FM - Teses de Doutoramento
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- Fatty liver disease : Relevance of nutritional determinants and nutritional care in the general population and in HIV patientsPublication . Policarpo, Sara; Cortez-Pinto, Helena Maria Ramos Marques Coelho; Mendes, Catarina Ferreira Murinello de Sousa Guerreiro Fragoso
- Mechanisms of t follicular regulatory cell differentiationPublication . Ribeiro, Filipa; Graça, Luis Ricardo Silva; Fonseca, Válter Bruno RibeiroRespostas imunológicas humorais protetoras coordenadas pelas células B, quer em resposta a infeções ou à vacinação, requerem a participação de células T. Entre estas, as células T foliculares auxiliares (Tfh) emergiram como elementos-chave, fornecendo ajuda às células B e facilitando a produção de anticorpos de alta afinidade. Por outro lado, as células T foliculares reguladoras (Tfr), originárias de células T reguladoras (Treg) tímicas nos tecidos linfoides periféricos, assumem o papel de orquestradoras. Elas modulam a magnitude das recções dos centros germinativos (GC) para evitar o aparecimento de clones de células B auto-reactivas, ao mesmo tempo que a produção de anticorpos protetores é otimizada. Deste modo, manter este delicado equilíbrio entre a produção de anticorpos e a autoimunidade depende imensamente das subpopulações celulares Tfh e Tfr. Apesar da extensa investigação sobre células Tfh desde a sua descoberta, as células Tfr só ganharam destaque recentemente. Mais de uma década de investigação sobre as células Tfr tem vindo a revelar algumas disparidades na sua biologia em comparação com as células Tfh, desafiando a suposição da sua estreita semelhança. Apesar da sua importância (uma vez que a sua ausência está associada ao desencadeamento de doenças autoimunes), persistem lacunas significativas na compreensão da biologia das células Tfr, proporcionando um vasto campo para exploração e descoberta. A presente tese pretende iluminar o mundo intrincado das células Tfr, procurando refinar a sua definição e elucidar o seu processo de diferenciação. A nossa investigação sobre as células Tfr durante a resposta imunitária em murganho proporcionou uma caracterização mais completa do seu fenótipo e das moléculas que governam a sua trajetória de desenvolvimento. Apesar da ontogenia reguladora partilhada, descobrimos diferenças significativas no fenótipo e na diferenciação das células Tfr em comparação com as células T reguladoras efetoras. Simultaneamente, demonstrámos que duas moléculas expressas na membrana e amplamente utilizadas para identificar as células Tfh e Tfr, o ICOS e o PD-1, exibem padrões distintos durante a diferenciação destas populações de células em humanos. Enquanto o ICOS e o PD-1 podem ser utilizados de forma intercambiável para a identificação das células Tfh, o ICOS é expresso antes da expressão de PD-1 nas células Tfr. Esta observação levou à delimitação de três fases distintas de desenvolvimento das células Tfr nos tecidos linfoides humanos. Adicionalmente, as nossas descobertas sugerem que o OX40L e o IL-6 podem desempenhar papéis cruciais na indução do programa de diferenciação das células Tfr nas células Treg humanas. No seu conjunto, os resultados obtidos nesta tese fornecem novas perspetivas sobre a caracterização e diferenciação das células Tfr. Além disso, proporcionam oportunidades para investigações futuras na modulação da resposta dos GC e no papel de moléculas específicas, identificadas neste estudo, na função das células Tfr.
- Unveiling the mechanism of single nuclear inactivation in multinucleated cellsPublication . Faleiro, Inês; Almeida, Sérgio Alexandre Fernandes de; Gomes, Edgar RodriguesMaintaining genomic integrity is crucial to prevent the activation of tumorigenic mechanisms caused by DNA damage. To achieve this, specialized mechanisms must be timely and coherently activated in response to DNA damage. Collectively known as the DNA damage response (DDR), these cellular mechanisms detect damaged sites and activate downstream effectors to repair the DNA. If repair fails, cells can trigger senescence or apoptosis mechanisms to prevent the propagation of unhealthy cells. Among various forms of DNA damage, DNA double-strand breaks (DSBs) pose the most severe threat. Cells have evolved two core pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ), to repair DSBs. These pathways have notable differences, as HR requires homologous sequences and is limited to specific cell cycle phases with sister chromatids available, while NHEJ operates throughout the entire cell cycle, but with error prone ligation of broken DNA strands. Although proliferating cells' DDR mechanisms are well-characterized, our understanding of such mechanisms in post-mitotic cells, such as skeletal muscle cells, remains limited. Terminally differentiated skeletal muscle cells are among the longest-lived cells in the human body and lack the advantage of proliferative renewal. Thus, they must adapt to stress to ensure vital functions for the organism while handling DNA lesions and preserving cell viability. Differentiated muscle cells, unable to re-enter the cell cycle and employ accurate DNA repair via HR, rely on the more mutagenic NHEJ pathway to repair DSBs. Unfortunately, this mechanism leads to progressive accumulation of mutations, compromising genomic stability. In this study, we examined the DDR in differentiated skeletal muscle cells. Our findings demonstrate that myotubes exhibit a prolonged DDR, yet remain competent in repairing DSBs. Through live-cell microscopy and single-molecule kinetic measurements, we observed that myotubes respond to DNA damage by temporarily suppressing global gene expression and altering the epigenetic landscape of the damaged nucleus. Surprisingly, despite the prolonged activation of the DDR compared to their precursor cells, differentiated skeletal muscle cells show remarkable resistance to cell death. This suggests the existence of a specific pathway that helps these cells avoid the catastrophic consequences of DNA damage. Our study reveals evidence that autophagy, the cellular process responsible for clearing damaged cellular material, plays a vital role in the DDR of skeletal muscle cells. We discovered a novel mechanism employed by these cells, whereby unrepaired DNA is removed from the nucleus and processed through autophagy. This process operates in conjunction with DNA repair proteins and contributes to the apoptotic resistance phenotype observed in skeletal muscle cells. Our findings provide insights into the strategy employed by human skeletal muscle to preserve genetic integrity and the remarkable resistance of these cells to DNA damage-induced apoptosis, ensuring long-term organ function even after DNA damage occurs.
- Detection of circulating tumor cells in renal cancer : the impact of no-touch surgeryPublication . Palmela Leitão, Tito; Costa, Luís António Marques da; Lopes, Tomé Manuel Matos
- On the clinical potential of ultra long-term subcutaneous EEG monitoring in epilepsyPublication . Viana, Pedro; Petiz, Maria Isabel Segurado Pavão Martins Catarino; Richardson, Mark Philip
- An image-based applied game for neuropsychological rehabilitation : assessing its feasibility among elderly with vascular mild cognitive impairmentPublication . Ferreira-Brito, Filipa; Verdelho, Ana Isabel Figueira; Carriço, Luís Manuel Pinto da Rocha Afonso; Caneiras, Cátia Sofia Gabriel
- Innovative contributions towards therapeutic strategies for metastatic breast cancer treatmentPublication . Oliveira, Filipa; Castanho, Miguel Augusto Rico BotasBreast cancer is the most frequently diagnosed cancer worldwide and is the leading cause of cancer-related death among women. Despite the vast therapeutic arsenal available to fight this heterogenous and complex disease, it still poses a challenge in the clinic. In fact, even though the extensive screening programs combined with improvements in the therapeutic management have been resulting in an increase of the median overall survival in women with metastatic breast cancer (MBC), this specific disease remains incurable. Moreover, as the incidence of MBC is increasing, more patients will die from this disease. The therapeutic approach for MBC relies on chemotherapy, which is associated with insufficient selectivity, that ultimately leads to drastic side-effects, and resistance to treatment. Additionally, while this type of therapy can be partially effective regarding metastases development in bones, soft tissue and viscera, MBC tends to spread to lungs, liver, bones and brain. Therefore, brain metastases represent a major clinical concern when treating and managing MBC, since the majority of the chemotherapeutic drugs present very poor penetration in the central nervous system (CNS) due to the impermeability of the blood-brain barrier (BBB). Therefore, there is the urgent need for more effective and innovative strategies for MBC treatment. In this work, our goal was to develop a new therapeutic strategy for the treatment of MBC that would selectively target breast cancer cells and interfere with the development of brain metastases. Considering the vast level of complexity of breast cancer, we decided that our approach would have to focus on molecular features that are widely found in cancer cells and tend to remain constant throughout time. Cancer cells exhibit a cell membrane with a net negative charge. Therefore, we decided to focus on the use of peptides with anticancer activity, because since these are generally cationic, their mode of action relies on the inevitable electrostatic interaction with cancer cells. In the first part of this work, we studied the anticancer activity of PvD1 – a plant defensin isolated from Phaseolus vulgaris, the common bean – and vCPP2319 – a cellpenetrating peptide (CPP) obtained from the capsid protein of the Torque teno douroucouli virus. The results showed that both peptides exhibited selective anticancer activity. The mode of action was also studied for both peptides and the results suggested that both peptides could potentially hinder the formation of metastases. Therefore, we concluded that these are promising molecules that can work as templates for the design of innovative approaches for MBC treatment. Nevertheless, peptides are still associated with some disadvantages as instability, immunogenicity, or limited ability to cross biological barriers such as the BBB. Hence, the second part of this work focused on the study of the interaction between extracellular vesicles (EVs) and vCPP2319. The goal was to use EVs as a potential drug delivery system (DDS) for vCPP2319. We isolated EVs from breast cancer cells MDA-MB-231 and studied the interaction between these and vCPP2319. As the results revealed the establishment of a strong interaction, we decided to test the ability of these EVs to deliver vCPP2319 across an in vitro model of the human BBB. The results revealed that, in contrast to what was observed for the peptide alone, vCPP2319 was able to transmigrate through the BBB model when combined with EVs. In line with these results, EVs did show the ability to transmigrate through the BBB, even without the peptide. In parallel with the present work, there were contributions that crossed roads with collaborations with other labs. One of those works was a literature review on the use of biophysical techniques to investigate the potential of bacteriocins as anticancer peptides (ACPs). Additionally, the anticancer activity of a peptide found in the venom glands of three different wasps, the protonectin (PTN), was studied. Aiming to explore alternative approaches for the use on EVs in breast cancer treatment, these vesicles were evaluated as a possible target for breast cancer treatment using the PvD1. Finally, there was also the participation in a project where several conditions were evaluated for the optimization of the production of EVs from mesenchymal stem cell cultures in bioreactors. All these works are in line with the main project that supports this thesis, adding on its contribution for new therapeutic strategies for metastatic breast cancer treatment. Overall, this thesis shows that ACPs are a very promising source of templates for the development of new molecules for MBC treatment and that EVs have the potential to work as a DDS with the ability to deliver its cargo across the BBB.
- Unravelling mitochondrial dynamics and turnover in the synapsePublication . Cardanho-Ramos, Carlos; Epifânio, Vanessa Alexandra dos Santos MoraisNeurons rely on mitochondria for ATP production and Ca2+ homeostasis, particularly at the synapse. As subcompartmentalized cells, they have different pools of mitochondria in each compartment. Achieving a non-uniform distribution requires mitochondria not only to be transported, but also to be retained in regions with high energy demands and high levels of Ca2+ . One of the goals of this thesis was to understand what makes mitochondria travel back and forward in a neuron and which mechanisms were activated to retain this organelle at the synapse. To address this, mouse primary neuron cultures, with fluorescent labelled mitochondria, were used to study the impact of the actomyosin cytoskeleton in mitochondrial transport and anchoring at the synapse. Taking advantage of a proteomic screen comparing synaptic with non-synaptic mitochondria, we selected two candidate proteins related with organelle movement: cell division control protein 42 (cdc-42), involved in actin polymerization; and myosin-VI, capable of anchoring mitochondria to actin cables. In the course of this project, a paper was published addressing all our questions, forcing us to focus on a different topic. In the other project of this thesis, we focused on understanding how mitochondria replicate in neurons. It is assumed that mitochondria are generated in the cell body and travel to the synapse to exert their functions. However, considering the rate of mitochondrial transport in neurons, the time it would take for a single mitochondrion to travel from the cell body to the synapse exceeds the half-life of most mitochondrial proteins. Our goal was to understand whether mitochondrial replication occurred in the periphery of neurons and which mechanisms were involved. We developed a technique to assess mitochondrial replication in mouse primary neuron cultures using 5-bromo-2′- deoxyuridine (BrdU)- and 5-ethynyl-2´-deoxyuridine (EdU)-labelling, two thymidine analogues that are incorporated into mtDNA upon replication. Most BrdU/EdU staining was observed in the cell body, but staining was also present to a lower extent in the periphery. Using microfluidic devices, where axons can be isolated from the cell body, we were able to add EdU only to the axonal side, without interfering with the cell body. In these conditions, EdU staining was only present in axons, confirming that mitochondrial replication in neurons can occur away from the cell body. We hypothesized that mRNA and local translation must be at place in the periphery of neurons in order to provide all the necessary machinery for mitochondria to replicate. To test this, we assessed mitochondrial replication upon inhibition of both nuclear-encoded and mitochondrial-encoded protein translation. Mitochondrial replication in neurons decreased when nuclear-encoded protein translation was inhibited. However, no differences were observed upon inhibition of mitochondrial-encoded protein translation. Taking advantage of the proteomic screen comparing synaptic with non-synaptic mitochondria, two candidate proteins related with protein translation were found upregulated in the synaptic fraction – eukaryotic elongation factor 1 alpha 1 (eEF1a1), involved in nuclear-encoded protein translation; and mitochondrial translation elongation factor Tu (TUFM), involved in mitochondrial-encoded protein translation. We performed loss and gain of function assays with our candidate proteins and assessed their impact on mitochondrial replication. When eEF1A1 was downregulated, we observed a decrease in mitochondrial replication in the periphery of neurons. This effect was rescued by reintroducing eEF1A1. Regarding TUFM, no differences were observed. Our results confirm that mitochondrial replication can occur in the periphery of neurons, and that this process requires nuclear-encoded protein translation, mediated by eEF1A1. Understanding how mitochondrial replication occurs in neurons, particularly at the level of the synapse, provides novel lines of research to tackle the pathophysiological mechanisms underlying neurodegenerative diseases.
- Dietary patterns, body composition and physical activity : progression of the health status in hemodialysis patientsPublication . Garagarza, Cristina; Mendes, Catarina Ferreira Murinello de Sousa Guerreiro Fragoso; Ferreira, Manuel Aníbal Antunes
- Adult outcomes of juvenil idiopathic arthritisPublication . Oliveira Ramos, Filipa; Fonseca, João Eurico Cabral da; Santos, Maria José Parreira dos