FC - DQ | Dissertações de Mestrado e Teses de Doutoramento
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- Insights into Acacia spp.: A Comprehensive Phytochemical Analysis for its Potential Industrial ApplicationsPublication . Pedro, Soraia Inês; Anjos, Ofélia Maria Serralha dos; Alba, Maria Eugénia Gallardo; Gominho, Jorge Manuel Barros D'AlmeidaThis study explored the bioactive and nutritional potential of various Australian Acacia species, recognized as problematic invasive plants in several regions worldwide. The research aimed to develop analytical methods to identify and quantify bioactive compounds in different biomass fractions of these plants. Initially, flowers of Acacia dealbata, Acacia mearnsii, and Acacia retinodes were harvested and analyzed, focusing on their chemical composition, antioxidant potential, and enzyme inhibition activities. The analysis included the identification of phenolic compounds using High-performance liquid chromatography coupled to a diode array detector (HPLC/DAD), as well as Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance (FTIR-ATR) and Fourier Transform Raman Spectroscopy (FT-RAMAN). The results indicated that extracts obtained during the early flowering stage exhibited higher bioactivity, likely due to the presence of chalcones, suggesting potential therapeutic applications, including treatments for dementia, diabetes, and microbial infections. Subsequently, the study evaluated the phenolic profile, antioxidant, and antimicrobial properties of seed pods from several Acacia species, including A. melanoxylon, A. longifolia, and A. cyclops. Liquid chromatography coupled with high-resolution mass spectrometry (LC-ESI-HRMS/MS) successfully identified key flavonoids, with A. pycnantha and A. cyclops displaying the highest concentrations of total phenolic compounds. The seed pod extracts demonstrated significant antibacterial activity, particularly against Klebsiella pneumoniae and Bacillus cereus, indicating potential for future industrial applications. The study also explored the potential of Acacia seed pods as a source of nutrients for animal feed and soil fertilization. Notably, A. retinodes seed pods were rich in proteins, fibers, and minerals such as potassium, calcium, and magnesium, suggesting their suitability for mineral supplementation, although further studies are required to assess bioaccessibility and toxicity. Finally, the nutritional composition and mineral content of leaves from eight Acacia species were analyzed to evaluate their potential as ruminant feed and mineral sources for soils. The extracts were found to be non-cytotoxic in Caco-2 cells, indicating their suitability as an alternative food source for animals. The leaves also exhibited high levels of proteins, fibers, and minerals, particularly calcium, phosphorus, and potassium. In conclusion, this research demonstrated that Acacia species contain promising bioactive compounds with significant therapeutic potential, particularly in antioxidant and antimicrobial applications. Additionally, the nutritional and mineral composition of Acacia species suggests promising applications in animal nutrition and soil fertilization. These findings pave the way for the sustainable and multifunctional use of Acacia species, contributing to mitigating their ecological impact while exploring their economic value.
- From production to purification: Towards an integrative process for recombinant pre-miRNA-29b as biopharmaceuticalPublication . Carapito, Ana Rita Mugeiro; Sousa, Fani Pereira de; Martins, Mara Guadalupe Freire; Sponchioni, MattiaRecent 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.
- G-quadruplex targeting by ligands as a lung cancer therapeutic strategyPublication . Figueiredo, Joana Patrícia Rodrigues; Cruz, Carla Patrícia Freire Madeira Alves da; Mergny, Jean-LouisG-quadruplexes (G4s) are non-canonical four-stranded nucleic acid secondary structures that can form in guanine-rich regions in the human genome and transcriptome. DNAs G4s are found in critical regulatory regions of the human genome such as the end of telomers and the promoter regions of several oncogenes. These structures have been implicated in the control of key cellular events including replication, transcription, genome stability, and epigenetic regulation. On the other hand, RNAs G4 are found within non-coding regions and are implicated in crucial RNA metabolism events, including the regulation of RNA processing and translation. Due to their biological relevance and structural features, G4s are considered suitable therapeutic targets. The prevalence of G4 folding in the cancer context and its stabilization can be used as an anti-tumor treatment strategy in different cancer types. Several synthetic small molecules with high specificity for G4s DNAs and RNAs relative to double-stranded DNA (G4 ligands) have been developed and evaluated for their therapeutic potential. Over the years, thousands of small molecules have been reported as G4 ligands with in vitro and/or in vivo anti-tumor activity. Often, but not always, G4 ligands have a planar aromatic core for π-π stacking with G-quartet and a positively charged or basic group(s) to interact with the phosphate backbone of nucleic acid. Different scaffolds containing these chemical features, such as quindolines, acridines, naphthalene diimides, and phenanthrolines have proved to have G4 binding properties and anti-tumor activities. These small organic molecules interacting with G4s may affect cancer cell growth in different ways, including inhibition of telomerase or interference with telomere function, modulation of oncogenes expression by stabilizing G4 in their promoter and regulating microRNA biogenesis (miRNAs) by stabilizing G4 in microRNA precursors (pre-miRNAs). Lung cancer (LC) is the leading cause of cancer-related death worldwide and is divided into two major subtypes, small-cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). Although therapeutic options such as surgery and chemoradiotherapy prove efficacy in the early stage of the disease, their effectiveness is limited in advanced LC where most patients are diagnosed. Although immune checkpoint inhibitors have shown promising clinical outcomes, the high level of molecular heterogeneity in LC makes treatment difficult and new therapeutic strategies are needed. The prevalence of G4 formation in important regulatory systems in LC including proto-oncogenes (e.g. MYC, BCL-2, KRAS, KIT, and VEGF) and telomere regions make then attractive targets. Similarly to other cancer types, G4 ligand-induced stabilization promotes changes in telomere maintenance and decreases oncogene expression levels. Ongoing efforts have been made to harness G4 ligands for inducing G4 stabilization in the LC context. Different classes of G4s ligands may lead to in vitro antiproliferative activity and, in some cases, in vivo anti-tumor effects. Overall, this thesis aims to develop novel G4 ligands derivatived from 1,10-phenanthroline, which can bind/stabilize G4 structures present in important regulatory regions in LC acting as anti-tumor agents. The chemical synthesis and screening of heterocyclic compounds featuring phenyl, quinoline, naphthalene, acridines, and phenanthroline scaffolds were assessed to explore their stabilization effect towards G4 structures in c-MYC, KRAS and VEGF promoters, human telomeric motif, and pre-MIR150 RNA G4. The acridine moiety exhibited the highest G4 stabilization, followed by phenanthroline. Additionally, most compounds proved greater anti-tumor efficacy in LC cells than in non-malignant cells. Subsequently, a novel class of phenanthroline derivatives was synthesized and structurally characterized. In addition, to assess their potential to bind/stabilize G4-forming sequences, several biophysical techniques, including förster resonance energy transfer (FRET) melting assay, circular dichroism (CD) studies and fluorescence titrations were employed. The in vitro anti-tumor activity of these compounds has been evaluated against LC cell lines via MTT assay. Sixteen derivatives of 1,10-phenanthroline were synthesized, featuring substituents at positions 2 and 9 with either amine or amide side chains, by direct condensation reactions. Among these derivatives, those bearing methoxyaniline and ethane-diaminium side chains have been shown to stabilize various G4 topologies derived from human telomere sequences. These included hybrid, parallel, and antiparallel G4 structures, with a marked preference for the hybrid topology of the F21T sequence (ΔTm =7.2 – 12 °C, by FRET melting). Additionally, the ligands displayed promising cytotoxic profiles and exhibited greater efficacy against A549 than H1299 LC cells. Additionally, novel ten 1,10-phenanthroline-2,9-bistriazoles derivatives were synthesized via cooper catalyze azide/alkyne cycloaddition reactions. Through biophysical assessment, three promising G4 ligands against KRAS G4 sequences were identified, exhibiting increases in melting temperature (ΔTm =4.7 – 11.2 °C, by FRET melting) and binding affinities ranging between 10-6 and 10-9 M. Evaluation of anti-tumor activity revealed that the compound bearing a phenyl ring linked to the triazole moiety exhibited a potent inhibitory effect on A549 and H1299 cancer cell growth (IC50 = 14.6 and 10.9 μM, respectively). Overall, the experimental results demonstrated that the synthesized compounds have the potential to bind/stabilize different G4-forming sequences and may serve as promising scaffolds for the development of G4 ligands. Additionally, this study provides invaluable insights into the structure-activity relationships for this class of compounds to maximize their activity. Finally, to the best of our knowledge, we have identified, for the first time, a G4-forming region within the human precursor of MIR150 (pre-MIR150). The G4-forming region folds into a parallel RNA G4 and has the potential to interact with nucleolin (NCL), which is a protein overexpressed on the cell surface of cancer cells involved in several cellular processes, including tumorigenesis, angiogenesis, and signaling pathways. The thermal stability of G4 increases in the presence of a commercial phenanthroline ligand (PhenDC3), and the formation of ternary complex G4/PhenDC3/NCL was observed. Moreover, the G4 structure in pre-MIR150 recognized NCL-positive LC cancer cells, and liquid biopsies when fluorescently labeled, can be used as a probe.
- Vessel passivation strategies to reduce vessel pickingPublication . Coelho, João Pedro Rodrigues; Vaz, Álvaro Frederico CamposIn recent decades, the paper industry has faced several challenges, both in terms of decarbonisation of the industrial sector and in terms of innovation and concomitant evolution of the digital era. One of the problems associated with the paper industry, and in particular the study case, Eucalyptus globulus (hardwood), is the presence of vessel elements in the wood structure. This problem stands out not in production (paper industry) but in the use (consumer) of the final product. In the case of printing from paper produced with this pulp, the poor adhesion of these elements to the surface of the paper can cause them to come loose and contaminate the printing system, a phenomenon called vessel picking and ink refusal. It is a problem commonly found in printing industries that need to make large quantities of prints and associated with the paper industry for the production of paper. During the printing process, this phenomenon occurs due to the stickiness of the inks involved in the printing process and can cause vessel elements torn from the paper surface to contaminate the printing machine circuit. This contamination can result in a drop in production due to the stopping of printing machines for cleaning, which can result in increased costs. Although it is not a directly felt problem in the paper industry, there is a continuous need to optimize the characteristics of a product, reducing the rate of complaints due to this problem. There is also a need to follow the evolution of the digital era and technologies, including printing technologies that increasingly print faster and with more colours, posing other types of challenges to the paper structure while at the same time accompanying the decarbonisation of the sector. This thesis aimed to understand the phenomenon allowing the creation of papers with a low level of vessel picking and following the studies carried out over the last decades on the subject, some approaches to this problem were attempted. The physical separation of vessel elements was attempted, using the Jacquelin method and also the Britt-Jar method. Some adaptations were made to separation methods (Britt-Jar) and others were created for the same purpose (adapted Strainer). The objective was to create a pure vessel fraction (>90%) to be able to use different analytical means and better understand the differences in behaviour between fibres and vessels, and to create a pulp fraction sufficiently rich in vessels to enable the vessel picking study. Morphological studies were carried out on the characteristics of the pulp and after achieving satisfactory separation levels (>90%) it was possible to carry out more concrete studies on the surface and global chemistry of the vessel elements and fibres. It was also possible to advance two treatment hypotheses, mechanical and enzymatic. With these studies it was possible to discover similarities and differences that could justify the different interaction between the elements (fibres and vessels) and thus try to better understand the vessel picking effect. It was also possible to perceive the effect of the treatments on the different pulp elements. The results reveal differences between vessels and fibres and point to a greater hydrophobicity of the vessels in relation to fibres (μ-FTIR spectrum with differences in the peak relative to the CH bonds and the COC ether group; it is possible that the presence of a higher concentration of hydrocarbons may be related to the greater hydrophobicity shown by the vessels, in agreement with surface free energy values, where the vessels present a higher dispersive/polar energy ratio, and with the higher values of water contact angle for the vessels (more hydrophobic)), a higher content of lignin and extractives in the vessels (low O/C content in XPS and EDX observed in the vessels can be explained by a higher content of lignin and extractives), a higher content of hemicelluloses in the vessels (having a higher content of total acids and hexenuronic acids) and a greater porosity for the vessels in relation to fibres (vessels with a higher content of mesopores and lower content of micropores). The use of enzymatic treatments revealed to have a positive effect on some of the characteristics presented by fibres and vessels (reduction in hemicellulose content and superficial reduction in the content of extractives and hemicelluloses) and also on the selectivity of enzyme action (greater effect on vessels, probably due to difference in porosity). In this work, paper with different quantities of vessels was also simulated and treatments were carried out on them. The effect of beating was studied, the effect of two enzymes (xylanase and an enzymatic cocktail containing cellulase and laccase) and the two treatments simultaneously were studied. Beating proved to be the most effective method in reducing vessel picking (>90%), although both enzymes, with greater relevance for the enzymatic cocktail, showed some effectiveness (vessel picking decrease: xylanase ≈ 38%; enzymatic cocktail ≈ 53%). Enzymatic treatments mainly revealed changes in the behaviour of the vessels and consequently in their integration into the paper structure. A preferential attack on the vessels was detected, probably due to the greater porosity of the vessels; there was a consequent increase in porosity with the enzymatic treatment, where the effect was more pronounced in the vessels; reduced hydrophobicity, proven by water contact angle measurements as well as by the increase in the polar component of the sample’s surface energy. However, the combination of the two methods proved to be the most effective methodology (>91%), and there may be a balance between beating and the use of enzymes, resulting in a reduction in operating costs. Within the tests carried out, enzymatic tests were also carried out directly on a fraction of vessels, which revealed an effectiveness of 94% in reducing vessel picking. In short, the problem of vessel picking can be controlled through beating, which is not a 100% effective solution. Enzymatic treatments improve the beating effect, proving to be efficient in terms of reducing vessel picking, however, it is necessary not to neglect side effects in the tensile properties. From the point of view of the paper production industry, when subjected to a raise in vessel problems, enzymatic treatments should be considered, before beating, presenting a good solution to mitigate the vessel picking problem.
- Caracterização fitoquímica dos extratos de casca e raízes de Alchornea cordifoliaPublication . Lufuankenda, Nsinga; Rodilla, Jesus Miguel LopezA pesquisa de substâncias naturais de plantas resultou na descoberta de vários medicamentos úteis para o tratamento de diferentes doenças, impulsionando o desenvolvimento de tecnologias de extração e separação, métodos espectroscópicos para elucidação da estrutura e metodologias sintéticas fundamentais da Química orgânica analítica. Este trabalho teve como objetivo caracterizar os compostos fitoquímicos presentes nos extratos da casca e raízes de Alchornea cordifolia, pertencente à família Euphorbiaceae. Foram utilizadas técnicas de extração sólido-líquido com solventes de polaridade crescente (n-hexano, acetona e etanol) e o extrato de hexano foi submetido à desceração e a cera obtida foi primeiramente submetida ao processo de hidrólise com metanol em presença de NaOH obtendo a parte neutra e as partes ácidas (1 e 2) e depois as partes ácidas foram esterificadas com diazometano. A análise por Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-EM) de cera do extrato de hexano da casca da parte aérea identificou quinze compostos fitoquímicos, incluindo triterpenoides, fitoesteróis e álcoois graxos. A caracterização por RMN dos extratos sugeriu a presença de substâncias bioativas, incluindo flavonoide, triterpenoide, ésteres, compostos fenólicos. Recomenda-se continuar o estudo com mais técnicas espectroscópicas (IV, CG-EM, HPLC) e investigações de actividades biológicas, como antimicrobiana e antidiabética.
- Avaliação da condutibilidade térmica de estruturas celulósicas ultra-porosasPublication . Martins, Joel Pais; Vieira, Joana Magalhães Gonçalves Costa; Pires, Luís Carlos Carvalho; Costa, Ana Paula Nunes de Almeida Alves daEste estudo teve como objetivo o desenvolvimento de estruturas celulósicas de elevada porosidade, através do processo de espuma, para a introdução de ar nas mesmas, o que faz com que estas possam ter uma boa capacidade para criar resistência à passagem de calor, e consequentemente sejam bons isolantes térmicos. Para se fazer a avaliação da condutibilidade térmica, também foi necessário desenvolver um sistema para a medição desta propriedade que fosse flexível à espessura das estruturas celulósicas produzidas. Produziram-se três tipos de estruturas a partir de fibras obtidas de papel reciclado. A primeira, considerada a referência, composta apenas pelas fibras celulósicas tal qual, a segunda, onde se adicionou o aditivo de resistência, o amido catiónico, e finalmente, a terceira, que para além do aditivo de resistência, se procedeu a uma ligeira refinação das fibras recicladas. Para a produção de cada uma das amostras, colocaram-se as fibras em suspensão e adicionou-se um surfactante, que permitiu criar espuma e assim introduzir ar dentro da rede fibrosa, fazendo assim, com que as estruturas obtidas apresentassem uma elevada porosidade. As amostras preparadas foram de seguida testadas para determinar a sua capacidade de isolamento térmico e as suas propriedades mecânicas. Os ensaios das propriedades mecânicas permitiram verificar uma melhoria da resistência das estruturas quando se fez a refinação das fibras e ainda se adicionou o aditivo de resistência na preparação das amostras. Os ensaios para determinação da condutibilidade térmica foram efetuados usando a sua espessura máxima, obtida logo após a secagem, e também realizados com uma compressão de 50%, para estudar o efeito da compressibilidade nesta propriedade. Os resultados obtidos permitiram verificar que estas estruturas têm uma capacidade enquanto isolantes térmicos que se aproxima dos materiais mais comuns usados no isolamento térmico de edifícios.
- Electrochemical oxidation as a strategy for wastewater reusePublication . Farinon, Daliany Maria; Fernandes, Annabel Dias Barrocas; Afonso, Alexandra Isabel GodinhoThis study aims to electrochemically oxidize (EO) industrial wastewater, namely olive mill wastewater (OMW) and membrane concentrate of textile wastewater (MCTW), to produce treated effluents that can be reused in hydroponic agriculture (treated OMW) and as an anti-fouling agent in membrane processes (treated MCTW). OMW treatment was performed using a boron-doped diamond (BDD) anode suitable for mineralizing organic compounds. Three different applied current intensities were studied, 300, 500, and 700 mA. EO removed up to 98% of the chemical oxygen demand (COD). The biodegradability of the treated OMW increased considerably, especially at 500 and 700 mA. The specific energy consumption increased with the applied current intensity. Considering the overall results, the OMW treated at 500 mA was considered the most suitable for hydroponic cultivation. However, it must be diluted in fresh water to adjust pH and electrical conductivity. MCTW treatment used a mixed metal oxide (MMO) anode, which is prone to generating active chlorine species from chloride oxidation. Two different MCTW samples were studied: ultrafiltration retentate (MCTW_1) and reverse osmosis retentate (MCTW_2). With both samples, hypochlorite (ClO-) was generated by EO, leading to treated MCTW samples rich in oxidizing agents with potential for industrial reuse. COD removal was 61% for MCTW_1 and 68% for MCTW_2. Further process optimization is required to minimize energy consumption and chlorate formation. The obtained results showed that EO is a promising process for treating industrial wastewater. It enables the reduction of the pollutant load and the reuse of the treated wastewater in a circular economy approach.
- Efeitos genómicos do triclosano a nível cardiovascular humanoPublication . Caetano, Catarina Pereira; Oliveira, Maria Elisa Cairrão RodriguesO triclosano (TCS) é um agente antimicrobiano sintético amplamente utilizado em produtos de uso diário, sendo encontrado em diversos itens de higiene pessoal e doméstica. Durante a pandemia de COVID-19, o uso de desinfetantes tornou-se indispensável para o acesso a muitos espaços públicos, sendo o TCS um componente comum nestes produtos, o que reforça a relevância de investigar os seus efeitos. Estudos recentes indicam que o TCS atua como um disruptor endócrino, interferindo nas atividades estrogénicas e androgénicas, com potencial para contribuir para o desenvolvimento de patologias a nível reprodutor, endócrino, neurológico e cardiovascular. No entanto, os seus impactos a nível humano ainda carecem de maior compreensão e investigação. Com isto, o objetivo deste trabalho consistiu no estudo dos potenciais efeitos do triclosano no sistema cardiovascular. Para atingir este objetivo foram delineados os seguintes objetivos específicos: 1) Análise do efeito direto do TCS na artéria umbilical humana (HUA) sem endotélio; 2) Análise dos possíveis mecanismos envolvidos no efeito do TCS na HUA, principalmente o envolvimento dos nucleótidos cíclicos e dos canais de Ca2+; 3) Realização com sucesso do isolamento e cultura HUASMCs; 4) Análise dos efeitos do TCS na viabilidade das HUASMCs por ensaio de MTT; 5) Análise da expressão de genes envolvidos nas propriedades contráteis das HUASMCs, usando o Real-Time PCR. Os resultados indicaram que o TCS promove relaxamentos de curto prazo de maneira dependente da concentração em HUAs desprovidas de endotélio, após contração com diferentes agentes vasoativos (não genómico). Quanto aos ensaios genómicos, verificou-se que o TCS pode estar associado à redução da síntese de óxido nítrico. Nos testes de viabilidade celular, apenas as concentrações mais altas de TCS 50 e 100 µM resultaram em uma diminuição significativa da viabilidade, apontando para um efeito tóxico do TCS nas Células Musculares Lisas da Artéria Umbilical Humana (HUASMCs). Em síntese, o TCS parece exercer um impacto negativo sobre o sistema cardiovascular. No entanto, os seus efeitos genómicos ainda não estão completamente esclarecidos, sobretudo no que diz respeito à incubação com TCS 1 µM, onde foi observada uma resposta bifásica. Dessa forma, são necessários estudos complementares para aprofundar o entendimento do mecanismo de ação do TCS no contexto vascular.
- O valor de Arbutus unedo L. como ingrediente bioativo para aplicação cutânea: avaliação da variabilidade inter-regional em território nacionalPublication . Pires, Carolina Pinho; Oliveira, Ana Cristina Palmeira de; Oliveira, Ana Sofia DominguesNa sociedade atual, a sustentabilidade tem sido uma preocupação crescente, refletindo-se nas escolhas de produtos cosméticos, nomeadamente para o cuidado da pele. O uso de extratos de algumas espécies de plantas aromáticas e medicinais têm tido destaque nas indústrias cosmética e farmacêutica devido às propriedades benéficas que possuem, que são valorizadas na saúde humana. Destes, o medronheiro (Arbutus unedo L.) ganhou destaque pelas suas propriedades antioxidantes, anti-inflamatórias e regenerativas. Embora o fruto seja bem conhecido, as folhas são frequentemente consideradas resíduos, apesar de serem ricas em compostos bioativos. Assim, este projeto analisou três extratos aquosos de folhas de A. unedo, provenientes de diferentes regiões de Portugal continental (Proença-a-Nova, Odemira e Gouveia), com o objetivo de avaliar o seu potencial como ingredientes bioativos para uma possível futura utilização em formulações cosméticas. Para isso, testou-se a biocompatibilidade dos extratos in vitro em células da pele e as propriedades antioxidantes, antimicrobianas, anti-inflamatórias e cicatrizantes dos extratos. Além disso, foi realizada uma caracterização química através das técnicas de UHPLC-PDA-MS/MS e GC-MS, para identificar os compostos bioativos que estão presentes nas folhas e compará-los, de modo a avaliar a possível junção de produções, com o objetivo de escalar a oferta considerando uma futura aplicação em formulações. A atividade antioxidante foi avaliada através do método de captação de radicais livres para o DPPH. A viabilidade celular foi testada em fibroblastos de ratinho, queratinócitos humanos e macrófagos de ratinho, quando expostos aos diferentes extratos aquosos, utilizando o ensaio MTT. A atividade anti-inflamatória foi medida em macrófagos, através da quantificação da produção de óxido nítrico (NO) após a estimulação com lipopolissacarídeo (LPS). A atividade antimicrobiana foi avaliada através do ensaio de microdiluição em caldo contra um painel de bactérias Gram-positivas e negativas, leveduras e fungos, com foco na aplicação cutânea. Foi avaliado também o potencial anti-acne recorrendo a um ensaio enzimático que mediu a atividade da enzima lipase, e o potencial cicatrizante foi analisado em fibroblastos segundo um ensaio de migração celular (scratch-wound). Os resultados indicaram que os principais compostos fenólicos encontrados foram derivados do ácido gálico e flavonoides na sua forma glicosilada (por exemplo, quercetina). Todos os extratos exibiram uma capacidade antioxidante muito forte, com índices de atividade antioxidante entre 4,41 e 6,61. Em termos de atividade antimicrobiana, Cutibacterium acnes foi a estirpe mais suscetível a todos os extratos (CMI 0,5 mg/mL). Todos os extratos demonstraram atividade anti-lipase, sendo o extrato de Odemira o mais eficaz na inibição da enzima. Relativamente à viabilidade celular, todos os extratos mostraram ser biocompatíveis a uma concentração máxima de 0,5 mg/mL para todas as linhas celulares testadas. No entanto, nenhum extrato demonstrou atividade antiinflamatória significativa e não se revelaram potenciais cicatrizantes. Conclui-se que o estudo dos extratos deve ser aprofundando, especialmente em termos de atividade anti-inflamatória, avaliando por exemplo outras vias para confirmar a ausência deste potencial. Relativamente à diferença entre eles, seria interessante juntar alguns extratos atendendo às características idênticas que apresentaram em determinadas bioatividades. Entretanto, a elevada atividade antioxidante e baixa toxicidade, aliadas à suscetibilidade de C. acnes aos extratos e ao potencial anti-lipase, indicam que estes são promissores para uma futura aplicação cutânea, particularmente associada ao tratamento de acne.
- Development of pH-responsive nanoparticles for the protection and delivery of RNAs to cancer cellsPublication . Graça, Tiago Samuel Silva; Sousa, Fani Pereira de; Mendonça, Patrícia Vitorino; Baptista, Bruno Miguel CameiraNanoparticles are receiving increased attention due to their significance in delivering biopharmaceuticals for gene-based therapies. At the same time, RNA is being studied to develop new and advanced treatments. However, the successful therapeutic application of these biomolecules is highly dependent on their effectiveness in reaching target cells. The development of pH-responsive nanoparticles may improve delivery efficiency in certain diseases that present a microenvironment with deregulated pH, such as lung cancer. In this work, it is explored the pH responsiveness and efficiency of a copolymer composed of poly(2- (diisopropylamino)ethyl methacrylate)) (PDPA) and poly(oligo(ethylene oxide)methyl ether methacrylate) (POEOMA) for the encapsulation and release of small RNAs (sRNA). The encapsulation process relies on the electrostatic interaction between the negatively charged sRNA and the protonated tertiary amine groups of the PDPA segment. Three copolymers with different architectures (linear or 4-arm star-shaped) and compositions (4-arm star PDPA-b-POEOMA or 4-arm star POEOMA-b-PDPA) were synthesized by atom transfer radical polymerization. The sRNA and miR-29b-duplex encapsulation efficiencies were studied by UV spectroscopy and agarose electrophoresis. The best formulations were further characterized regarding the size and surface charge of the polyplexes, by dynamic Light Scattering (DLS) and electrophoretic Light Scattering (ELS), respectively. Depending on the copolymer, concentration, and pH of the formulation, the systems achieved complexation efficiencies ranging from 35% to 84%. The polyplexes presenting higher encapsulation efficiencies had sizes ranging from 92 to 104 nm. It was also possible to understand the changes in the nanoparticle’s structure at different pH values with TEM, which are in accordance with their release potential and profile, studied by UV spectroscopy and confirmed by agarose electrophoresis. Additionally, the nanoparticles of the different conformations were submitted to several biologically relevant conditions to evaluate their protection capacity, where the 4-arm star, POEOMA core, and copolymer proved to have superior properties than their linear and conformational inverse counterparts. Moreover, cell viability, via MTS assays was employed where all three architectures proved biocompatible. This research demonstrates the potential of pH-sensitive polymers as promising RNA delivery systems, and how the different conformations inside the same copolymer block combination can influence its overall performance as an effective delivery vehicle.