ability of cryogels to purify the mRNA was tested. In this phase, the NaCl concentration in the elution buffers was varied in order to achieve the separation between mRNA and pDNA used for its production. As the main results, the synthesis of the ligand 1-(2-propenyl) thymine was successfully achieved. Furthermore, in this task, it was also possible to isolate the unreacted thymine and a by-product of the reaction in which di-alkylation of thymine occurred (1,3-bis(2- propenyl) thymine). The fact that it was possible to isolate these two compounds makes the present work fit in with the principles of circular economy, reducing the costs of the synthesis of the ligand by reusing the isolated thymine, while eliminating the waste of by-products by the possibility of using di-alkylated thymine as a cross-linking agent instead of N, N'-Methylenebisacrylamide. However, traces of a second by-product (3-(2- propenyl) thymine) were found, and it was not possible to fully isolate the ligand of interest due to its structural similarity to this by-product, thus the isolation step needs to be optimized in the future. Regarding the characterization of the cryogels, the functionalized ones did not show significant changes in rehydration capacity compared to the non-functionalized one. Differences in morphology were visualized, which may denote the presence of the ligand in the polymeric matrix of the cryogels. Also, they may have resulted from the conditions used in the synthesis process of cryogels. Furthermore, through the EDXA, it was possible to confirm that there was functionalization of the cryogels, however, this method is not the most indicated for this type of confirmation, other methods, such as elemental analysis and solid-state NMR, can be used in the future to corroborate the results at the functionalization level. The results of the chromatographic assays demonstrate that the ProT functionalized cryogels were able to bind and elute mRNA and pDNA. Moreover, they showed potential to separate pDNA and mRNA, demonstrating a higher affinity for mRNA. The control cryogel (with no ProT ligand incorporated) also demonstrated the ability to bind and elute both mRNA and pDNA, however, it showed no aptitude to separate these two biomolecules. Thus, ProT functionalized cryogels appear to be a novel approach to overcoming challenges in mRNA purification.
    2023-03-07Master thesis Open access Show more
  • As Relações Transatlânticas: O Papel da Missão Diplomática de Portugal em Ottawa
    Publication . Almeida, Lyandra Victoria França de; Terrenas, João David Malagueta
    O presente relatório é resultado da reflexão feita a partir do estágio curricular realizado na Embaixada de Portugal em Ottawa. As missões diplomáticas desempenham um papel fundamental nas relações bilaterais entre países, sendo responsáveis por representar o país de origem no país de destino, promovendo os interesses nacionais e estabelecendo e mantendo relações de cooperação. Neste sentido, torna-se necessário compreender a atuação da Embaixada de Portugal em Ottawa enquanto principal missão diplomática em território canadense. Tomando como ponto de partida o estágio curricular realizado nesta instituição e adotando uma abordagem qualitativa, operacionalizada através da observação participante e da análise documental, este relatório tem como objetivo explorar as relações Portugal-Canadá, com ênfase no papel desempenhado pelo corpo diplomático português no desenvolvimento das relações bilaterais e na promoção dos interesses nacionais e de sua comunidade no estrangeiro. Para cumprir com os objetivos propostos, o relatório apresenta um enquadramento conceitual da diplomacia econômica, comercial e cultural, consideradas as vertentes mais relevantes para este estudo, bem como uma breve análise acerca das especificidades das missões diplomáticas e da evolução da diplomacia portuguesa. Seguindo a natureza conceitual e contextual deste relatório, é explorada a definição e a presença da diáspora dentre as diretrizes da política externa de Portugal, e a forma como constituiu-se a comunidade portuguesa em território canadense ao longo dos anos. Por fim, é apresentada a Embaixada, objeto principal deste estudo, suas respectivas competências em cada uma das vertentes da diplomacia aqui abordadas, e como são executadas as suas atividades em conjunto com os principais acordos e agências representativas do país.
    2023-03-07Master thesis Open access Show more
  • Programação Visual em Desenvolvimento e Design de Jogos Digitais
    Publication . Ferreira, Lucas de Azevedo Lopes; Silva, Bruno Miguel Correia da
    Esta dissertação surge da necessidade de procurar um modo de substituir as várias formas de programação tradicionais para um método alternativo, direcionado especialmente a pessoas que recentemente ingressaram na área de desenvolvimento de jogos e/ou para artistas que, apenas desejam criar programas e jogos usando objetos de 2D e 3D para dar vida e controlo às suas criações. Neste sentido, esta tese começa por apresentar uma textualização sobre o tema, seguindo por uma parte prática, onde é feita a comparação entre duas linguagens de programação visual. Nesta segunda parte, vão ser apresentados os vários passos que tomei para desenvolver um simples jogo digital completamente funcional em dois programas diferentes, mas mantendo a mesma lógica de código (ou o mais próximo possível) e as mesmas características técnicas. O principal objetivo desta tese é apresentar um estudo detalhado sobre a programação visual, como uma introdução a qualquer um que tenha interesse nesta área para desenvolver um jogo simples, sem a necessidade de aprender e/ou usar programação tradicional. Com base nas informações aqui partilhadas, o leitor poderá expandir os seus conhecimentos e ser capaz de construir vários tipos de sistemas que lhe podem ser úteis durante a sua jornada profissional.
    2023-03-07Master thesis Open access Show more