FC - DQ | Dissertações de Mestrado e Teses de Doutoramento
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- Obesogens-induced deregulation of periprostatic adipose tissue: a driven force in the onset and progression of prostate cancer?Publication . Feijó, Mariana Pombal ; Socorro, Sílvia Cristina da Cruz Marques; Correia, Sara Carina de Lima; Kiss-Tóth, EndreProstate cancer (PCa) is a hormone-dependent cancer whose development and progression are strongly influenced by the tumour microenvironment and exogenous factors, such as environmental influences. The periprostatic adipose tissue (PPAT), by its anatomical proximity and functional crosstalk with prostate cells, emerged as a key driver of tumour growth, particularly in obesity, with the secretome of “obese” PPAT being associated with enhanced tumour aggressiveness. On the other hand, epidemiological and experimental studies have implicated endocrine-disrupting chemicals (EDCs) as environmental risk factors for PCa. Given the hormone dependency of PCa, it is predictable that it is a cancer highly susceptible to the influence of environmental exposures, namely EDCs and specifically those with obesogenic properties (i.e. obesogens), which are capable of disrupting both endocrine and metabolic pathways. Notably, based on their mechanisms of action and the cellular and molecular alterations they induce, obesogens may promote tumorigenesis either directly by acting on prostate cells or indirectly by inducing adipose tissue dysfunction. However, the extent to which obesogenic compounds drive these alterations and the consequent impact on prostate tumorigenesis remain largely unknown. Moreover, despite the well-established effects of obesogens on adipose tissue, no study has characterised their actions on PPAT. Addressing these gaps is critical to understanding how environmental factors intersect with adipose tissue biology, influencing interorgan communication between the prostate and adipose tissue and PCa development. Based on this scientific rationale, this doctoral thesis hypothesises that obesogen-induced PPAT dysfunction represents a driving force in the initiation and progression of PCa. Tributyltin (TBT) is a well-characterised obesogenic EDC and a potent regulator of adipogenesis, widely used in experimental settings to investigate the effects of obesogens. Therefore, using the obesogen model TBT this thesis aimed to: (i) characterise the morphological and secretory alterations of PPAT following TBT exposure; (ii) assess the impact of TBT-induced PPAT dysregulation on prostate cell fate, metabolism, oxidative and inflammatory status, and response to chemotherapeutic drugs; and (iii) identify the molecular targets and signalling pathways mediating the crosstalk between dysregulated PPAT and prostate cells. First, it was demonstrated that in vivo exposure to TBT (50 μg/kg) besides increasing rat body weight, enhanced PPAT somatic index and altered its functional phenotype. TBT treatment promoted a shift in rat prostate cells toward a glycolytic and lipogenic metabolic profile and stimulated oncogenic signalling pathways, including increased phosphorylated/total protein kinase B (pAKT/AKT) ratio and androgen receptor expression. Moreover, macrophage infiltration and a shift in macrophage polarisation towards a pro-inflammatory phenotype were observed both in the prostate and PPAT of TBT-exposed animals, suggesting that TBT can perturb the local prostate-PPAT immune status, contributing to an environment permissive to prostate carcinogenesis. These findings confirmed the effects of TBT on prostate cells, supporting the hypothesis and the investigation into the contribution of PPAT-mediated effects in altering prostate cell behaviour. Culture of the PPAT from rats exposed to TBT clearly demonstrated that TBT induced a dysregulation of the PPAT secretome. TBT-treated PPAT (TBT-PPAT) displayed increased leptin/adiponectin ratio and C-C motif chemokine ligand 7 (CCL7) levels. This adipokine/chemokine profile induced by TBT mimics that observed in obesity and is concurrent with a metabolic reprogramming associated with enhanced glucose, free fatty acids, and lipid peroxidation. Importantly, ex vivo exposure of rat PPAT to TBT (100 nM) recapitulated the findings obtained in vivo concerning the features of its secretome, which are capable of having an impact on prostate cell fate. The results obtained in the subsequent preclinical approaches using co-cultures and conditioned media (CM) assays confirmed the ability of TBT-PPAT enhance the viability, proliferation and migration, as well as apoptosis resistance, in all the studied prostate cell line models, namely non-neoplastic prostate epithelial cells (PNT1A), and androgen-sensitive (22Rv1) and androgen-insensitive (DU145 and PC3) PCa cells. Notably, the TBT-PPAT secretome increased the expression of CCL7 receptor, the C-C motif receptor 3 (CCR3), in prostate cells, which, together with the enhanced CCL7 secretion observed in our experimental setting, raised curiosity about the role of the CCL7-CCR3 axis underlying the pro-tumorigenic effects of TBT-PPAT. The use of a CCR3 antagonist significantly reduced TBT-PPAT induced migration across all cell lines, allowing to implicate the CCL7 and CCR3 in the observed responses of prostate cells. Other molecular targets beyond the CCL7-CCR3 axis were also highlighted. It is the case of tribbles homolog 1 (TRIB1), a pseudokinase involved in tumorigenesis and lipid homeostasis that was overexpressed across all studied cell models exposed to TBT-PPAT-CM. Cell fate alterations observed upon TBT treatment were accompanied by metabolic changes with distinct outcomes in non-neoplastic and neoplastic cell lines: in PNT1A, enhanced fatty acid β-oxidation and synthesis indicate a plausible shift toward a cancer-like metabolic profile; in 22Rv1, the unaltered metabolic and oxidative status suggests the activation of alternative signalling pathways sustaining TBT-PPAT effects; in DU145 and PC3, the distinct metabolic responses observed underscore the differential responsiveness of androgen-insensitive PCa cell subtypes to adipose-derived cues. The investigation in the present thesis was extended to a clinically relevant setting. Human PPAT obtained from patients submitted to radical prostatectomy or prostatic adenomectomy (Millin’s procedure) was treated ex vivo with TBT (100 nM) to confirm if the human tissues resemble the pro-tumorigenic cues identified in controlled experimental models. This approach demonstrated that human PPAT is a target of obesogenic dysregulation and showed that the secretome of obesogen-dysregulated PPAT can significantly enhance the viability of prostate cells. Moreover, the presence of human TBT-PPAT reduced the sensitivity of PCa cells to docetaxel and cabazitaxel, suggesting that obesogenic dysregulation contributes to PCa resistance to taxane-based chemotherapy. Overall, the scientific evidence gathered in this thesis identifies PPAT as a key target of obesogenic EDCs, which disrupt PPAT function and its crosstalk with prostate cells, thereby contributing to the initiation and progression of PCa. These findings open new avenues for developing interventions aimed at modulating PPAT activity to counteract its tumour-promoting effects and emphasise obesity as a critical modulator of PCa aggressiveness.
- Biointeraction studies of recombinant HPV16 oncoproteins to discover therapeutic inhibitorsPublication . Gomes, Diana Vanessa Duarte ; Sousa, Ângela Maria Almeida de; Passarinha, Luís António PaulinoCervical cancer (CC) remains a significant global health burden, particularly in low- and middle-income countries, where it is a leading cause of cancer-related mortality among women. Annually, more than 600,000 new cervical cancer cases are diagnosed worldwide, with a 53% mortality rate. The development of CC is strongly associated with a persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, which are responsible for 70% of cases. This persistent HPV infection leads to uncontrolled proliferation of infected cells via downregulation of epithelial differentiation, disruption of epidermal development, and evasion of innate immune responses. The oncogenic potential of HR-HPV types is primarily attributed to the expression of E6 and E7 oncoproteins, which are integral to cancer phenotype maintenance in HPV-infected cells. Despite the implementation of preventive strategies, such as prophylactic vaccination and screening, which have contributed to a reduction in disease incidence, there remains an urgent need for targeted therapeutic interventions aimed at addressing persistent HPV infection and advanced cervical lesions. The HR-HPV E6 protein is composed of 158 amino acids and 2 zinc-binding motifs, and its most important function is escaping cell death by the induction of p53 degradation. In fact, E6 interacts with the LxxLL motif of the E6-associated protein (E6AP), an E3 ubiquitin ligase, inducing p53 degradation through the proteasome pathway and, consequently, blocking p53-dependent apoptosis. The HR-HPV E7 protein has around 98 amino acids, with an N-terminal region intrinsically disordered, whereas the C-terminal features a well-structured zinc-binding site. One of the most important protein regulation mechanisms by the E7 protein is through the retinoblastoma (pRb)/E2F system. The E7 protein binds to the pRb through the motif LxCxE, displacing E2F complexes, allowing its release and, as a result, the transcription of S-phase genes. This ensures that the infected cells remain in an S-phase-competent state, leading to hyperproliferation. Considering their crucial role in driving cellular transformation and cancer maintenance, both proteins are primary targets for developing targeted HPV therapies. However, the rational design and optimization of therapeutic strategies may depend on the ability to obtain both proteins. Unfortunatelyss, isolating E6 and E7 proteins from human cells is not feasible, so recombinant technology is a great alternative. Nonetheless, challenges persist related to the low solubility and tendency of these proteins to aggregate. Overcoming these issues is essential for enabling structural studies, biophysical characterization, and high-throughput screening of potential inhibitors. Thus, this thesis proposes the implementation of a biotechnology platform, starting with the biosynthesis towards the purification of the HPV16 E6 and E7 proteins, to ultimately discover inhibitory drugs that will be able to block the oncogenic effects triggered by both targets through biophysical and in vitro approaches. Firstly, we established a biotechnological platform for the recombinant expression and purification of HPV16 E6 in Escherichia coli BL21, employing a dual-tagged construct to enhance yield and address solubility issues. A two-step chromatographic process: affinity chromatography, followed by immobilized-metal affinity or size exclusion chromatography, obtained highly pure protein fractions (~98%). Circular dichroism confirmed that the secondary structure of His6-MBP-E6 protein is preserved in both purification strategies. Additionally, thermal shift assay (TSA) suggested potential additives for protein stabilization, such as maltose, glycerol, and detergents that can be incorporated in further characterization studies. In parallel to obtain the E6 protein, we focused on identifying natural compounds that may disrupt the E6/E6AP interaction using virtual screening. Three candidates selected from in silico studies with favorable predicted binding affinities were evaluated in HPV-negative and HPV-positive cell lines. Taxifolin and lucidin protected p53 from E6-mediated degradation and reactivated its transcriptional activity, leading to apoptosis induction. Nevertheless, neither compound affected cell cycle progression; thus, we extended the search for E6 inhibitors targeting directly the p53 binding site by exploring four distinct families of small molecules (barbiturates, dihydropyrimidinones/thiones, acetanilides and steroid derivatives). The screening was achieved through a combination of computational strategies and biophysical assays using the recombinant E6 protein previously produced. Fluorescence-based studies suggested that 4 compounds displayed a moderate affinity for the E6 protein. These candidates (CMP44, CMP50, CMP10c and CMP11c) were further characterized in HPV-positive and HPV-negative cell lines. The results demonstrated that CMP50 and CMP11c effectively rescued p53 from E6 degradation, reducing the proliferation and migration of HPV-positive cancer cells. Additionally, both compounds reactivated p53-dependent transcriptional factors, activating an apoptotic pathway and inducing cell cycle arrest, which suggests their therapeutic potential against HPV. Finally, considering the aim is also to study the E7 protein and potential inhibitors, we explored and characterized the dual-tagged recombinant HPV16 E7 protein. Following a similar workflow to what we had built for the E6 protein, we explored several chromatographic approaches, including affinity, anion-exchange, mixed-mode, and sizeexclusion chromatography, to obtain the His6-MBP-E7 protein. A thorough analysis of buffer compositions and additives indicated that a pH between 6.0 and 7.0, as well as sugars and detergents, maximized E7 solubility and minimized sample heterogeneity. In this process, we obtained the target protein with high yield, a purity of around 98% as confirmed by LC-MS/MS analysis. Additionally, the inclusion of detergents in the final formulations was studied, where the inclusion of DM (η-Decyl-β-D-Maltopyranoside) or η-Dodecyl-β-D-Maltopyranoside (DDM) led to a monodisperse sample. The optimized protocol is suitable for downstream structural and functional characterization, as well as for future inhibitor screening studies. Collectively, the work presented in this thesis establishes a reproducible platform for the production and study of the HPV16 E6 and E7 oncoproteins, which can be used for expressing proteins from other HPV types. Additionally, by integrating computational, biophysical, and cellular approaches, this research identified specific anti-HPV drugs, which can be the starting point for structural studies aiming at obtaining structures of the protein-inhibitor complexes.
- Non-canonical DNA secondary structures as a therapeutic strategy for lung cancerPublication . Miranda, André Filipe Rodrigues ; Cruz, Carla Patrícia Alves Freire Madeira; Mergny, Jean-Louis; Oliveira, Paula Alexandra Martins deNucleic acids store and control genetic information and, beyond the canonical B-form DNA double helix, can adopt a variety of non-canonical architectures, including G-quadruplex (G4s), i-motifs (iMs), triplexes, R-loops, and Z-DNA/Z-RNA. These structures are widespread across genomes and transcriptomes and play important biological roles (transcription, replication, translation, and genome stability). They are recognized as regulatory hotspots enriched at promoters, enhancers, telomeres, untranslated regions, and viral genomes and their involvement in cancer, viral infection, neurodegeneration, and inflammatory disorders. Their in vivo functions have also opened therapeutic avenues both as drug targets and therapeutic tools. As drug targets, these non-canonical structures can be modulated to alter cellular behavior, while as therapeutic agents, nucleic acids can be engineered to adopt non-canonical folds that bind key cellular partners and exert biological effects. This dual role is unusual for other approaches and positions non-canonical structures at the forefront of modern drug discovery. These structures are emerging as important tools in oncological research and therapeutic development. Proto-oncogenes encode proteins essential to normal physiology; however, when mutated or amplified become oncogenes, they drive unchecked growth and survival. The transcription factors (TFs) are a particularly important class of proto-oncogenes that bind to DNA and recruit co-regulators, orchestrating broad gene-expression programs. Their sweeping control of transcription makes them powerful cancer drivers but also challenging drug targets. Unlike enzymes with well-defined catalytic pockets, TFs often rely on flexible, intrinsically disordered regions for interactions, contributing to their reputation as “undruggable.” Numerous TFs have been reported as dysregulated in cancer and are frequently correlated with poor prognosis and resistance to chemotherapy. Other TF are gaining relevance, such as B-MYB (MYBL2), a transcription factor from the MYB family required for normal cell-cycle progression, which acts as an oncogene when overexpressed or deregulated. Functionally, the B-MYB protein serves as a master cell-cycle regulator by integrating into multiprotein assemblies, most notably the DREAM and MMB complexes, that coordinate cell-cycle gene expression. Beyond these physiological functions, B-MYB is upregulated in multiple cancers, such as breast and lung, and its overexpression is correlated with aggressive disease, treatment resistance, and unfavorable prognosis. The molecular mechanisms linking B-MYB to tumorigenesis include gene amplification, cell cycle deregulation, genomic instability, apoptosis suppression, post-transcriptional and post-translational modifications, or can contribute to epithelial-to-mesenchymal transition. Thus, B-MYB TF can be seen as a relevant clinical biomarker and one of the main orchestrators of carcinogenesis; however, until now, no pharmacological therapy against B-MYB has been developed, also related to its “undruggable” profile. Also, it’s known, by bioinformatic analysis of the human genome, that oncogene promoter regions are G/C rich around transcription start sites (TSS), which allows the formation of structures called G4 or iM. The G4s are formed by the self-association of four guanine bases in a quasi-planar arrangement via Hoogsteen bonds and are described as having a gene regulatory function, namely at the transcriptional level, while the iM is formed by Hoogsteen bonds between protonated and deprotonated cytosines. Motivated by these observations, the central objective of this thesis was to explore alternative routes to target the B-MYB oncogene using non-canonical nucleic-acid structures: first as drug targets and later as therapeutics. The thesis started by the identification of G-rich sequences at the B-MYB promoter capable of forming G4 structures. The identification of G-rich sequences was performed using the G4Hunter algorithm, and their conservation across mammalian species was also verified. The experimental validation of their formation was performed by combining 10 biophysical and biochemical methods. Later, the in-cell relevance of G4 structures was evaluated employing the G4access method, which reveals that from the predicted sequences, only the most stable one (B-MYB 43R) was shown to be significantly formed in cells, evidencing a potential impact on the transcription of this gene to its location closest to the TSS. Next, the ability of C-rich sequences to form iM structure was evaluated. Again, the iM-forming sequences were predicted using the G4Hunter algorithm and the experimental validation started by circular dichroism (CD) and nuclear magnetic resonance (NMR) to determine if the sequences fold into an iM structure. Then, stability parameters such as pHT (pH transitional midpoint) were determined by acquiring spectra at different pH values (between pH=5 and pH=8; 0.25-unit increments). The thermal stability and thermodynamic parameters were also calculated using the denaturation and renaturation melting curves. Then, the in-cell formation was assessed using iM-CUT&Tag experiments in HEK293T, which revealed the formation of the iB-MYB 43 that was characterized to have the highest Tm and the highest pHT among the studied sequences. After this characterization, the interaction of small molecules with iB-MYB structures was assessed. As a general tendency, the ligands did not affect the CD spectral shape; however, some of them evidenced changes in secondary structure or thermal destabilization. After evaluating the capacity to form G4 or iM, we continued to validate the G4 formation within a cellular environment using a G4-triggered fluorogenic hybridization probe. This strategy circumvents the limitations of the antibodies and small-molecule probes, which show a lack of structural selectivity labeling diverse DNA and RNA G4s indiscriminately, and cannot specifically target the desired G4. The molecular probe was composed of G4-recognizing light-up ligand (acridine derivative) with an antisense oligonucleotide that hybridizes adjacent region of B-MYB G4. Before probe synthesis using a click chemistry approach, the ligands were photophysically characterized, biophysically evaluated against G4, and further validated spectroscopically and in cells. Cellular studies confirmed the co-localization between the molecular probe and B-MYB G4 in-cell, offering promise for future applications in cancer research in terms of targeted therapies and monitoring of G4. Although G4s are highly dynamic and topologically diverse, promoter G4s are commonly parallel, whereas antiparallel forms are underreported and less characterized. In earlier work, we identified a B-MYB promoter sequence (B-MYB 26RA), that forms an antiparallel G4. Thus, a biophysical pipeline, starting in solution NMR spectroscopy alongside in-cell NMR studies, was made to predict a three-dimensional model (antiparallel quadruplex-duplex junction) and to assess the formation in a live complex environment of B-MYB 26RA. Interestingly, B-MYB 26RA evidenced an ionic sensitivity to K+ and Na+, increasing their topological dynamics, and demonstrated that, when associated with PhenDC3 ligand, a conformational shift from hybrid to antiparallel topology happens. Then, we moved to B-MYB 43R G4 to discuss the therapeutic relevance and the capacity to be targeted by small molecules. Thus, using spectroscopic methods, the G4 formation and its interaction with a panel of G4-stabilizing ligands were confirmed. From the tested ligands, PhenDC3 and TMPyP4 demonstrated the highest binding affinity and stabilization and revealed that both ligands inhibited proliferation and migration in two lung cancer cell lines (A549 and H1299), with PhenDC3 showing potent cytotoxic and cytostatic effects. Furthermore, PhenDC3 upregulated B-MYB expression despite its strong phenotypic effects, highlighting the complexity of G4-targeting mechanisms and supporting the growing evidence that ligands do not always downregulate their target genes. Finally, we explored the use of Polypurine Reverse-Hoogsteen (PPRH) hairpins as an alternative therapeutic approach to target the B-MYB promoter. PPRH are DNA oligonucleotides that fold into intramolecular hairpins and bind complementary polypyrimidine, forming stable DNA triplexes, impeding transcription, and displacing the G-rich strand, inducing G4 formation, enabling dual-level regulation of oncogene expression. Thus, using a bioinformatic tool (TFO Searching Tool), a PPRH was designed against the G4-forming region at the B-MYB promoter (B-MYB 43R), and then experimentally assessed the triplex formation using biophysical methods. The biological effects of designed PPRH, evaluated in lung cellular models (A549, H1299 and MRC-5), demonstrated a low cell viability and clonogenic capacity accompanied by mRNA expression reduction and proteomic profile alterations. Thus, the combination of triplex-based approaches with G4 biology could be a future venue for therapy. Overall, this thesis establishes non-canonical nucleic-acid structures as both actionable targets and therapeutic agents. As a target, the formation of G4 and iM structures at the promoter region of the B-MYB oncogene; was provided new insights about structure and topological dynamics according to the surrounding environment; was validated their formation in-cell, as well as explored the interaction with small molecules. On the other side, the therapeutic potential was explored using PPRH, which revealed a selective and powerful tool to target B-MYB. Together, these findings provide fundamental insights and open new avenues for translational research on the B-MYB oncogene.
- Development and validation of a method for the determination of cocaine and its metabolites in hair using QuEChERS and GC- MS/MSPublication . Brito, Hugo Bernardo Marques Antunes de; Alba, Maria Eugénia Gallardo; Pires, Bruno Miguel Pinheiro; Rosado, Tiago Alexandre PiresCocaine (COC) is one of the most widely consumed illicit stimulants worldwide, with a high addictive potential and severe toxicological effects. Hair analysis offers unique advantages in forensic toxicology, as it allows the documentation of chronic use and, in some cases, acute exposure. In this study, a miniaturised version of the QuEChERS (Quick, Easy, Cheap, Rugged, and Safe) extraction method was developed for the determination of COC and its major metabolites — anhydroecgonine methyl ester (AEME), ecgonine methyl ester (EME), cocaethylene (COET), benzoylecgonine (BEG), and norcocaine (NCOC) — in hair samples. The procedure involved 3 mL of formic acid (5%) in acetonitrile (ACN) and 625 mg of NH4HCO2 as solvent and partitioning salt, respectively, followed by a dispersive solidphase extraction (d-SPE) step using 175 mg of MgSO4 and 55 mg of primary secondary amine (PSA). Samples were analysed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Following optimisation through design of experiments (DoE), the method was validated according to the guidelines of the Society of Hair Testing (SOHT). The working range was set at 0.05–5 ng/mg for all analytes, except AEME (0.5–5 ng/mg). Recoveries ranged from 20–29% (AEME), 24–27% (EME), 33–42% (COC), 43–77% (COET), 25– 48% (BEG), and 27–37% (NCOC). The method was successfully applied to authentic hair samples, in which COC was detected in almost all cases, often accompanied by metabolites at concentrations above the lowest limit of quantification (LLOQ). The µ-QuEChERS method proved to be a sustainable, sensitive, and multi-analytical approach for the determination of cocaine and metabolites in hair, with strong potential for application in clinical and forensic toxicology.
- Otimização do bioprocesso de obtenção da vacina de DNA minicircular contra o cancro do colo do úteroPublication . Ferreira, Beatriz Morais; Costa, Matilde Bogalheiro; Sousa, Ângela Maria Almeida deO cancro do colo do útero é um dos tumores mais prevalentes entre as mulheres a nível mundial, estando maioritariamente associado à infeção persistente pelo Vírus do Papiloma Humano (HPV), sobretudo pelos genótipos de alto risco HPV-16 e HPV-18. Nestes casos, as oncoproteínas virais E6 e E7 desempenham um papel central na transformação maligna, ao promoverem a degradação das proteínas supressoras de tumor p53 e pRB, respetivamente. As vacinas de DNA têm emergido como uma estratégia promissora para a prevenção e tratamento de diversas doenças infeciosas e oncológicas. Esta abordagem baseia-se na introdução de DNA exógeno em células hospedeiras, com o objetivo de induzir a expressão de antigénios específicos capazes de desencadear uma resposta imunitária preventiva e terapêutica. Entre os vetores não virais utilizados, o DNA plasmídico (pDNA) tem sido o mais explorado, devido o seu baixo custo e facilidade de produção. No entanto, a presença de elementos bacterianos, como genes de resistência a antibióticos, pode desencadear respostas imunitárias indesejadas e reduzir a segurança da sua utilização. Como alternativa, o DNA minicircular (mcDNA) constitui uma geração mais avançada de vetores, resultante de uma recombinação intramolecular do plasmídeo parental (PP) numa cultura bacteriana, que se divide em duas moléculas filhas, o miniplasmídeo (mP) (constituído somente pelos genes procariotas necessários durante o processo de produção) e o mcDNA (sendo formado exclusivamente pela cassete de expressão eucariótica). Esta característica confere-lhe vantagens significativas face ao pDNA convencional, incluindo maior segurança, menor dimensão, eficiência acrescida de transfeção, maior estabilidade intracelular e expressão génica mais duradoura, traduzindo-se num elevado potencial terapêutico. Contudo, a sua aplicação clínica requer o desenvolvimento de métodos de produção e purificação robustos, capazes de cumprir os requisitos das entidades reguladoras. Neste contexto, o presente trabalho teve como objetivo explorar a cromatografia de interação hidrofóbica (HIC) como estratégia para a purificação de pDNA e mcDNA. Numa primeira fase, foram estudadas as condições de separação entre pDNA e RNA, seguindo-se a avaliação da seletividade entre mcDNA e RNA. Observou-se que o mcDNA tende a eluir em fases mais precoces, o que pode estar associado à sua menor hidrofobicidade relativamente ao RNA. Os ensaios de quantificação permitiram avaliar a pureza e recuperação dos vetores de DNA em função das estratégias exploradas, verificando a sua conformidade com os parâmetros definidos pelas agências reguladoras para as condições otimizadas. Contudo, não foi possível obter uma separação clara entre isoformas de DNA nem entre mcDNA e PP. Em suma, os resultados demonstram que a HIC constitui uma abordagem promissora para a purificação de mcDNA, fornecendo uma base sólida para otimizações futuras e para a consolidação deste vetor como ferramenta terapêutica de nova geração.
- Effects of Clobetasol on Human Skin Lipids: An Ex Vivo Study - Insights into barrier disruption and regenerative potentialPublication . Ramos, Ana Carolina da Silva; Oliveira, Rita Manuela Palmeira de; Paraskevopoulou, AnnaThe skin is the largest organ of the body, playing a vital role in the protection of the human organism. The stratum corneum (SC) is the outermost layer, composed of corneocytes embedded in a lipid matrix. Lipid ratio disruptions lead to an increase in water loss and a weaker skin barrier, causing different skin diseases. One of them, atopic dermatitis (AD), is a chronic inflammatory skin condition, impacting 15% to 20% of children and 1% to 3% of adults worldwide. As a common treatment for AD, glucocorticosteroids are used, but their transient efficacy and non-specific action often requires additional treatments, increasing the risks for adverse effects. In this project, the effect of glucocorticosteroids on the lipids of SC was investigated. To achieve this, donated human skin from plastic surgery (abdomen) was cultivated for 10 days, while applying topically clobetasol 0.05% in a suitable vehicle (propylene glycol:ethanol (7:3)) for 7 consecutive days. The viability of skin explants was tracked by the TTC assay. To gain a better understanding of the skin barrier and lipid alterations, the skin and the SC lipids were analysed using various methods, including transepidermal water loss measurement, infrared spectroscopy, alongside Liquid Chromatography with Mass Spectrometry. The results showed an increase in transepidermal water loss after treatment with clobetasol. In terms of skin viability, lipid composition, and lipid arrangement, the skin remained mostly stable. This demonstrates that short-term clobetasol treatment does not markedly alter the lipid composition of the SC. Overall, these findings help us understand how corticosteroids alter the skin barrier and SC lipids, and may inform future studies focused on creating safer, more targeted approaches to treating AD.
- Propriedades biológicas de óleos essenciais de plantas aromáticas: avaliação do potencial desinfetante do óleo essencial de Thymus zygis contra Listeria monocytogenesPublication . Sousa, Beatriz Fernandes; Ferreira, Susana Margarida Paraíso; Duarte, Ana Paula Coelho; Coimbra, Alexandra TeixeiraA contaminação de alimentos durante as etapas de produção e conservação, nomeadamente pelo contacto com superfícies, representa um risco significativo para a segurança dos alimentos. Esse risco é agravado pela presença de patógenos como Listeria monocytogenes, bactéria capaz de sobreviver em condições adversas e provocar listeriose. Nesse contexto, as plantas aromáticas, ricas em compostos bioativos, podem ser utilizadas frescas, secas ou na forma de óleos essenciais (OEs), apresentando propriedades bioativas relevantes, como atividade antimicrobiana. Esses OEs podem constituir alternativas naturais e seguras aos conservantes e desinfetantes químicos, contribuindo para a conservação e segurança dos alimentos. Thymus zygis é uma planta amplamente conhecida e utilizada na culinária, cujo OE apresenta atividades biológicas significativas. Considerando isto, este trabalho teve como objetivo principal investigar o potencial biológico de OEs provenientes de diversas plantas aromáticas, com atenção às suas propriedades antioxidantes, antimicrobianas e citotóxicas, bem como avaliar a eficácia do OE de T. zygis na redução de L. monocytogenes em suspensão, biofilmes, e em superfícies de aço inoxidável, visando a sua utilização como desinfetante natural. Sete OEs provenientes das plantas Murraya koenigii, Artemisia dracunculus, Carum carvi, Trigonella foenum-graecum, Zingiber officinale, Salvia lavandulifolia e Thymus mastichina, foram inicialmente avaliados quanto às suas propriedades antioxidantes, antimicrobianas e citotóxicas. Embora todos apresentassem forte atividade antioxidante, apenas o OE de T. foenum-graecum revelou baixa toxicidade para células humanas, enquanto os restantes se mostraram tóxicos e com reduzida ação antimicrobiana. Devido a estes resultados, o estudo concentrou-se no OE de T. zygis, analisando a sua eficácia como agente antimicrobiano e desinfetante. Constatou-se que tanto o OE quanto os seus compostos voláteis exerceram efeito bactericida sobre L. monocytogenes. Ensaios realizados segundo a norma EN 1276:2009 indicaram que o OE, na concentração de 2 µL/mL, apresentou atividade como desinfetante eficaz, enquanto a 1 µL/mL apenas foi eficaz em superfícies limpas, com aumento de atividade ao longo do tempo. Apesar de não atingir plenamente os critérios da norma EN 13697:2001, os resultados demonstram claramente o potencial do OE de T. zygis e dos seus compostos voláteis como agentes antimicrobianos em superfícies de contacto com alimentos, capazes de reduzir biofilmes formados por esta bactéria mesmo a baixas concentrações e em curtos períodos de exposição. Assim, pode-se concluir que OE de T. zygis demonstrou potencial para uso como desinfetante natural e agente anti-biofilme contra L. monocytogenes.
- Análise Comparativa entre os ensaios Freelite® e N-Latex® na Quantificação Sérica de Cadeias Leves Livres em doentes com Gamopatias MonoclonaisPublication . Francisco, Catarina Isabel Ribeiro Silva; Monteiro, Andreia Sofia dos Reis; Tomaz, Cândida Ascensão TeixeiraO Laboratório de Patologia Clínica do Centro Hospitalar Universitário da Cova da Beira (CHUCB), da Unidade Local de Saúde (ULS) Cova da Beira, constitui uma unidade especializada na recolha e análise de amostras biológicas com vista à obtenção de dados laboratoriais rigorosos, fundamentais para a formulação de diagnósticos e de estratégias terapêuticas. O presente trabalho descreve a atividade desenvolvida nas secções de Microbiologia, Hematologia e Imunoquímica, com especial ênfase na realização de um projeto de investigação na área da Imunoquímica. Nos últimos anos, o interesse clínico e científico relativamente às gamopatias monoclonais tem vindo a intensificar-se, refletindo a maior sensibilidade dos métodos de diagnóstico laboratorial e o aumento da sua prevalência, principalmente em populações envelhecidas. Estas patologias resultam da proliferação anómala de plasmócitos, responsáveis pela produção excessiva de imunoglobulinas monoclonais podendo manifestar-se de forma assintomática, como a Gamopatia Monoclonal de Significado Indeterminado, ou evoluir para doenças malignas, como o Mieloma Múltiplo. A sua deteção precoce e correta caracterização são fundamentais para uma adequada vigilância clínica e intervenção terapêutica atempada. Neste contexto, o presente projeto de investigação teve como objetivo a comparação analítica, dos métodos atualmente disponíveis no Laboratório de Patologia Clínica do Centro Hospitalar Universitário da Cova da Beira, para a quantificação de cadeias leves livres (CLL) no soro de doentes com gamopatias monoclonais. Foram avaliados o método Freelite® (The Binding Site), que utiliza anticorpos policlonais, e o método N Latex® (Siemens), baseado em anticorpos monoclonais. O estudo incluiu, para a análise estatística, resultados da quantificação das CLL em amostras de soro de 108 doentes, dos quais 64 eram do sexo masculino e 44 do sexo feminino, entre outubro de 2024 e abril de 2025, com idades compreendidas entre os 41 e 96 anos. Os dados foram estratificados por tipo de gamopatia monoclonal (Gamopatia Monoclonal de Significado Indeterminado IgM, Gamopatia Monoclonal de Significado Indeterminado não-IgM e Mieloma Múltiplo). Para a análise estatística utilizaram-se os métodos de regressão de Passing-Bablok, o coeficiente de Spearman, método de Bland-Altman e o coeficiente kappa de Cohen. Os resultados revelaram discrepâncias analíticas relevantes entre os métodos, com situações em que os valores de CLL foram classificados como normais por um método e patológicos pelo outro. A concordância entre os métodos foi globalmente fraca, com particular destaque para o subgrupo MGUS não-IgM, que apresentou as maiores divergências, especialmente na determinação da razão κ/λ. Apesar de limitações decorrentes do tamanho amostral em alguns subgrupos, os resultados obtidos foram consistentes com a literatura, tendo em conta as diferenças metodológicas. Os resultados evidenciaram uma fraca correlação e baixa concordância entre os dois métodos, sublinhando a não intercambialidade dos mesmos, alertando para potenciais implicações clínicas no diagnóstico, monitorização e decisões terapêuticas e no seguimento de doentes com gamopatias monoclonais.
- Avaliação de novas combinações de fármacos em cancro do pulmão de não pequenas célulasPublication . Valente, Fabiana Duarte; Sousa, Ângela Maria Almeida de; Santos, Adriana Oliveira dosO cancro do pulmão de não pequenas células (CPNPC) continua a ser uma das principais causas de mortalidade oncológica, sendo a resistência adquirida aos inibidores da tirosina-quinase (TKIs) um dos maiores obstáculos à eficácia terapêutica. O osimertinib, TKI de terceira geração dirigido a mutações ativadoras do EGFR, é um exemplo de fármaco que apresenta eficácia clínica significativa, mas enfrenta limitações devido ao desenvolvimento de resistência. Paralelamente, a sinvastatina tem demonstrado, em investigação pré-clínica, propriedades antitumorais relevantes, incluindo indução de apoptose e modulação de vias de sinalização associadas à progressão tumoral. Neste contexto, a coentrega destes fármacos em lipossomas surge como uma estratégia inovadora a ser desenvolvida para aumentar a eficácia terapêutica e reduzir a toxicidade sistémica da sinvastatina. Neste estudo, foram desenvolvidas e caracterizadas formulações lipossomais contendo sinvastatina ou osimertinib. Ambas as formulações apresentaram diâmetro hidrodinâmico nanométrico (=90 nm), baixo índice de polidispersidade (PDI <0,2) e estabilidade físico-química adequada, garantindo consistência entre diferentes lotes. O método de encapsulamento revelou-se eficiente, com recuperações superiores a 80% para ambos os fármacos e eficácias de encapsulamento de aproximadamente de 100%. A avaliação da viabilidade celular revelou que a sinvastatina apresentou uma redução significativa do IC50 quando encapsulada em lipossomas (1,160 µM) comparativamente à forma livre (1,770 µM). O osimertinib mostrou uma diminuição ligeira do IC50 em lipossomas em relação à forma livre (0,02076 µM vs. 0,02569 µM). Notavelmente, a combinação dos dois fármacos induziu uma redução mais pronunciada do IC50 quando encapsulada em lipossomas (0,01562 µM) comparativamente à forma livre (0,1566 µM), evidenciando um efeito sinérgico significativo que supera a contribuição de cada fármaco isoladamente. Estes resultados sugerem que a encapsulação lipossomal potencia a eficácia antiproliferativa das combinações de sinvastatina e osimertinib na linha celular H1975. Estes resultados fornecem uma prova de conceito sólida para a utilização de lipossomas como veículos de coentrega de sinvastatina e osimertinib, evidenciando a sua aplicabilidade enquanto estratégia combinada no CPNPC. A abordagem demonstra potencial para maximizar a eficácia terapêutica, reduzir efeitos adversos e constituir uma base para futuros estudos pré-clínicos e clínicos.
- Study of the bioaccessibility, biodisponibility and cytotoxicity of Salvia divinorum: method development and in vitro evaluationPublication . Calado, Sara Agostinho; Alba, Maria Eugénia Gallardo; Duarte, Ana Paula Coelho; Pires, Bruno Miguel PinheiroSalvia divinorum is a psychoactive plant traditionally used in spiritual and medicinal contexts in Mexico, whose main active constituent is salvinorin A (SA). This diterpene exhibits a unique pharmacological profile, acting as a potent and selective agonist of the ?-opioid receptor, which distinguishes it from classical psychedelic substances. Due to these properties, the plant has attracted attention both as a recreational drug, particularly among young people in Europe and the United States, and within scientific research, given its potential therapeutic applications in the management of pain, depression, anxiety, and addiction. However, adverse effects and the absence of clear regulation raise toxicological concerns. The present study aimed to investigate the bioaccessibility and bioavailability of SA, with particular focus on absorption through the oral mucosa, one of the common routes of consumption. To this end, an in vitro model simulating the oral digestive process was developed, employing artificial saliva. Plant extraction was carried out from dried S. divinorum leaves, subjected to maceration and subsequent incubation with artificial saliva at 37 °C under agitation for 3 hours. The resulting extracts were analysed by highperformance liquid chromatography coupled to diode array detector (HPLC-DAD), confirming the presence of SA, but not its metabolite salvinorin B (SB). In parallel, human tongue carcinoma cells (UPCI:SCC154) were used to assess both permeability and cytotoxicity of the extract. The MTT assay revealed a dose- and timedependent effect, with reduced cell viability after prolonged exposures. The bioaccessibility assay further demonstrated that oral digestion markedly decreases the fraction of SA available for systemic absorption. The results confirmed that SA is effectively absorbed through the oral mucosa, accounting for the rapid and intense effects reported in the plant’s consumption. Nevertheless, a significant cytotoxic impact was also observed in cellular models, underscoring the need for caution in repeated or chronic use. From an analytical perspective, HPLC-DAD proved to be a robust technique for quantifying SA in complex matrices. In conclusion, S. divinorum combines considerable pharmacological potential with risks associated with uncontrolled use. The evidence gathered in this study highlights the need for further clinical investigations to evaluate safety and efficacy, as well as the development of formulations that could enable therapeutic applications of SA while minimising its adverse effects.