FCT: DQ - Teses de Doutoramento
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- ALIMENTOS FUNCIONAIS E NUTRACÊUTICOS DIRIGIDOS À PREVENÇÃO DAS DOENÇAS CARDIOVASCULARES E NEURODEGENERATIVASPublication . Gomes, Romina Melissa Conceição Cunha; Bandarra, Narcisa; Bispo, Ana; Duarte, Maria PaulaDada a importância do metabolismo lipídico na Doença de Alzheimer (DA) e a estreita correlação desta patologia com a saúde cardiovascular, o trabalho realizado visou avaliar o papel de alimentos específicos sobre o perfil lipídico de eritrócitos e cérebro num modelo ani- mal de DA (murganhos transgénicos 5xFAD (B6SJL/Tg6799). Os alimentos selecionados foram a cavala (Scomber colias) devido à sua abundância, elevados teores de ácido docosahexaenóico (DHA) e outros ácidos gordos polinsaturados da família ómega-3, independentemente da es- tação do ano considerada, e também de vitamina B12. Foi também selecionada a quinoa, como fonte de vitamina B9, para estudar o eventual benefício da combinação da cavala com a qui- noa, e assim identificar um possível efeito sinérgico que potenciasse o efeito da cavala. Os murganhos foram divididos em quatro grupos de estudo (8 animais/grupo), deno- minados por: Controlo (C), Cavala (CV), Quinoa (Q), Cavala mais Quinoa (CVQ). Ao grupo C foi fornecida uma dieta comercial padrão para roedores (AIN-93M), ao grupo CV foi fornecida uma dieta AIN-93M suplementada com 10% de cavala, ao grupo Q foi fornecida uma dieta AIN-93M suplementada com 5% de quinoa, e ao grupo CVQ foi fornecida uma dieta AIN- 93M suplementada com 10% de cavala e 5% de quinoa. Às dietas CV e CVQ foi ainda adicio- nado 6,2 % de óleo de cavala, de modo a obter uma dose-alvo de 1,2% de DHA. Os resultados obtidos, mostraram uma alteração do perfil lipídico nas membranas dos eritrócitos dos murganhos cujas dietas continham cavala, com um aumento superior a 100% em ácido eicosapentaenóico (EPA) e de cerca de 97% (dieta CV) ou 57% (grupo CVQ) em DHA. O aumento em EPA e DHA foi acompanhado por uma diminuição de cerca de 50% em ácido araquidónico. Esta alteração do perfil lipídico sugere que a incorporação da cavala na dieta, possa ter um impacte positivo na redução da inflamação. O enriquecimento em DHA e EPA, traduziu-se num aumento do índice ómega-3 para valores superiores a 8%, isto é, para valores considerados ideais para a saúde cardiovascular e que também têm sido associados a uma maior saúde cerebral. Os resultados mostraram ainda um aumento de cerca de 25% dos níveis de DHA, bem como da razão n-3/n-6, no cérebro dos murganhos cuja dieta foi suplementada apenas com cavala. Deste modo, os resultados obtidos no modelo animal utilizado, sugerem que a incor- poração de cavala na dieta possa trazer benéficos para a saúde cardiovascular e cerebral em doentes que sofrem de DA. Em relação aos parâmetros analisados, os resultados não eviden- ciaram efeitos de sinergia entre a cavala e a quinoa, visto que a dieta suplementada apenas com cavala foi sempre a que permitiu obter os resultados mais favoráveis. Este trabalho visou igualmente o desenvolvimento de extratos enriquecidos em liso- fosfatidilcolina-DHA para serem utilizados na suplementação/fortificação de alimentos, vi- sando o enriquecimento neural com DHA. Por ser abundante e rica em DHA, especialmente nos seus fosfolípidos, foi utilizada como matéria-prima a sarda (Scomber scombrus). A fração lipídica polar foi extraída e posteriormente hidrolisada com a lipase de Rhizomucor miehei, para converter enzimaticamente a fosfatidilcolina (PC) em LPC. Os resultados mostraram que a abordagem seguida permitiu alcançar o objetivo proposto, tendo após a hidrólise, quase me- tade da PC do extrato de sarda sido convertida em LPC.
- Exploiting human macrophage galactose-type lectin interactions towards cancer therapiesPublication . Lima, Carlos David Lourenço; Marcelo, Filipa; Corzana López , FranciscoA MGL é uma lectina oligimérica do tipo C presente em células imunitárias, com uma forte especificidade para resíduos de GalNAc em glicoproteínas e lípidos. O seu reconhecimento seletivo de antigénios tumorais contendo GalNAc torna-a um alvo terapêutico promissor. Esta tese apresenta a caracterização estrutural e engenharia do seu domínio de reconhecimento de hidratos de carbono (MGL-CRD) para apoiar o desenvolvimento de novas terapias. Inicialmente, mapeou-se a interação entre a coordenação de cálcio, a ligação ao ligando e o pH. A RMN revelou que o ligando inverte a hierarquia intrínseca de preferência por cálcio da MGL para priorizar a ligação. A interação MGL-GalNAc mimetizando o ambiente fisiológico (pH 7,4) e tumoral (pH 6,2) revelou afinidades comparáveis. Em conjugação com a forte especificidade para antigénios Tn/STn, este comportamento reforça o seu potencial terapêutico. Com base nestes dados, desenvolveu-se o primeiro conjugado MGL-fármaco para entrega direcionada. Criou-se uma variante com uma cisteína no terminal C (mMGL) para conjugação sítio-específica com o fármaco vcMMAE. O conjugado preservou a estabilidade térmica e a afinidade de ligação ao αGalNAc. O mMGL-vcMMAE validou a sua especificidade em tecidos de cancro colorretal (CCR) e demonstrou maior atividade citotóxica que o vcMMAE livre em linhas celulares de CCR expressando antigénios Tn/STn, suportando o potencial desta estratégia de entrega. Para otimizar a estabilidade e a ligação do mMGL, criaram-se três mutantes (mMGL-Mut1, mMGL-Mut5, mMGL-Mut6). Uma mutação no sítio de ligação (Mut1) diminuiu a estabilidade térmica, mas aumentou a resistência a proteases. As variantes estabilizadas estruturalmente (mMGL-Mut5, mMGL-Mut6) exibiram uma capacidade de ligação semelhante, mas com uma maior estabilidade térmica no caso do mMGL-Mut5. Por fim, ligandos GalNAc triméricos comprovaram a sua capacidade de ligação e aglomeração (clustering) de múltiplos MGL-CRD. Globalmente, este trabalho fornece as informações essenciais para o desenho racional de terapias baseadas na MGL.
- Polyhydroxyalkanoate production process: real-time monitoring, extraction optimization and framework for sustainability assessmentPublication . Rodrigues, Ana Marta da Silva; Lourenço, Nídia; Reis, Maria D’Ascensão; Costa, NunoThe advantageous properties of conventional plastics have made them a major commodity worldwide, with yearly global productions currently exceeding 300 million tons. Despite their benefits, their recalcitrancy to biodegradation combined with the inefficient collection and recycling mechanisms have resulted in an alarming accumulation of plastic waste in the environment. Polyhydroxyalkanoates (PHA) arose as a potential alternative to conventional plastics. PHA are biodegradable aliphatic polyesters that accumulate intracellularly in microorganisms as carbon and energy reserves, and present properties similar to those of conventional plastics. Despite their environmental advantages, the widespread use of PHA is conditioned by their high production costs, which, so far, prevented them from being cost-competitive with conventional plastics. Research efforts in the last decades have managed to reduce PHA process production costs, but further action is still required. Considering this, the goal of the present work was to develop strategies to, potentially, make PHA production more sustainable by exploring three different approaches. The first approach focused on developing predictive models based on near-infrared (NIR) spectroscopy for the real-time monitoring of critical operation parameters in a pilot-scale fedbatch PHA accumulation stage of a three-stage PHA production process using mixed microbial cultures (MMC) and fruit waste as feedstock. These parameters were the carbon substrate concentration and the intracellular PHA content, as their real-time monitoring would allow to minimize or even prevent microbial PHA consumption between feed-pulses during PHA accumulation, and, consequently, contribute to productivity increase. Good predictive models were obtained for intracellular PHA content, total fermentation products concentration (present in the fermented fruit waste), and butyrate (one of the main fermentation products) concentration. The best model for intracellular PHA quantification presented an external validation coefficient of determination (R2) of 0.98 and a root mean square error of prediction (RMSEP) of 1.75 wt.% PHA in a range of 16.6-49.8 wt.% PHA, while the best model for total fermentation products presented an external validation R2 of 0.97 and a RMSEP of 0.11 gCOD/L in a range of 0.00-1.98 gCOD/L. The best predictive model for butyrate quantification presented an external validation R2 of 0.97 and a RMSEP of 0.12 gCOD/L in a range of 0.00-1.4 gCOD/L. These results demonstrate the potential of NIR spectroscopy to be used in the real-time monitoring and optimization of PHA production from real and complex feedstocks. In the second approach, efforts were directed towards downstream processing, which represents one of the most cost-intensive steps in PHA production. PHA extraction methods, using chemicals with lower environmental impact than organic solvents, were first optimized for a MMC produced in the pilot-scale system described in the first approach, and then for a MMC produced from paper-mill wastewater, also at pilot-scale. This approach focused on PHA extraction methods based on the chemical digestion of non-PHA cell matter (NPCM), due to their potential to be more sustainable and scalable than other processes. For the fruit wastefed biomass, a central composite rotatable design of experiments was performed to determine the optimal conditions for NPCM digestion with either sodium hydroxide (NaOH) or sodium hypochlorite (NaClO). These conditions were found to be a concentration of 0.28 M and a digestion time of 4.8h for NaOH, and a concentration of 8.97% (w/v) and a digestion time of 3.4h for NaClO, having resulted in high purity polymers without significant molecular weight reduction, but distinct thermal behaviors. Afterwards, these conditions were used to assess the impact of intracellular PHA content, biomass concentration and biomass pre-treatment on the PHA extraction performance. The results suggested that the higher the PHA content in the biomass, the higher the purity of the extracted polymers. Conversely, increasing biomass concentration during extraction negatively affected polymer purity. Additionally, biomass pretreatment by drying at 60 °C resulted in polymer degradation, as evidenced by reduced molecular weight and altered thermal properties, highlighting that the use of fresh biomass is preferable to preserve polymer quality. In the extraction of PHA from biomass produced with paper-mill wastewater, a few challenges were found, namely its low PHA content (< 30%) and its recalcitrant impurities, including calcium carbonate (CaCO3) and lignin-related compounds. After determining that the previously developed chemical digestion-based methods using NaOH, NaClO and hydrogen peroxide (H2O2) were ineffective for this biomass, a modified approach was developed to specifically address its complex impurities. This strategy introduced a pre-treatment step aimed at the removal of CaCO₃ and lignin-derived compounds, based on incubation with acetic acid (CH₃COOH) and H₂O₂. The in situ formation of peracetic acid enables lignin degradation, enhances cell disruption, and generates low-toxicity byproducts, while the acidic conditions simultaneously promote the dissolution of CaCO₃. This method was developed and optimized at lab-scale and, afterwards, successfully applied at pilot-scale, where ca. 7 kg of fresh biomass, containing 1.7 kg of solids (with ca. 29% of PHA and 53% of inorganics) were subjected to a first incubation with CH3COOH at 20% and H2O2 at 6% (w/v), at room temperature for 2 hours, and then a second incubation with NaClO at 0.5 g/gorganics, at room temperature for 3 hours. At these conditions, 0.3 kg of dry polymer with a PHA purity of 70% and 4% of inorganics were obtained. This polymer presented properties comparable to those of the polymer extracted using the benchmark protocol (PHA dissolution in chloroform and precipitation in ethanol), in terms of molecular weight and thermal behavior. These results show that the developed PHA extraction method has the potential to efficiently recover PHA from biomass produced with complex wastes, and to be used in large scale. The third approach entailed the development of a framework to assess the sustainability of PHA, and other bioplastics, as well as conventional plastics, in the environmental, economic and societal dimensions, so that these materials can be fairly compared. This life-cycle sustainability assessment (LCSA) framework was built based on information present in the literature and information provided by a group of stakeholders directly involved in the life-cycles of bioplastics and conventional plastics, who responded to a survey elaborated in this context. This work, particularly the insights gathered from stakeholders, made it evident that the economic dimension cannot be the only vector for evaluating the sustainability of plastics/bioplastics. Additionally, an integrated analysis such as LCSA has the benefit of providing a broader overview of the advantages/disadvantages of products and potential trade-offs between the three sustainability dimensions and between its life-cycle stages. The results of this approach provide guidance to perform an overall sustainability assessment of plastics/bioplastics.
- Percrystallization Of Pharmaceutical Compounds Using Inorganic MembranesPublication . Sequeira, Catarina Bonnet; Crespo, João; Duarte, Maria Teresa; Henriques, AntónioConventional active pharmaceutical ingredient (API) manufacturing remains structurally complex, typically requiring crystallization, filtration, drying, and, often, additional particle-conditioning steps. This sequence increases solvent consumption, energy demand, equipment footprint, material handling, contamination risk, and variability in critical quality attributes of the final solid. The challenge is particularly important for small-molecule active pharmaceutical ingredients (APIs), for which crystal size, morphology, polymorphic form, residual solvent content, and powder properties strongly influence downstream manufacturability and product performance. Although continuous and membrane-based crystallization technologies have advanced process control, they still generally produce wet solids that require further isolation and drying. This thesis investigates percrystallization (PerX) as an intensified membrane-based solution to this problem. In PerX, a solution permeates through a porous membrane and solvent evaporates on the permeate side, generating localized supersaturation and enabling the direct formation of dry crystals, while simultaneously recovering solvent, in a single step. This work develops this concept beyond the aqueous systems that dominate the PerX literature by establishing the role of solvent–membrane–solute interactions in organic media, applying the process to ibuprofen as a model API, and extending it to sugar systems to assess broader process versatility. The results demonstrate that PerX can successfully process pharmaceutical-relevant solvent systems, including ethanol, methanol, acetone, and acetonitrile, with high ibuprofen recovery efficiencies above 95% and near-complete solvent recovery. Across all tested conditions, PerX consistently produced ibuprofen in its stable polymorphic Form I, with purity above 99.9%, residual solvent levels within ICH Q3C limits, and low moisture content without the need for a separate drying step. In addition to integrating crystallization and drying, the process also enabled particle engineering: compared with commercial ibuprofen, the PerX-derived material exhibited agglomerates containing smaller, predominantly plate-like crystals instead of the heterogeneous elongated rod to needle-like particles. This morphological change, together with agglomerate formation, resulted in markedly improved flow properties, enhanced tabletability, and reduced tablet friability. Crystal size was strongly correlated with the solvent evaporation flux, showing that PerX offers predictable control over particle attributes through operating conditions. Preliminary application to challenging sugar systems further supported the broader applicability of the concept as a direct route to dry crystalline solids. Overall, this thesis establishes percrystallization as a promising intensified solution-to-powder platform that integrates crystallization, separation, drying, and solvent recovery in a single step, while enabling control over crystal critical quality attributes. By reducing downstream process fragmentation and improving solid-state and powder properties, PerX offers a strong foundation for continuous, efficient, and sustainable pharmaceutical manufacturing.
- Advanced water-soluble soft nanomaterials encapsulating luminescent inorganic and organic emitters and hydrophobic drugs as novel nanocarriers: from bioimaging to drug delivery and environmental applicationsPublication . Duarte, Frederico Gonçalo do Vale; Lodeiro Y Espiño, Carlos; Marques, Elisabete; Cuerva de Alaíz, CristiánFluorescent molecular probes have become indispensable tools across chemical, envi- ronmental, and biomedical sciences, yet current sensing technologies often lack the sensitivity, multifunctionality, and adaptability required to address today’s analytical challenges. With in- creasing concerns regarding environmental pollutants, toxic metal exposure, and real-time di- agnostics, there is a growing demand for highly responsive, cost-effective, and versatile fluo- rescent systems capable of reliable performance across diverse settings. This doctoral thesis addresses this need by developing innovative molecular platforms that combine advanced photophysical design with practical sensing capabilities. It presents the conception, synthesis, and application of next-generation multifunctional fluorophores, show- ing how strategic chromophore engineering can improve detection accuracy, environmental responsiveness, and functional versatility. Central to this research is the construction of eighteen dansyl-based fluorophores in- tegrating tailored receptor architectures, macrocyclic frameworks, and dual-chromophoric as- semblies to enhance optical responses and enable finely tuned sensing pathways. Key advance- ments include (i) the development of solvatochromic and aggregation-induced emission (AIE) systems capable of reporting subtle changes in microenvironmental polarity and water con- tent; (ii) the design of single-chromophore and dyads that exploit ICT, PET, and CHEF mecha- nisms for selective detection of toxic ions such as Hg²⁺, Cu²⁺, and cyanide; (iii) the incorporation of disulfide (S–S) and thiol (–S–) functionalities that induce fluorescence quenching with emis- sion restored upon cleavage and serve as recognition sites for soft-soft selective Hg²⁺ detection; (iv) the design of macrocycle-based dansyl platforms that enhance light absorption and fluorescence intensity while alkyl side chains promote mesomorphic organization, improving stability in aggregated environments; (v) the fabrication of polymeric particles and films providing water stability, temperature-responsive sensing by incorporating the fluorophores which can also act as stabilizers during metallic nanoparticle synthesis; and (vi) the discovery of fluor- ophores exhibiting intrinsic antimicrobial activity, demonstrating multifunctionality beyond sensing. Overall, this thesis shows how multifunctional dansyl-based fluorophores can be rationally designed to combine tunable photophysics, selective sensing, material fabrication, and biological activity. These findings provide a versatile framework for developing next-generation fluorescent materials with applications in environmental monitoring, analytical chemistry, and biomedical diagnostics.
- Marine waste streams valorization for the production of novel bioactive 3D structures and their evaluation for potential biomedical applicationsPublication . Batista, Miguel Pedro Lopes; Bronze, Maria do Rosário; Duarte, Ana; Gaspar, FrédéricThe underutilization of marine byproducts contributes to waste accumulation and economic loss. The scientific community has recently focused on extracting valuable polymers like collagen for high-value products in food, cosmetics, and biomedical applications. However, current recovery methods are time-consuming, chemically intensive, and energy demanding. This dissertation focused on using natural deep eutectic solvents (NADES) to extract marine collagen from fish processing waste. After extraction, this work intended to process the collagen-NADES extract to develop new porous materials. Hence, the main scope of this thesis was to: i) Develop a novel extraction using NADES to intensify the conventional process and charac- terize the protein purity and structure. ii) Perform a comparative life cycle assessment (LCA) of conventional and NADES-enhanced processes. iii) Implement an unexplored route for collagen drying materials using supercritical CO2. iv) Prepare a porous material from chitosan and collagen-NADES extract loaded with hydroxy- tyrosol (HT). Results revealed that the NADES-enhanced process yielded pure type I collagen with 2.5 times higher efficiency while reducing the extraction time from 96 h to 1 h compared to the conventional approach. The LCA of both methods indicated that producing 1 kg of purified marine collagen using this NADES approach reduces 14% of the conventional process’s environmental impact. However, the LCA also showed that extraction processes involving NADES must be deeply scrutinized due to the impact of NADES component production. Drying the gelled collagen extract with supercritical CO2 was a suitable technique to produce collagen aerogels showing a relatively dense mesoporous network with a specific surface area and pore volume of 201–203 m2/g and 1.08–1.15 cm3/g, respectively. Physicochemical characterization confirmed collagen purity and that the production process does not impact protein tertiary structure. Alternatively, incorporating chitosan and HT improved freeze-dried collagen material's mechanical robustness and biological performance. HT showed a controlled release profile, up to 70% after 10 h. Both fabricated 3D structures produced from the collagen-NADES extract exhibited suitable features for potential topical biomedical applications. This dissertation contributes to the circular economy, addressing fish processing waste and the environmental footprint of collagen extraction activities. The present work also promotes biomedical innovation and the valorization of marine byproducts, showcasing the versatility of collagen materials and the therapeutic potential of marine waste.
- Development of polymeric dressings for advanced wound care based on bacterial cellulose and Fu- coPolPublication . Esmail, Asiyah; Freitas, Maria Filomena; Serra, Ana TeresaBacterial cellulose (BC) is a biopolymer characterised by high purity, mechanical re- sistance, water-holding capacity and biocompatibility, making it a strong candidate for wound dressing applications. However, high production costs and lack of intrinsic bioactivity remain major limitations. This thesis addressed these challenges by (i) producing BC from sustainable feedstocks and (ii) developing BC:FucoPol (FP) composites with bioactive properties. BC was produced from unconventional substrates, including polyethene terephthalate (PET) monomers (terephthalic acid and ethylene glycol), styrene and food waste (stale bread hydrolysate and waste apple pulp). BC production from plastic-derived monomers was feasible but yielded ≤1 g/L, requiring glucose supplementation. Food wastes performed far better: stale bread hydrolysate resulted in a production of 2.40 g/L. In comparison, waste apple pulp (APPsup) yielded up to 3.38 g/L, surpassing the production in standard glucose-supplemented Hestrin–Schramm medium (1.5-2.1 g/L). The BC obtained from APPsup also exhibited favour- able mechanical and barrier properties, making it the most promising feedstock among the tested substrates. To confer bioactivity, FP, an exopolysaccharide with antioxidant, photoprotective and wound-healing properties, was incorporated into BC through three strategies. In-situ compo- sites, obtained by adding FP to the culture medium during biosynthesis, enabled homogene- ous FP distribution and retained thermal stability (Tdeg = 317–343 °C), though structural dis- ruption occurred above 1 wt.% FP. Ex-situ Fe³⁺-crosslinked composites, produced by solvent immersion followed by ionic gelation, reinforced the membranes' mechanical performance (Storage modulus (G') up to 57.1 kPa) and promoted cell adhesion and spreading. Still, FP remained in a gelled, less diffusible state. In contrast, simple ex-situ impregnation (BC_IMFP), without Fe³⁺-induced gelation, preserved FP’s bioactivity. Although it reduced the membrane's thermal stability (276 °C vs. 340 °C), it strongly enhanced keratinocyte viability (up to 159%) and significantly improved wound closure (28.3 ± 7.5% in 24 h). Overall, this work demonstrates that BC can be sustainably produced from waste and petrochemical-derived substrates, and the resulting membranes can be functionalised with the bioactive polysaccharide FP to generate multifunctional wound dressings. Each formulation provides distinct advantages for different uses: neat BC membranes can be used for fluid man- agement, the Fe³⁺-crosslinked composites offer improved mechanical reinforcement and pro- mote cell adhesion, and the BC_IMFP displayed fast wound closure ability. These findings es- tablish BC/FP systems as versatile dressings, bridging waste valorisation to advanced thera- peutic applications.
- From Biomarkers to Transcriptomics: Assessing the Impact of PFOA, TBBPA, and EE2 on the Sentinel Species Mytilus galloprovincialisPublication . Copeto, Sandra Cristina Marinheiro Ferreira; Diniz, Mário; Motta, Carla; Silva, MarcoEmerging contaminants, particularly endocrine-disrupting chemicals (EDCs), are in- creasingly detected in marine ecosystems, food, and consumer products, posing risks to both wildlife and human health. Among them, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA), and 17α-ethinylestradiol (EE2) stand out due to their environmental persistence, bioaccumulation potential, and ability to interfere with hormonal regulation, oxidative balance, and cellular homeostasis. However, their toxicological effects on marine bivalves, key sentinel species, remain poorly understood. Mussels such as Mytilus galloprovincialis play a central ecological role and are widely used as bioindicators because of their capacity to accumulate contaminants through filter feed- ing. Assessing their biochemical and transcriptomic responses provides critical insight into the sublethal and realistic effects of EDCs at multiple levels of biological organization. This is par- ticularly relevant given the high seafood consumption in Europe, which represents a major human exposure pathway to these contaminants. Laboratory exposures of M. galloprovincialis were conducted using environmentally relevant concentrations of PFOA (1–100 μg·L⁻¹), TBBPA (1–100 μg·L⁻¹), and EE2 (10–300 ng·L⁻¹). A multi-biomarker strategy was applied to evaluate oxidative stress (SOD, CAT, GST, TAC, MDA), protein degradation (UBI), apoptosis (caspase-3), neurotoxicity (AChE), and endocrine disruption (VTG). In parallel, transcriptomic analyses were performed to identify differentially expressed genes and disrupted pathways related to stress responses, detoxification, lipid me- tabolism, and endocrine regulation. The results revealed concentration-dependent alterations in antioxidant defences and cellular homeostasis, with PFOA and TBBPA inducing oxidative stress and apoptosis, while EE2 primarily triggered estrogenic and gametogenic disruptions. Transcriptomic analysis confirmed the modulation of pathways associated with xenobiotic metabolism, energy balance, apoptosis, and non-genomic estrogen signaling. Together, these results indicate that M. gal- loprovincialis exhibits coordinated biochemical and molecular responses upon exposure to EDCs, reinforcing its relevance as a sentinel species. The study highlights potential risks of contaminant biomagnification through the food chain and provides mechanistic evidence to support ecotoxicological monitoring frameworks and regulatory measures aimed at protecting marine ecosystems and public health.
- The cephalopod skin as a source of proteins: lessons from adaptation and ecophysiologyPublication . Padrão, Inês Rodrigues; Roque, Ana Cecília; Costa, PedroColeoid cephalopods are organisms with high ecological and economical relevance, distinguished by rapid and reversible changes in body coloration, skin patterning and texture. This is achieved by the interplay between pigmentary chromatophores and structural iridophores and leucophores, forming a layered tissue that supports camouflage, signaling and environmental sensing. The skin of cuttlefish and squid have been extensively investigated at the structural and protein levels, while the molecular basis of skin function in Octopus vulgaris remains comparatively less understood. Expanding the knowledge in this area is not only relevant for evolutionary and ecological biology, but also provides a foundation for blue biotechnology, where marine- derived proteins are increasingly recognized as sustainable sources for innovation. The main goal of this thesis is to address this gap by integrating morphological, biophysical and transcriptomic approaches. Light microscopy, transmission electron microscopy (TEM) and focused ion beam-scanning electron microscopy (FIB-SEM) provided insights into the integrated function of chromatophores, iridophores and leucophores in camouflage. Skin protein content analysis and transcriptomics confirmed the presence of reflectins and crystallins, and suggested novel skin sensory roles. Secondly, a reflectin from O. vulgaris (Rov1) was identified, expressed and characterized. This protein was shown to be intrinsically disordered, aggregation-prone and thermally stable with moderate pH-responsive assembly dynamics. Lastly, comparative transcriptomics between the skin of O. vulgaris and the polar squid Galiteuthis glacialis revealed distinct strategies. Octopus skin preferred reflectins and neural associated transcripts for fast camouflage. Squid, however, relied on crystallin-like proteins, chemosensory receptors and muscle structure for transparency, environmental sensing and locomotion. By advancing the molecular and structural characterization of cephalopod skin, this work contributed with new insights into dynamic coloration and sensory integration. These findings also highlight the relevance of cephalopod-derived proteins as candidates for future exploration within blue biotechnology.
- Transdermal Delivery of Pharmaceuticals with Ionic Systems and NanomaterialsPublication . Faísca, Francisco Luís de Aragão; Branco, Luís; Lima, SofiaA administração transdérmica de fármacos constitui uma alternativa promissora às vias convencionais. No entanto, a maioria dos antibióticos, devido à sua elevada polaridade e baixa lipofilicidade, apresenta limitações significativas na capacidade de atravessar eficazmente a barreira cutânea. Esta tese explora a utilização de sais e líquidos iónicos derivados do próprio fármaco (API-OSILs) como estratégia química para melhorar a solubilidade, lipofilicidade e permeabilidade cutânea de antibióticos. Avalia-se ainda a sua integração em plataformas avançadas de administração, incluindo nanopartículas lipídicas e microagulhas dissolúveis. A cefuroxima e a estreptomicina foram selecionadas como sistemas modelo para investigar como a conjugação com iões orgânicos poderá potenciar a administração transdérmica. O trabalho está organizado em quatro estudos principais: (1) síntese e avaliação de sais de cefuroxima (CFX-OSILs), (2) desenho e triagem de sais orgânicos de estreptomicina (STP-OS), (3) formulação destes compostos em nanopartículas lipídicas sólidas e (4) incorporação em dispositivos de microagulhas dissolúveis. Para explorar o potencial da modificação iónica na melhoria da administração transdérmica de antibióticos, a tese inicia-se com a síntese de sais orgânicos de cefuroxima utilizando catiões de piridínio e imidazólio. Estes sais aumentaram a solubilidade original, até 200 vezes superior à do fármaco de base a 37 °C, e modificaram o comportamento de partição, sendo que catiões mais lipofílicos aumentaram a afinidade pela fase orgânica. Entre eles, [PyC10Py][CFX]2 manteve atividade antibacteriana, enquanto outros apresentaram eficácia reduzida. Os ensaios de citotoxicidade revelaram maior toxicidade apenas em sais contendo cadeias longas de alquilo. Embora não fossem candidatos finais à administração transdérmica, este estudo forneceu contributos metodológicos relevantes para a modificação iónica e estabeleceu uma base para aplicar esta estratégia a antibióticos mais desafiantes e clinicamente relevantes. Dando seguimento a esta abordagem, o segundo estudo aplicou uma estratégia semelhante de modificação iónica à estreptomicina, um antibiótico aminoglicosídeo particularmente desafiante. Foram sintetizados oito sais orgânicos de estreptomicina (STP-OS), contendo contra-iões sulfonato e carboxilato, que originaram sólidos estáveis e foram caracterizados sistematicamente. Em estudos de permeação através de bicamadas fosfolipídicas, [STPH3][p-TolSO3]3 apresentou a maior permeabilidade cutânea, [STPH3][C6SO3]3 exibiu um equilíbrio entre permeabilidade e solubilidade, enquanto [STPH3][GlyCOO]3 mostrou maior solubilidade mas permeação negligenciável. Os ensaios antimicrobianos confirmaram a manutenção da atividade contra estirpes suscetíveis. Em conjunto, estes resultados demonstram que a modificação iónica pode ser uma ferramenta versátil para ajustar a solubilidade, lipofilicidade e interação com membranas da estreptomicina, estabelecendo uma base química para a sua incorporação em sistemas avançados de administração transdérmica. Com base nos resultados promissores da modificação iónica, o terceiro estudo investigou a incorporação de STP-OS selecionados: [STPH3][p-TolSO3]3, [STPH3][C6SO3]3 e [STPH3][GlyCOO]3, em paralelo com o sal convencional de sulfato, em nanopartículas lipídicas sólidas (NLS) utilizando Softisan® como matriz lipídica. As nanopartículas resultantes apresentaram cerca de 150 nm de diâmetro, índices de polidispersidade baixos (~0,2) e potenciais zeta negativos (~–30 mV), indicadores de estabilidade coloidal. As eficiências de encapsulação variaram entre 35 e 62%, com cargas de fármaco entre 4-6%, demonstrando que todos os sais eram globalmente compatíveis com o veículo lipídico. Géis liofilizados obtidos a partir destas NLS mostraram-se adequados para aplicação dérmica, com manuseamento e consistência favoráveis. Os ensaios de CIM revelaram maior potência antimicrobiana das formulações em NLS comparativamente aos sais livres, reforçando o valor da encapsulação em nanopartículas. Importa salientar que a formulação bem-sucedida de sais com perfis distintos de solubilidade e permeabilidade demonstrou que a identidade do OSIL não comprometeu a formulação, permitindo ainda afinar o comportamento de administração. O quarto e último estudo incorporou os mesmos três STP-OS em microdispositivos dissolúveis (MAPs) compostos por PVP/PVA. O sal de sulfato convencional apresentou menor solubilidade na matriz polimérica, resultando em menor carga de fármaco quando comparado com os dispositivos contendo STP-OS. Apesar destas diferenças, todos os MAPs apresentaram excelentes propriedades mecânicas, com >99% de inserção confirmada por tomografia de coerência ótica e microscopia. Estudos ex vivo em pele de leitão de espessura total revelaram que, embora a carga total de fármaco variasse, os perfis de libertação e deposição foram comparáveis entre formulações. Curiosamente, os OSILs apresentaram ligeiramente maior retenção nas camadas cutâneas. Estes resultados evidenciam a importância da solubilidade na matriz de formulação e demonstram como a modificação iónica pode expandir as opções de formulação em sistemas de administração transdérmica. Em conjunto, os quatro estudos desta tese demonstram que os API-OSILs representam uma plataforma química para modular a solubilidade, lipofilicidade, permeabilidade e perfis de administração de antibióticos. Além disso, estas modificações podem ser com sucesso transpostas para sistemas avançados de administração, como nanopartículas lipídicas e microagulhas dissolúveis. Embora a extensão da melhoria dependa da molécula, como observado na modulação limitada da estreptomicina altamente hidrofílica, os resultados fornecem uma prova de conceito de que a modificação iónica constitui uma estratégia viável e adaptável para potenciar e otimizar a administração transdérmica de fármacos.