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From production to purification: Towards an integrative process for recombinant pre-miRNA-29b as biopharmaceutical
Publication . Carapito, Ana Rita Mugeiro; Sousa, Fani Pereira de; Martins, Mara Guadalupe Freire; Sponchioni, Mattia
Recent advances in RNA research have greatly demonstrated the potential of RNA-based therapies, offering innovative ways to target a variety of diseases with enhanced specificity. Unlike traditional small-molecule drugs, RNA therapeutics like small-interfering RNA and microRNA (miRNA) can precisely regulate gene expression and target specific biological pathways. For instance, recent studies demonstrate that miRNA-29 regulates some pathological routes associated with Alzheimer's disease (AD), a neurodegenerative disorder affecting millions of people around the world. MiRNA-29 plays a crucial role in processes like amyloid-β peptides (Aβ) formation, which contributes to memory loss and neuronal cell damage. Low levels of miRNA-29 are linked to increased production and activity of the enzyme β-secretase (BACE1), which leads to higher production of Aβ and, consequently, β-plaque formation. Given its important functions, restoring or increasing miRNA-29 levels in AD patients can be a promising strategy for AD treatment. Current research is investigating the use of recombinant miRNA-29b precursor (pre-miRNA-29b) to silence BACE1 expression and decrease Aβ levels, aiming to develop novel approaches to slow the progression of AD.
Given this, biopharmaceuticals production and subsequent purification constitute an important process that needs to be in full accordance with criteria established by regulatory entities. Regarding RNA production, the standard technique is through chemical synthesis, however this strategy comes with some disadvantages, namely the biomolecules length that can be correctly produced and the low production levels. Recombinant production is the alternative method that is more cost-effective and applicable for large-scale production. Escherichia coli is the most widely used and studied host, nonetheless, Rhodovulum sulfidophilum (R. sulfidophilum) presents interesting characteristics considering nucleic acids production, that comprise the ability to secrete nucleic acids to the extracellular medium, without secreting RNases. The recovery of recombinantly produced nucleic acids from the extracellular medium might be a great advantage for the further downstream processing because the contaminants, such as cell debris and endotoxins, are not present, as usually are in the intracellular samples.
The downstream process is the most expensive stage of the whole bioprocess, and usually, several chromatographic steps are required to achieve the intended purity and quality. Given this, there is a high demand for specific and efficient purification strategies. Multimodal chromatography is currently under thorough research as it can lead to the same specificity for target compounds as it is observed for affinity chromatography, without using biological ligands that greatly increase the process cost. Ionic Liquids (ILs) are molten salts that can present this multimodal character when used as ligands immobilized onto the stationary phase, being recently studied for nucleic acids purification. The different moieties of the cation in an IL can allow the exploitation of different types of interactions with the target molecule. Therefore, this Doctoral Thesis explores a promising and alternative recombinant host for the production of pre-miRNA-29b, as well as its purification using newly synthesized resins, aiming for the development of a whole bioprocess.
Initially, a DNA vector was designed to produce the target pre-miRNA-29b in R. sulfidophilum. In this study, the impact of the plasmid on bacterial growth was analysed and compared to the non-transformed strain. The transformed strain has shown a global growth about 5 times lower than the non-transformed strain, but this did not impact negatively the target RNA production. An optimization of the extracellular extraction protocol was also conducted during this study, comparing a protocol with ethanol or isopropanol as precipitation agents. The results proven that the protocol using isopropanol as precipitation agent was more efficient reaching a concentration of 0.7 μg of pre-miRNA-29b per liter of medium.
After successfully developing an efficient pre-miRNA-29b production system, it became important to develop a purification strategy. For this, four different silica-based Ionic Liquids (SILs) were synthesized and evaluated on their ability for nucleic acids separation. An initial screening of binding and elution conditions with a low molecular weight RNA sample was performed by using both ionic and hydrophobic conditions, to select the more promising support for further purification assays. The support SSi[C3C3NH2Im]Cl was selected, proving to be highly efficient in separating different species of DNA (genomic and plasmidic) from RNA. Then, it became important to test this support regarding its ability for pre-miRNA-29b purification from other types of RNAs. Given that this approach is more challenging due to the high physical and chemical similarity among small RNAs, four different ILs were employed in this stage to act as competition agents aiming to enhance target selectivity. From the four tested ILs, the 1-ethylimidazolium chloride was proven to have a higher impact on the pre-miRNA-29b selectivity improvement, achieving 76% purity.
Since the aim is to develop a method suitable for a biopharmaceutical production and purification, it would be needed to achieve higher purity levels than what was verified with SSi[C3C3NH2Im]Cl. Therefore, a commercial multimodal resin, Capto Q ImpRes was chosen to analyse and compare its performance to the previous newly synthesized support. Both intra- and extracellular RNA samples were tested regarding pre-miRNA-29b purification, and a Design of Experiments (DoE) was established for each sample. Screening assays with intracellular RNA sample were made to identify the factors to be implemented in the DoE. Sodium chloride concentration and pH were defined, and it was possible to establish conditions that uncover a balance between the recovery and purity of the target. For intracellular RNA samples, the DoE effectively identified optimal purification parameters, attaining recovery rates up to 73% and purity levels around 78%. However, these two responses shown to be almost inversely proportional. Nonetheless, it was possible to achieve the optimal point with a recovery of 48.21% and purity of 51.15%. On the other hand, purifying extracellular pre-miRNA-29b presented substantial challenges, exhibiting significant inconsistencies in purity and difficulties in identifying the target molecule, probably due to the low concentration of pre-miRNA in the extracellular extract and the difficulty in detection.
To search for an alternative that could lead to an improved selectivity for the biopharmaceutical under study, a set of several oligonucleotides were designed to interact with the target. Initially, 13 oligonucleotides were designed to interact through base complementarity with different regions of the target. Each oligonucleotide was linked to a carbon chain (6 or 12 carbons) with an amino group, resulting in 26 different ligands. After data analysis, some ligands showed higher specificity for their target sites, while others demonstrated versatility in recognizing multiple sites. Based on this, 4 oligonucleotides were identified as the most promising for further experimental testing.
Overall, in this doctoral thesis, a bioprocess was developed starting with the upstream stage with recombinant production of the pre-miRNA-29b, followed by the downstream stage exploiting different approaches applicable to target purification. The work opens the route for the development of an integrated production and purification process for pre-miRNA-29b, with great potential for large-scale translation.
Design of Communication and Control for Swarms of Aquatic Surface Drones
Publication . Christensen, Anders Lyhne; Oliveira, Sancho; Postolache, Octavian; Oliveira, Maria João de; Sargento, Susana; Santana, Pedro; Nunes, Luis; Velez, Fernando J.; Sebastião, Pedro; Costa, Vasco; Duarte, Miguel; Gomes, Jorge; Rodrigues, Tiago; Silva, Fernando
The availability of relatively capable and inexpensive hardware components has made it feasible to consider large-scale systems of autonomous aquatic drones for maritime tasks. In this paper, we present the CORATAM and HANCAD projects, which focus on the fundamental challenges related to communication and control in swarms of aquatic drones. We argue for: (i) the adoption of a heterogeneous approach to communication in which a small subset of the drones have long-range communication capabilities while the majority carry only short-range communication hardware, and (ii) the use of decentralized control to facilitate inherent robustness and scalability. A heterogeneous communication system and decentralized control allow for the average drone to be kept relatively simple and therefore inexpensive. To assess the proposed methodology, we are currently building 25 prototype drones from off-the-shelf components. We present the current hardware designs and discuss the results of simulation-based experiments involving swarms of up to 1,000 aquatic drones that successfully patrolled a 20 km-long strip for 24 hours.
LTE-Advanced Radio and Network Optimization: Basic Coverage and Interference Constraints
Publication . Velez, Fernando J.; Sousa, Sofia; Acevedo Flores, Jessica; Robalo, Daniel; Mihovska, Albena; Prasad, Ramjee
In cellular optimization, the UL and DL the values from carrier-to-noise-plus-interference ratio (CNIR) from/at the mobile station are very important parameters. From a detailed analysis of its variation with the coverage and reuse distances for different values of the Channel Quality Indicator (CQI) and given ITU-R propagation models, an evaluation of the possible range for the reuse factor of LTE-A is performed for the DL. By considering CQI and reference CNIR requirements recommended by 3GPP, DL peak bit rates along with the Transport Block Size assumed for single stream and bandwidths of 10 and 20 MHz, PHY and supported throughputs are analysed. HetNets with Carrier Aggregation are considered, where macro cells operating at 800 MHz provide coverage and small cells (SCs) operating at 2.6 GHz provide throughput enhancement at hotspots. A clear decrease is shown for the supported throughput for the longest coverage distances in NLoS propagation conditions. In the given range of coverage distances, the same maximum value occurs for the supported throughput for K=3 and 7, both for macro and SCs.
Concepção e Planeamento de Redes sem Fios IEEE 802.16
Publication . Carvalho, Vitor; Santos, Dany; Marcos, Rui; Costa, Rui; Velez, Fernando J.; Sebastião, Pedro; Rodrigues, António; Riscado, José; Romão, José; Cavaleiro, Victor
Esta comunicação aborda aspectos de projecto e planeamento celular duma rede sem fios WiMax (IEEE 802.16) para interligar ponto-a-ponto a Faculdade de Ciências da Saúde da Universidade da Beira Interior (FCS/UBI) ao Hospital Amato Lusitana (Castelo Branco), uma rede ponto-multiponto para ligar a FCS/UBI/Hospital Pêro da Covilhã a toda a área urbana do centro da cidade da Covilhã e uma rede móvel celular dedicada aos serviços públicos de emergência e de segurança, abrangendo toda a região da Beira-Interior, fazendo-se a avaliação detalhada da existência de linha de vista no concelho da Covilhã. Efectuou-se o planeamento tendo em conta aspectos relacionados com a relação portadora-ruído, C/N, e a relação portadora-interferência, concluindo-se ser necessário usar sectorização e considerar sobreposição de micro-células à estrutura macro-celular (cell overlay). Utilizaram-se ferramentas GIS para auxiliar no processo de determinação das posições para as estações de base e da respectiva área com linha de vista, de forma a assegurar um expoente de propagação baixo. Dos resultados para a ligação ponto-a-ponto Covilhã-Gardunha-Castelo Branco, conclui-se que é possível assegurar comunicação com qualidade a 3.5 GHz, mas a 5.8 GHz, como a distância máxima para a qual C/N<(C/N)min é de 25 km, é necessário aumentar o ganho de pelo menos uma das antenas para assegurar a qualidade da ligação.
Protótipo do VABAMPAC - Validação por Código de Barras da Administração de Medicamentos aos Pacientes
Publication . Coelho, Denis; Marques, Susana; Lajes, Tiago; Velez, Fernando J.
Uma barreira de segurança adicional, impedindo erros de dosagem e da escolha do fármaco no momento de tomada dos remédios pelos doentes, reduz a complexidade do trabalho na linha da frente dos cuidados médicos e aumenta a qualidade e a segurança do processo.