FCT: DCV - Teses de Doutoramento
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- Towards understanding of genetic susceptibility linking preterm birth and periodontitisPublication . Costa, Joana Morais Couceiro da; Quintas, Alexandre; Fernandes, Maria Alexandra; Mendes, JoséPreterm Birth (PTB) is a major public health challenge involving a strong inflammatory component, as in chronic diseases such as Crohn’s disease and periodontitis (PD). The traditional clinical approach treats these as distinct conditions based on their symptoms. However, evi-dence suggests a potential shared genetic predisposition underlying some chronic diseases. Interestingly, several of these – especially inflammatory ones – have also been considered risk factor for PTB. This thesis investigates the hypothesis that PTB and PD may stem from a com-mon genetic susceptibility to inflammatory dysregulation rather than a direct causal relationship. A case-control study was conducted in a Portuguese cohort, focusing on inflammatory-related genetic variants. An initial panel of 73 variants in 36 genes was selected via literature review and bioinformatic tools. Two KCNN3 variants, rs1218585 and rs1218584, were associ-ated with increased spontaneous PTB (SPTB) odds. Haplotype analysis further supported the association, aligning with the role of KCNN3 in regulating contractility through potassium channels. In the PD context, a novel association was found with IL1RN rs4251961, highlighting its potential role in modulating inflammatory responses in periodontal tissues. Furthermore, in line with this thesis hypothesis, a composite inflammatory phenotype including both SPTB and PD was analyzed, revealing a consistent association with the TLR1 rs5743618. Although not as robust, IL1RN rs4251961 also showed a moderate association, potentially supporting a broader inflammatory susceptibility. Despite the limited sample size, this study supports a common genetic predisposition to complex inflammatory conditions, manifesting as SPTB and PD. These findings advance understanding of the genetic basis of inflammatory-driven diseases and may inform future preventive and therapeutic strategies.
- GENOMICS OF NONTUBERCULOUS MYCOBACTERIA (NTM): DEVELOPMENT OF A LABORATORY TOOL FOR RAPID AND PRE- CISE DIAGNOSIS AND SURVEILLANCE OF NTM INFECTIONSPublication . Carneiro, Sofia Coutinho Carvalho Baptista; Macedo, Ana; Gomes, João; Mota, LuísIn Portugal, the impact of nontuberculous mycobacteria (NTM), which are responsible for emerging diseases worldwide, is still unknown, and it is imperative to improve epidemiolog- ical surveillance and knowledge of antimicrobial resistance. The main goal of this dissertation was to understand the dynamics of NTM infections in Por- tugal and improve their monitoring. Despite not being a notifiable disease, the data available at the LNR-TB between 2014 and 2024 showed that species belonging to the Mycobacterium avium (MAC) and Mycobacterium abscessus (MABC) complexes were the main culprits. To enhance our genomic knowledge of these most prevalent complexes, we used whole ge- nome sequencing (WGS) studies. The genetic characterisation and identification of resistance markers of the MABC isolates showed a progressive acquisition of resistance to different drugs in vivo and allowed us to identify "dominant circulating clones", which are associated with a worse prognosis, high resistance and cross-border dissemination. With regard to the MAC isolates, genomic analysis made it possible to discriminate between species, subspecies and lineages, suggesting a possible need for taxonomic revision within the complex. Analysis of the resistance profiles revealed differences between species and subspe- cies, which reinforces the importance of accurate identification for appropriate therapeutic ap- proaches. In the final chapter, we intend to develop an amplicon methodology to identify and discrimi- nate between MABC and MAC species and detect resistance mutations directly in clinical sam- ples. Once optimised, this approach could be integrated into clinical practice, allowing for a more accurate and personalised diagnosis, reducing the time needed to make therapeutic de- cisions. This study was a pioneer in the application of WGS in NTM surveillance in Portugal, reinforces the need to implement a mandatory reporting system and demonstrates the importance of integrating genomic approaches into laboratory routine to improve the diagnosis and treat- ment of NTM infections.
- Coleoid venoms: predicting cephalotoxin function and biotechnological applications from ecological and evolutionary traitsPublication . Gonçalves, Cátia Vanessa Caetano; Costa, Pedro; Grosso, Ana Rita; Karlsen, OddThe ocean’s immense biodiversity offers almost limitless but little explored bioactives for safer and more cost-effective alternatives to synthetic drugs. However, addressing marine biodiversity, especially invertebrates, while producing validated data for industry is challenging. Albeit often neglected, commercial venomous species such as cuttlefishes and octopuses can provide varied bioactives for the purpose of bioprospecting. Cephalotoxins, secreted by the salivary glands of coleoids, are little-known substances with at least analgesics properties. Benefitting from their economic and ecological relevance, our case studies are the Octopus vulgaris and the Sepia officinalis. The integration of the toxin-delivery apparatus (i.e., anterior and posterior salivary glands) ecophysiology, combined with toxicology and molecular biology provided comprehensive information on the function and targets of these cephalopods bioactives. Altogether, the microanatomy revealed that the two species secrete different substances. Despite generally similar anatomy between the two salivary glands, characterised by branched glandular tubules bearing mucous and secretory epithelia, the octopus’ posterior salivary gland (PSG) is more complex structurally than its anterior counterpart (ASG), which also extends to the simpler cuttlefish PSG. Transcriptomics confirmed the high diversity of venom-peptides secreted by both cephalopods, of which bioactives with potential neurotoxic and endocrine-disrupting effects (e.g., pro-insulins) are highlighted, together with proteins with inhibitory functions. However, the cuttlefish PSG presented more putative cephalotoxin variants than octopus. Seven full coding sequences of interest, such as three cysteine-rich venom proteins, a venom insulin, a cephalotoxin, a chitinase and a hyaluronidase, were isolated and validated. These bioactives exhibited different patterns of sequence variation within venomous taxa, suggesting potential specificity among octopus and cuttlefishes. The crude protein extracts of both PSG were able to activate human G protein-coupled receptors of interest, indicating its great potential to aid in xiv bioprospection, albeit yielding low combined cytotoxicity, which shows an interesting path towards safer drugs targeting the human druggable proteome.
- Nanoheaters – Localized hyperthermia for precise gene deliveryPublication . Ferreira, Daniela Filipa Cardoso; Baptista, Pedro; Fernandes, Maria AlexandraGene therapy relies on the precise transfection of nucleic acid effectors into cancer cells, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASO). Therefore, novel therapeutic approaches are widely needed to deliver silencing moieties with maximal trans- fection efficiency and minimal toxicity. This thesis explored the use of mild hyperthermia me- diated by gold nanoparticles (AuNPs) or magnetic nanoparticles (MNPs) to enhance nucleic acids delivery with spatiotemporal control over laser irradiation or magnetic modulation, re- spectively. First, the photothermal effect of AuNPs under visible light irradiation was verified, leading to enhanced cellular uptake. The potential of mild photothermy via AuNPs was demonstrated by effectively silencing the GFP gene in colorectal carcinoma cell line (HCT116) and breast adenocarcinoma cell line (MCF-7), with comparable gene silencing efficiency to commercial transfection reagent, but without cytotoxicity. Improving gene silencing strategies in 3D cell cultures is important since it provides in vitro models that closely resemble the in vivo tumor microenvironment (TME). Then, it was shown that mild photothermy mediated by AuNPs functionalized with ASO decreases c-MYC oncogene expression in HCT116 cells and 7-day spheroids, respectively. Localized magnetic hyperthermia mediated by immobilized MNPs on the cell mem- brane through bioorthogonal chemistry improves the transfection of anti-GFP in MCF-7 cells, with similar efficacy and less cytotoxicity compared to standard transfection reagent. Taking advantage of this approach, an effective IDO1 gene silencing was obtained through the trans- fection of siRNA in dendritic cells derived from an acute monocytic leukemia cell line (THP- 1). Moreover, the upregulation of pro-inflammatory cytokines IL6, TNFA, and IL12 genes and the downregulation of anti-inflammatory IL10 gene might contribute to a more immunogenic state in a TME context. In summary, this thesis highlights nanoparticle-mediated mild hyperthermia as a promising strategy for controlled and efficient nucleic acids delivery that might pave the way for improved gene therapy applications in more complex cancer models.
- Simplified Chip Prototyping for Improved Screening of Gene-silencing Therapeutics Using 3D Cell ModelsPublication . Oliveira, Ana Beatriz Brito de; Baptista, PedroCancer research has long been hindered by the limitations of conventional 2D culture models, which fail to replicate the intricacies of the tumor microenvironment. This lack of complexity prevents accurate evaluation of cancer therapeutics, including gene-silencing approaches. The shortcomings of standard cancer models in recapitulating the dynamic microenvironment of tumors prompted the development of more physiologically relevant models. While Tumor-on-Chip systems show promise in addressing these challenges, their widespread adoption is limited due to technical complexity and high fabrication costs. To overcome these barriers, this thesis presents a strategy to simplify the prototyping of ToC devices by focusing on accessible materials and fabrication processes. This work outlines a pathway to create efficient, reproducible, and cost-effective biochips suitable for cancer modeling and screening gene- silencing therapeutics. The objectives include synthesizing and characterizing gold nanoparticle conjugates, evaluating their gene silencing efficiency in 2D and 3D models, streamlining the biochip fabrication, and translating these findings to the final device. The results provided insights into the differential behavior of nanoparticle uptake and resultant silencing efficiency between models. While 2D cultures exhibit faster uptake kinetics, 3D spheroids better mimic diffusional barriers and tumor heterogeneity, making them a more reliable model for evaluating nanoparticle-based therapies. The developed ToC device successfully reproduced these complexities, yielding promising results. Still, future work should aim to incorporate fluidic channels and immune cells to further enhance tumor microenvironment recapitulation, bridging the gap between preclinical models and in vivo complexity to improve therapeutic predictions.
- Cytochromes in Geobacter: fostering sustainable sources of energy productionPublication . Castillo Portela, Maria do Pilar; Salgueiro, Carlos; Malvankar, NikhilGeobacter bacteria congregate interesting features for application in sustainability: they are able to couple the oxidation of toxic organic compounds, such as toluene, to the reduction of Fe(III) and toxic Mn(IV), Cr(III), U(VI). Furthermore, they can associate with electrodes in Microbial Fuel Cells (MFC) to produce electrical energy. These features are possible because of the chain of multiheme cytochromes poised at the different cellular compartments that relay the electrons to the cell’s exterior – Extracellular Electron Transfer (EET). The applications are promising but challenges still lay ahead, especially since the EET chain components and their interactions are not well-characterized. This work focused on three main aspects of Geobacter – exploration of long-range ET; assessment of redox partnership in the context of oxidative stress; manipulation of the redox properties of cytochromes. In the long-range ET realm, we studied the redox partnership of triheme periplasmic cytochromes PpcA-E with native OmcS nanowires and determined for the first time that PpcA-E can directly provide reducing power to OmcS, clarifying this ET route. We worked on the heterologous expression system in E. coli for OmcZ and OmcS to, respectively, improve it and develop it to, in the future, facilitate sample obtention for biochemical studies. In the context of oxidative stress, we determined that PpcA-E provide reducing power to MacA peroxidase, specifically to its HP heme, elucidating this redox partnership. The role of PpcA-family periplasmic cytochromes is pivotal in various ET pathways and, as such, we studied how their redox properties can be manipulated to fine tune the ET directionality of future Geobacter-engineered strains with minimal components. Using protein engineering and PpcA from G. metallireducens as a framework, we have shifted its midpoint reduction potential value 71% towards the one of PpcA from G. sulfurreducens. These studies contributed to the elucidation of two redox partnerships important in Geobacter’s ET chain, to the establishment of the foundations for the heterologous overexpression of two nanowires, and to the creation of a mutants’ library of PpcA periplasmic cytochromes, a pivotal component of the ET chain.
- Studying the role of glycan-based biomarkers in cancer : Towards prognostic and therapeutic applicationsPublication . Lourenço, Rita Adubeiro; Videira, Paula Alexandra Quintela; Silva, Zélia Maria Cordeiro da; Sousa, Rui Pedro Romero AmandiGlycosylation is a complex post-translational modification that impacts most proteins in the human body and is critical for several biological processes. Disruptions in glycosylation are often associated with diseases, particularly cancer, where altered glycan structures drive tumour development and progression. One key alteration is the increase in sialylated glycans like the abnormal sialyl-Tn (STn), typically absent in healthy tissues, making it a promising cancer biomarker and therapeutic target. However, the exact role of STn in cancer progression is still unclear and remains under active investigation. This thesis aimed to clarify the role of STn in cancer, more specifically in triple-negative breast cancer (TNBC) and colorectal cancer (CRC). A systematic review of the literature revealed that STn is generally associated with poor survival. However, its prognostic value varies across cancer types and cohorts. Mucins, along with proteins like CD44 and beta 1 integrin (ITGB1), were identified as key carriers of STn in cancers, all of which are involved in tumour progression. In TNBC, only ~25 % of the cases express STn. This subgroup is associated with reduced survival and with an immunosuppressive environment. STn shows a negative association with the c-Myc, despite promoting higher cell proliferation in vitro. In CRC, STn expression is present in most cases and correlated with important molecular features such as microsatellite instability (MSI) and with glycosylation-related genes, such as the ST6GALNAC1 gene, which explains the expression of STn and correlated with the consensus molecular subtype 3 (CMS3). Additionally, this work presents the development of an interactive platform integrating visualisation tools of glycomic and glycosylation-related transcriptomic data from cancer cell lines and tissues. It provides a tool to visually explore glycosylation traits, which significantly contributes to cancer glycosylation research and accelerates the identification of glycan-based biomarkers that can be used in the clinic. Overall, this work's findings elucidate the impact of STn on cancer progression, rein-forcing its significant but complex role as a cancer biomarker for prognostic and targeted therapeutic purposes.
- GLYCOVID-19: unravelling the impact of glycosylation and therapeutics in SARS-CoV-2 infectionPublication . Barreira, Daniela Sofia Ferreira; Videira, Paula Alexandra Quintela; Soares, Helena Isabel MartinsIn early 2020, the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a widespread pandemic. In response to its rapid transmission and mortality rates, extensive research focused on developing therapies and vaccines to limit SARS-CoV-2 infection, treat patients, and alleviate the burden on healthcare systems. Among the mechanisms studied was the impact of glycosylation in SARS-CoV-2 infection, a post-translational modification involved in many biological processes, including pathogen-host interactions. In SARS-CoV-2 infection glycans may facilitate the initial viral attachment prior to ACE2 binding, and act as “shields” masking viral immunogenic epitopes, affecting both immune responses and vaccines’ effectivity. Glycans are often terminated by sialic acid, a carbohydrate involved in host-pathogen interplay, can serve as receptor or co-receptor for viral entry. Thus, this thesis aimed to investigate the role of sialylation in SARS-CoV-2 infection. To assess sialyation impacted on viral entry it was used a hyposialylated cell model, obtained through GNE knockout. Our findings showed that cellular sialylation acts as a restricting factor against SARS-CoV-2 infection. Additionally, we aimed to unveil whether sialic acids presented potential as a disease severity biomarker in pregnant individuals, a vulnerable population with a heightened mortality and morbidity risk after infection. The sialylation analysis of pregnant Individuals’ plasma demonstrated increased sialic acid levels post-vaccination com-pared to natural infection. We also systematically reviewed the therapeutic value of convalescent plasma (CP) ad-ministered to hospitalized COVID-19 patients. Our results demonstrated CP administration as safe, improving mortality rates and reducing the viral load. Concurrently, an anti-STn mono-clonal antibody was characterized to assess its application as a therapeutical agent for glycosylation-related disorders, such as cancer. Altogether, this thesis’ work contributes to further the understanding of sialylation in viral infections and in the immune response of vulnerable populations, while assessing potential therapeutic approaches.
- Structural and functional insights on the electrifying pathways of Geobacter sulfurreducensPublication . Fernandes, Tomás Monteiro; Morgado, Maria Leonor; Salgueiro, CarlosOs microorganismos electroactivos são capazes de transferir electrões para o meio extracelular, o que permite que sejam utilizados em tecnologias electroquímicas microbianas (TEM). No entanto, apesar do seu potencial, a aplicação e eficiência destas tecnologias é ainda limitada, principalmente devido aos baixos níveis de corrente produzidos. Consequentemente, é fundamental compreender os mecanismos que governam o fenómeno de transferência electrónica extracelular (TEE) de uma perspectiva estrutural e funcional, de forma a que estes processos possam ser optimizados. Geobacter sulfurreducens é uma bactéria Gram-negativa electroactiva modelo, sendo responsável pelas maiores densidades de corrente observadas em TEM. Os componentes mais relevantes das vias de TEE desta bactéria já foram identificados por estudos genómicos e proteómicos, no entanto, os mecanismos subjacentes a este processo não foram clarificados. O trabalho desenvolvido durante esta Tese resulta da aplicação de uma combinação de técnicas biofísicas e métodos computacionais que permitiram o estudo de vários componentes das vias de TEE de G. sulfurreducens. Os modelos dos complexos porina-citocromo desta bactéria foram previstos pelo AlphaFold 2, o que permitiu obter informação sobre a coordenação axial e disposição espacial dos hemos, a função, o enrolamento e a organização geral destes complexos. Esta informação serviu para definir novas estratégias de produção destas proteínas. Estudos de RMN e SAXS demonstraram que a proteína periplasmática ExtJ, parte do complexo porina-citocromo ExtHIJKL, forma homodímeros através de pontes dissulfureto intramoleculares, utilizando um resíduo de cisteína localizado no domínio flexível do C-terminal. A sua estrutura em solução indica que a proteína é homóloga a proteínas que ligam moléculas de flavina, no entanto, a proteína não interage com FMN. Previsões do AlphaFold-Multimer sugerem que a ExtJ poderá interagir com a porina ExtI. Com base nestes resultados, postula-se que o processo de dimerização possa ser controlado por mecanismos de quebra e formação de pontes dissulfureto, que por sua vez controlam a formação do complexo ExtI-ExtJ e a passagem de moléculas através desta porina. O citocromo extracelular PgcA possui uma estrutura difusa com três domínios citocromo ligados por secções desestruturadas. Estudos biofísicos dos domínios individuais e do citocromo completo demonstram que a proteína estabelece uma cadeia electrónica flexível, adoptando múltiplas conformações que promovem transferência electrónica a várias distâncias. O mecanismo descrito, sem precedentes em sistemas biológicos, poderá ser vantajoso durante a redução de metais. Finalmente, o citocromo trihémico periplasmático GSU0105 foi caracterizado por espectroscopias de RPE, RMN e UV-visível, tendo estas demonstrado que a proteína contém um grupo hemo que sofre uma conversão de spin-baixo para spin-alto dependente do estado redox. Ademais, o potencial de redução deste hemo é modulado pelo pH da solução, que controla indirectamente a conversão do estado de spin, ao promover diferentes conformações estruturais. Globalmente, estes resultados constituem um contributo importante para o nosso conhecimento actual dos mecanismos de TEE de G. sulfurreducens e para o desenvolvimento de TEM mais eficientes.
- TOWARDS THERAPEUTIC APPROACHES FOR HUMAN GLYCOSYLATION DISORDERS FROM TRANSCRIPTOMIC AND IMMUNOLOGICAL CHARACTERIZATION TO PATIENT-CENTRED OUTCOMES RESEARCHPublication . Smith, Carlota Moutinho Pascoal; Videira, Paula; Ferreira, VanessaGlycosylation is a post-translational modification that occurs ubiquitously in the human body and essential for all biological and physiological body functions. An impaired or altered glycosylation process affects homeostasis, leading to diseases like congenital disorders of glycosylation (CDG), cancer and immunological disbalance. This thesis focuses on two glycosylation disorders, namely the ultra-rare phosphomannomutase 2 (PMM2)-CDG and the low incident triple negative breast cancer (TNBC). These complex, long-term conditions are associated with fundamental challenges, poor patient outcomes and reduced well-being. In this context, patient-centric research constitutes an opportunity to generate inclusive methodologies and meaningful results answering urgent community needs. The aim of this thesis was to implement a public involvement framework and devise patient-oriented research on community-identified topics. Using a comprehensive approach of transcriptomics and validation techniques, this thesis validates patient-derived fibroblasts as a cellular model to study immunopathology, given the scarcity of other models, particularly in PMM2-CDG. This work allowed to proceed with further studies to unveil the mechanisms behind immunological involvement in PMM2-CDG, encompassing highly impactful and life- threatening manifestations. Glycosylation-related mechanistic defects were identified, specifically, dysregulated immune receptor downstream signalling and immune effectors, highlighting potential therapeutic targets and interconnections with other affected systems. Importantly, patient-reported outcomes measures that evaluate health related quality of life for specific PMM2-CDG symptoms were identified and analysed. This is a contribution to an urgent need that can aid in future clinical research stages, enabling the accurate measurement of targeted therapeutic benefits of investigational drugs. Lastly, using a methodology based in three different models, the role of a glycan epitope was characterized in TNBC which correlates with disease aggressiveness, contributing to patient stratification and the potential development of more targeted therapies. xiv In conclusion, this thesis illustrates the benefits of adopting patient-centric approaches in research, combining scientific progress in unexplored and urgent topics with desired and meaningful research outcomes for the community.