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.

Nucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the bio- manufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA puri- fication processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C3C3NH2Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromato- graphic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.

Nucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the biomanufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA purification processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C3C3NH2Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromatographic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.

The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

This paper addresses the mechanical characterization of polycaprolactone (PCL)–bioglass (FastOs®BG) composites and scaffolds intended for use in tissue engineering. Tissue engineering scaffolds support the self-healing mechanism of the human body and promote the regrowth of damaged tissue. These implants can dissolve after successful tissue regeneration minimising the immune reaction and the need for revision surgery. However, their mechanical properties should match surrounding tissue in order to avoid strain concentration and possible separation at the interface. Therefore, an extensive experimental testing programme of this advanced material using uni-axial compressive testing was conducted. Tests were performed at low strain rates corresponding to quasi-static loading conditions. The initial elastic gradient, plateau stress and densification strain were obtained. Tested specimens varied according to their average density and material composition. In total, four groups of solid and robocast porous PCL samples containing 0, 20, 30, and 35% bioglass, respectively were tested. The addition of bioglass was found to slightly decrease the initial elastic gradient and the plateau stress of the biomaterial scaffolds.

The microalgae Aurantiochytrium sp. is a strong alternative source of ω-3 fatty acids, including ocosahexaenoic acid (DHA). This work encompasses the optimization of SFE conditions to maximize the total extraction yield (ηTotal), DHA content (CDHA), total phenolics content (TPC), and antioxidant capacity (AOC) of the extracts produced from Aurantiochytrium sp. biomass. A full factorial experimental plan was performed, comprising three factors (pressure, temperature, and flow rate) and two levels (200−300 bar, 40−80 °C, and 6–12 gmin−1, respectively). The maximum and minimum experimental results were ηTotal = 2.1 and 13.4 wt.%, CDHA= 27.3 and 39.3 wt.%, TPC =1.19 and 2.24 mgGAE g−extract 1 , and AOC = 0.3 and 1.4 mg g− TEAC extract 1. Under the studied experimental conditions, increasing pressure up to 300 bar is the optimum to rise both ηTotal and CDHA. Temperature increase from 40 to 80 °C leads to opposing effects: it favors the concentration of phenolics in the supercritical extracts at the expenses of decreasing DHA content and total yield. Surface models were adjusted to ηTotal, CDHA and TPC data, and the goodness of the fits ranged from coefficients of determination of 0.752-0.711 (TPC) to 0.997-0.994 (CDHA). Under optimized conditions, supercritical extracts exhibited a DHA content more than 3.5-fold richer than fish oil, and 7.9-fold richer than the best alternative microalgae species (Pavlova lutheri) found in the literature.

In this study, Selective Laser Melting (SLM) was used to produce mono-material Ti64Al4V- and NiTi-cubic cellular structures with an open-cell size and wall thickness of 500 μm and 100 μm, respectively. Bioactive beta-tricalcium phosphate (βTCP) and polymer poly-ether-ether ketone (PEEK) were used to fill the produced structures open-cells, thus creating multi-material components. These structures were characterized in vitro in terms of cell viability, adhesion, differentiation and mineralization. Also, bio-tribological experiments were performed against bovine plate to mimic the moment of implant insertion. Results revealed that metabolic activity and mineralization were improved on SLM mono-material groups, when compared to the control group. All cell metrics were improved with the addition of PEEK, conversely to βTCP where no significant differences were found. These results suggest that the proposed solutions can be used to improve implants performance.

Aurantiochytrium sp. is an emerging alternative source of polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA), and squalene, playing an important role in the phasing out of traditional fish sources for these compounds. Novel lipid extraction techniques with a focus on sustainability and low environmental footprint are being developed for this organism, but the exploration of other added-value compounds within it is still very limited. In this work, a combination of novel green extraction techniques (high hydrostatic pressure extraction (HPE) and supercritical fluid extraction (SFE)) and traditional techniques (organic solvent Soxhlet extraction and hydrodistillation (HD)) was used to obtain lipophilic extracts of Aurantiochytrium sp., which were then screened for antioxidant (DPPH radical reduction capacity and ferric-reducing antioxidant potential (FRAP) assays), lipid oxidation protection, antimicrobial, anti-aging enzyme inhibition (collagenase, elastase and hyaluronidase), and anti-inflammatory (inhibition of NO production) activities. The screening revealed promising extracts in nearly all categories of biological activity tested, with only the enzymatic inhibition being low in all extracts. Powerful lipid oxidation protection and anti-inflammatory activity were observed in most SFE samples. Ethanolic HPEs inhibited both lipid oxidation reactions and microbial growth. The HD extract demonstrated high antioxidant, antimicrobial, and antiinflammatory activities making, it a major contender for further studies aiming at the valorization of Aurantiochytrium sp. Taken together, this study presents compelling evidence of the bioactive potential of Aurantiochytrium sp. and encourages further exploration of its composition and application.

This work addresses the effect of chaotropic anions (thiocyanate and tosylate) on the solubility of glycine, L-leucine, L-phenylalanine, and L-aspartic acid in aqueous solutions at 298.2 K. The salts used were NaSCN, KSCN, NH4SCN, and NaC7H7SO3 (sodium tosylate), with salt concentrations ranging from 0 to 2 molal. The pH of the saturated solutions was registered, and solid-phase studies were also performed. All the thiocyanate salts and sodium tosylate induced a salting-in effect, except in the systems composed of glycine in aqueous sodium tosylate solutions at 0.5 and 1 molal. For L-leucine, L-phenylalanine, and L-aspartic acid the salting effect of anions followed the order tosylate− > SCN− > NO3- > Cl−, in good agreement with the behavior predicted by the Hofmeister series. Differently, the relative solubility of glycine in aqueous nitrate solutions was higher than in those containing thiocyanate, followed by the chloride and, closing the rank, the solutions containing the tosylate anion, suggesting that the solubility change in this case is achieved by a different mechanism.

Aqueous solubility can be enhanced through hydrotropy, where an amphiphilic molecule (the hydrotrope) ag-gregates around the solute, boosting its solubility. To understand the impact of temperature on this phenomenon, the solubility of syringic acid was measured in aqueous solutions of different bio-based hydrotropes. Solubility curves were modelled using Gaussian processes, a powerful class of machine learning interpolators. This allowed for a thermodynamic analysis of excess solvation properties that was complemented using the COSMO-RS model. As expected, the absolute solubility of syringic acid increased with temperature. However, at low hydrotrope concentrations, this increase was not driven by interactions in the liquid phase. Thus, the solubility enhancement of syringic acid was found to be independent of temperature. Conversely, at high hydrotrope concentrations, increasing the temperature significantly decreased the solubility enhancement of the solute. These effects were interpreted considering different solubilization mechanisms, namely hydrotropy (low hydrotrope concentrations) and co-solvency (high hydrotrope concentrations).