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- Appendicular myological reconstruction and functional analysis of Lourinhasaurus alenquerensis (Camarasauridae, Sauropoda)Publication . Fonseca,Pedro Alexandre Simão da; Lopes,Pedro Daniel Mocho; Calés,Daniel Vidal; Faculty of SciencesIn recent years, advances in the study of the titanosauriform appendicular skeleton have shown key locomotor adaptations and their stepwise evolution. However, the knowledge on some of these adaptations, such as the appearance or degree of wide-gauge posture, on stem members of Titanosauriformes is still limited. The lectotypic specimen of Lourinhasaurus alenquerensis, a Late Jurassic camarasaurid, is the most complete sauropod individual found in Portugal, preserving a remarkably complete set of girdles and limb bones. As one of the few known early-branching macronarians, Lourinhasaurus alenquerensis is important to understand the evolution of locomotor novelties in the lineages leading up to Titanosauriformes. The detailed study of the morphology and osteological correlates of the appendicular skeleton of Lourinhasaurus alenquerensis allowed the reconstruction of its musculature. 3D scanning of the bones also allowed the analysis of its functional capabilities, highlighting key differences compared with Camarasaurus. Lourinhasaurus alenquerensis differs from Camarasaurus in having a longer forelimb, greater range of motion in the forelimb, reduced cingulo-axial musculature, and in the presence of the linea intermuscularis cranialis and medially deflected condyles on the femur. Some of these features are shared with titanosauriforms and are possibly related to the appearance of a wider-gauge stance. It is also possible that Lourinhasaurus alenquerensis had a more verticalized pre-sacral skeleton compared to Camarasaurus, perhaps suggestive of different dietary habits. Given the mosaic of locomotor features in Lourinhasaurus alenquerensis, it probably had a more specialised locomotion than what has traditionally been suggested for Camarasauridae, showing at once convergences with titanosauriforms and synapomorphic traits with its sister taxon, Camarasaurus.
- Efeito da variabilidade genética e da concentração plasmática da osteocalcina e sua relação com os níveis séricos de testosterona em HomensPublication . António,Mariana Filipa Tavares Pais; Barbosa,Ana Paula; Dias,Deodália; Faculdade de CiênciasCurrently, osteocalcin has been studied not only as a bone matrix protein but also as a hormone with potential endocrine actions in different physiological processes. In this context, it has been associated with the regulation of energy metabolism, gonadal function, and bone homeostasis, justifying the investigation of its role in various aspects of male health. The aim of this study was to evaluate the relationship between serum testosterone levels, biochemical, anthropometric, and densitometric parameters, and the genetic variability of osteocalcin (rs1800247) and its receptor GPRC6A (rs2274911), in a population of men attending endocrinology consultations. This was a cross-sectional retrospective study including 123 individuals, divided into four groups according to total serum testosterone concentration (≤2.5; >2.5–≤3.2; >3.2–<4; ≥4 ng/mL). Anthropometric, biochemical, and hormonal data, as well as bone mineral density, were analyzed. DNA extraction and genotyping allowed the identification of the polymorphisms under study, and statistical analysis was performed using different genetic models and tests according to the distribution of the variables. The results showed that higher testosterone levels were associated with favorable markers of bone metabolism, while reduced concentrations were related to dyslipidemia and greater insulin resistance. Consistent correlations were also observed with LH, FSH, SHBG, and estradiol. Regarding genetic polymorphisms, no significant associations were found with serum testosterone levels or bone mineral density, although trends of metabolic influence emerged in certain genotypes. In summary, this study reinforces the relevance of testosterone levels as regulators of bone and metabolic parameters and suggests that the genetic variability of osteocalcin and its receptor may indirectly contribute to these interactions. These findings highlight the importance of future studies in larger populations to clarify the role of these polymorphisms in metabolic and hormonal regulation.
- Identification of genes/proteins linking CFTR to CancerPublication . Fragoso,Mariana Sousa; Pankonien,Ines; Amaral,Margarida Sofia Pereira Duarte; Faculty of SciencesCystic Fibrosis (CF) is a life-threatening genetic disorder caused by pathogenic variants in the CF Transmembrane conductance Regulator (CFTR) gene, which encodes an anion channel for chloride (Cl-) and bicarbonate (HCO3-) transport across epithelial membranes. CFTR variants result in impaired ion transport and mucus clearance in multiple organs, namely in the airways. The approval of CFTR modulator (CFTRm) therapies has markedly increased the life expectancy of people with CF (pwCF). However, new challenges are emerging as the CF population ages, including an increased risk of developing cancer, particularly colorectal cancer (CRC). This project aimed at exploring possible factors linking CFTR dysfunction to cancer using patient-derived intestinal organoids (PDIOs). CF and non-CF PDIOs were cultured and analysed to: 1) assess CFTR protein expression and localisation by Western Blot (WB) and immunofluorescence (IF); 2) determine CFTR interactors by co-immunoprecipitation (Co-IP); 3) evaluate proliferation and stemness; and 4) analyse expression of cancer-related genes. WB and IF confirmed CFTR expression and correct apical plasma membrane (PM) localisation in non-CF organoids and its absence in CF organoids. CFTR was successfully pulled-down in non-CF organoids, verified by co-IP of its interacting partners NHERF1 and Ezrin, providing the basis for future interactome studies on CFTR-related cancer mechanisms. Further analyses revealed an increased LGR5 expression at both protein and mRNA levels in CF PDIOs, indicating an increase in the intestinal stem cell population possibly associated with CFTR dysfunction. Gene expression analysis showed subtle but non-significant differences for several cancer-related genes between CF and non-CF PDIOs, suggesting that CFTR dysfunction alone may not be sufficient to drive oncogenic activation under the studied conditions. Overall, the study established a robust framework for studying CFTR-associated molecular pathways in PDIOs and provides first evidence that CFTR dysfunction affects epithelial stemness, presenting a potential mechanism underlying the increased CRC risk reported in pwCF.
- Insights into the electrochemical reactions of Aryl KetonesPublication . Catarino,Carolina Maria Duarte; Coelho,Jaime Alfredo da Silva; Faculty of Sciences; Department of Chemistry and BiochemistryThe agricultural sector plays a vital role in national development, relying heavily on agrochemicals to address soil nutrient depletion and pest control. The synthesis of agrochemical metabolites is essential for assessing the safety profile of agrochemical active ingredients (AAIs), particularly in relation to their toxicity. Among the metabolic transformations observed in vivo, benzylic and aromatic oxidation, typically catalyzed by cytochrome P450 enzymes during phase I metabolism, are of notable relevance as they are common metabolic pathways for agrochemicals. Organic electrosynthesis has emerged as a sustainable alternative to conventional oxidation methods, offering selective and efficient transformations under mild conditions. In this study, the electrochemical reactivity of metrafenone and related substrates was explored through cyclic voltammetry and electrosynthetic methodologies. Controlled-potential electrolysis of metrafenone successfully yielded the target metabolite CL 379395 (72), with an isolated yield of 19%, alongside two additional oxidation products, a benzylic hydroxylation product (81), obtained with 6% qNMR yield, and an aromatic hydroxylation product (82), with 42% qNMR yield, encouraging further optimization of the electrolysis conditions. The successful generation of metabolite CL 379395 (72) demonstrates the potential of organic electrosynthesis as a valuable tool for accessing such compounds. For ASCENZA Agro, the outcome was productive, since, even with a moderate yield, they can produce the quantity required for toxicological evaluation.
- Optimization of intersectional genetics-based biosensors for human cell targeting purposesPublication . Félix,Sara Grilo; Gontijo,Álisson Marques de Miranda Cabral; Menezes,Juliane; Faculty of Sciences; Department of PhysicsGenetically targeting human cell types with precision is a central challenge in synthetic biology and biomedicine. Intersectional genetic circuits address this using multiple inputs, ensuring that only cells with a unique regulatory signature activate the circuit. A common implementation uses multi-split proteins that are reassembled in the target cell type by split-inteins. Here, we sought to improve the reconstitution of two split-proteins (the TetR*-KRAB transcriptional repressor and the Cre recombinase), and to create a stable human induced pluripotent stem cell (hiPSC) line to report Cre activity. Our lab had previously engineered a TetR*-KRAB that is splitable into 2 to 4 fragments using split-inteins. The 4x-split design, which had the highest potential for cell targeting, performed suboptimally likely due to one of the split-inteins used, NrdJ-1. To overcome this, we substituted NrdJ-1 by split-inteins IMPDH-1 or RmaDnaB, but found little improvement in performance, so further work, likely on split-site choice, is required to ameliorate TetR*-KRAB reconstitution. Regarding Cre, previous split-versions near its catalytic residue 173, rendered it non-self-complementing–a valuable trait for intersectional genetics–, but also non-functional, likely because of an extein scar left at the split site after reconstitution. Here, by modifying scar length, we found that this split site is extremely sensitive to even small (3-residue) extein scars, and that local endogenous aminoacids could not support reconstitution of a scarless split-Cre, suggesting a requirement for more context-independent splitinteins to overcome this limitation. Finally, we identified a hiPSC line carrying a stably-integrated Cresensitive fluorescent reporter cassette, which showed homogeneous reporter expression and robust responsiveness to Cre. Together, this work contributes to design principles for multi-split protein reconstitution in human cells and delivers an integrated Cre reporter hiPSC line that can accelerate the development of intersectional biosensors for precise cell-type targeting.
- Análise das necessidades e alternativas aos PFAS na indústria nacional : substituição em membranas de troca de protõesPublication . Cardoso, Bruno Alexandre Santos; Lourenço,Maria José Vitoriano; Alexandre,João Henrique Almeida; Faculdade de Ciências; Departamento de Química e BioquímicaThousands of chemical substances are used daily which, although largely unfamiliar to the public, play a crucial role in society due to their versatility and wide-ranging applications. Among these are PFAS (per- and polyfluoroalkyl substances), employed across many sectors and products, from medical devices to firefighting foams. In response to the growing concerns about the risks associated with PFAS, proposals are currently being discussed within the European Union to impose a universal restriction on their production, use, and/or placing on the market. If implemented, such a measure would ban over ten thousand substances, compelling European industry to identify and adopt suitable alternatives in order to maintain operations. This dissertation was structured in two main phases. The first examined the potential impact on the national textile and energy sectors of restricting PFAS. Analysis of available information, complemented by the engagement with key stakeholders, indicated that despite limited awareness of the current PFAS context, the energy sector would face the greatest challenges, particularly regarding Nafion proton exchange membranes used in fuel cells. The second phase focused on evaluating potential substitutes for Nafion. Ten candidates were identified and assessed, yet the applied methodology allowed to demonstrate that, in the medium term, none represents a viable alternative. These findings underline the urgent need for continued research and innovation to develop effective PFAS substitutes, including suitable alternatives to Nafion in fuel cell membranes.
- Exploring choline : quinone oxidoreductase from pathogenic bacterium Staphylococcus aureusPublication . Ribeiro,Inês Março Tomás de Castro; Pereira,Manuela Alexandra de Abreu Serra Marques; Calisto,Filipa; Faculty of SciencesStaphylococcus aureus is an opportunistic pathogen causing severe, life-threatening infections. Its metabolic versatility enables survival in diverse hostile environments. Choline: quinone oxidoreductase (ChoQO) contributes to its ability to grow under high salinity environments and may hold potential for medical and biotechnological applications. This work aimed to explore the molecular and cellular roles of ChoQO from S. aureus, with a focus on its contribution to energy metabolism and functional-structural relationships. ChoQO production and purification faced several challenges, nevertheless it was succeeded in the production of the protein after several troubleshooting procedures. Cloning of the betA gene into pET-28+ failed due to unsuccessful restriction-ligation cloning. This was overcome by constructing and ordering recombinant plasmid to a commercial company. Protein production at 37 ºC resulted in its presence in inclusion bodies, but lowering the temperature allowed the production of soluble protein. However, ChoQO from cytosolic and membrane fractions did not bind to His-Trap or Q-Sepharose columns, consistent with reported instability of this protein once removed from membrane environment. Flavoprotein-like spectra confirmed ChoQO presence, but no enzymatic activity was detected under the tested conditions, likely due to lack of purified protein. It was investigated the role of ChoQO by assessing the growth behaviors of a transposon mutant with an interrupted gene coding ChoQO. In growth assays, the transposon mutant grew similarly to the wild-type strain under standard conditions but showed impaired growth under osmotic stress. Supplementation with glycine betaine rescued this phenotype, highlighting the importance of the osmoprotectant and confirming ChoQO’s role in its biosynthesis pathway. Overall, this work provides new insights into ChoQO in S. aureus, highlighting its potential as a therapeutic target. Further biochemical studies are required to define functional domains and interaction partners, while complementary analysis involving betB and metabolomics approaches may deepen our understanding osmotic stress in S. aureus.
- Impacts of anthropogenic pressure on genetic and phenotypic diversity of the African elephant (Loxodonta africana) in southern AfricaPublication . Santos,Teresa Leonor Nunes dos Santos Nobre dos; Fernandes,Carlos Alberto Rodrigues; Mumby,Hannah S.; Gilbert,Marcus Thomas Pius; Faculty of SciencesA pressão antropogénica é uma força evolutiva cada vez mais influente e que pode moldar as caraterísticas genéticas e fenotípicas das populações e das espécies. O elefante da savana africana (Loxodonta africana) é uma espécie emblemática e carismática com a qual os seres humanos têm interagido directamente desde há milénios. Os elefantes têm sofrido especialmente com a fragmentação do habitat e a caça para obtenção de marfim. Uma das regiões africanas que, no passado recente, foi afetada por estas acções é a África Austral. As populações desta região sofreram alterações demográficas consideráveis mediadas pelo homem, e o comércio ilegal de animais selvagens ainda ocorre na região. Porém, a África Austral detém actualmente algumas das maiores populações do continente, que são indiscutivelmente muito importantes para a sobrevivência a longo prazo da espécie. Esta tese tem como objetivo investigar os impactos genómicos e fenotípicos das acções humanas nas populações de elefantes selvagens na África Austral e fornecer informação para acções de conservação. Especificamente, descrevemos a diversidade genómica contemporânea das populações da África do Sul e mostrámos que uma rápida velocidade de recuperação demográfica pode travar a erosão genómica. Também identificámos novas sequências de mitogenoma para áreas com informação genómica limitada em Angola e Moçambique, e verificámos uma diminuição do tamanho das presas obtidas através da caça de troféus na maior parte do continente. Por último, investigámos os artefactos e as fontes de enviesamento que podem surgir quando se combinam conjuntos de dados genómicos para realizar análises genómicas temporais. Esta tese oferece informações para esforços de conservação como translocações e identificação da proveniência de marfim, bem como sobre as melhores práticas na realização de estudos genómicos que recorrem a várias técnicas de sequenciação genómica.
- Plasmid diversity and evolution in the bacterial worldPublication . Rebelo,João Santos; Dionísio,Francisco André de Campos Pereira; Monteiro,Francisca; Faculty of SciencesAs bactérias estão distribuídas na natureza de forma desequilibrada em duas bioesferas distintas: uma “bioesfera comum”, composta por poucas espécies altamente abundantes, e uma “biosfera rara”, composta por muitas espécies presentes em baixíssimas abundâncias. Apesar de escassas em número, estas espécies bacterianas da biosfera rara desempenham funções ecológicas cruciais, como regulação do ciclo de nutrientes e degradação de compostos específicos, sendo essenciais para a estabilidade e funcionamento dos ecossistemas. O avanço das técnicas de sequenciação de nova geração permitiu revelar a diversidade e complexidade destas espécies raras, destacando a sua importância na resiliência e no equilíbrio das comunidades microbianas, especialmente em condições ambientais variáveis. Mecanismos de comunicação bacteriana (como por exemplo o quorum sensing), formação de biofilmes e comportamentos cooperativos mediados por seleção de parentesco desempenham papéis relevantes na regulação destas comunidades, permitindo que bactérias geneticamente relacionadas beneficiem umas das outras em ambientes estruturados, como intestino, solos e sedimentos. Frequentemente, as bactérias transportam plasmídeos. Os plasmídeos, elementos genéticos extracromossómicos e autorreplicativos, desempenham um papel central na ecologia, evolução e adaptação das comunidades bacterianas, estando presentes em praticamente todos os ambientes, desde solos a ambientes aquáticos. Capazes de codificar genes que conferem resistência a antibióticos, fatores de virulência ou tolerância a metais pesados, os plasmídeos contribuem para a rápida aquisição de características vantajosas pelos seus hospedeiros, mesmo em ambientes sob pressão seletiva. A capacidade de mobilidade e plasticidade genética faz dos plasmídeos elementos fundamentais para a regulação das comunidades microbianas, influenciando a dinâmica de aquisição de novas funções e promovendo a adaptação a ambientes em mudança.
- From molecules to manufacture : an analytical portfolio definition for enhanced Dry Powder Inhaler (DPI) screeningPublication . Veiga,Cíntia Leonora Semedo; Rodrigues,Mário Manuel Silveira; Almeida e Sousa,Luís; Faculty of SciencesA administração terapêutica de fármacos por inalação desempenha um papel fundamental no tratamento de doenças respiratórias, sendo os inaladores de pó seco um dos principais sistemas de administração. A otimização das formulações para inalação requer um conhecimento avançado das várias interações entre partículas, de modo a maximizar a deposição nos alvéolos pulmonares. Este estudo desenvolveu uma abordagem analítica para prever o desempenho aerodinâmico, analisando atributos-chave da formulação, como o tamanho das partículas, a energia de superfície, a agregação e a dinâmica de desaglomeração. Foi desenvolvida uma nova metodologia de Calorimetria de Solução (Solcal) para avaliar a coesão das partículas, demonstrando que a entalpia de dissolução é um preditor da fração de partículas finas (FPF). Para além disso, foi desenvolvido um modelo matemático que correlaciona, com elevada precisão, as propriedades das partículas e formulações com o desempenho aerodinâmico para sistemas homogéneos (um componente) Os resultados demonstraram que a concentração de ingrediente farmacêutico ativo e o teor de excipientes finos influenciam significativamente a performance aerodinâmica, com concentrações ótimas de excipientes finos a favorecer a desaglomeração até a um limite, que a partir do qual a coesão entre partículas prejudica a performance. As técnicas de engenharia de partículas, como a micronização por jet-milling e o wet-polishing, modificaram as forças de adesão e as propriedades de dispersão. Além disso, a incorporação de leucina em formulações sem carreador melhorou a dispersão e a performance aerodinâmica, reduzindo a coesão e estabilizando a energia de superfície. Este estudo desenvolveu um modelo preditivo e metodologias analíticas para otimizar formulações de DPIs. Estudos futuros deverão incluir a melhoria do modelo, através da inclusão de fatores específicos que tenham em conta o inalador e formulações mais complexas, contribuindo para o desenvolvimento de sistemas inovadores de administração pulmonar de fármacos.