FCT: DCV - Dissertações de Mestrado
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- Multi-Disease Biomarker Discovery for Drug Repurposing in Glioma A contribution towards glioma treatmentPublication . Galaricha, Eduardo Filipe Nepomuceno; Lopes, Marta; Martins, SusanaGliomas are the most prevalent primary Central Nervous System (CNS) tumors, comprising 80% of malignant adult cases and 30% of all brain tumors. They are classified into glioblastoma (GBM), astrocytoma, and oligodendroglioma. Among these, GBM and astrocytoma are the most aggressive, with poor prognosis and high recurrence. The absence of universally accepted treatment and the limitations of current options highlight an urgent unmet need. Drug repurposing, finding new indications for approved or clinical-stage compounds, offers a faster, lower-risk path to therapy. This study applies state-of-the-art methodologies such as sparse learning models and network- based analysis to identify molecular biomarkers shared by GBM and astrocytoma with translational potential. Incorporating various methodologies to enhance the efficacy of classification models, this study introduces Gene Network Identity Vector (GeNeIV), a novel feature selection method that emphasizes genes exhibiting similar network patterns in both aggressive glioma types. A total of 31 candidate genes were identified, including ITGAM, ADORA3, CDC20, as well as the ribosomal genes RPS16, RPS8, and RPL19. Many of these genes have been identified in the literature as either being related to gliomas or participating in pathways that are similarly impacted. These identified biomarker candidates were associated with 130 distinct pharmacological agents, many of which demonstrate potential for glioma treatment, including Theophylline, Ataluren, and Olaparib.
- Study of human gut microbiome dynamics in general population As part of the Citizen Science project Microbioma Comunidade PortugalPublication . Morais, Patrícia Costa; Gordo, Isabel; Xavier, Karina; Mota, JaimeThe human gut microbiome is highly diverse and plays a crucial role in health, influenced by factors such as diet, environment, and genetics. Although its composition has been extensively studied in several countries, the Portuguese microbiome remains poorly characterized. Moreover, only recently have a few longitudinal studies begun to emerge. This gap is addressed in this thesis through Microbioma Comunidade Portugal, a citizen science project involving 32 families, mainly from Oeiras municipality. Participants collected three fecal samples over time, and the family-based design allowed the inclusion of individuals across different ages and contributed to sustained motivation. Results revealed that these Portuguese cohort’s gut microbiota was dominated by Bacillota and Bacteroidota, consistent with other studies, and remained stable over time at the phylum level. The Bacillota/Bacteroidota ratio was consistent across time, age, and body mass index. Interestingly, alpha diversity increased overtime, possibly reflecting seasonal and/or behavior changes of the participants. Surprisingly, alpha diversity was higher in the elderly. Similar to other studies, intraindividual diversity was lower than interindividual diversity. Fecal metabolomic profiling did not show notable changes over time, and no correlations were found between key short-chain fatty acid-producing genera and the concentrations of these metabolites. Participants actively contributed feedback and engaged in laboratory visits and events, enhancing scientific literacy and trust in science. This study constitutes the first longitudinal analysis of the Portuguese gut microbiome, with an adherence rate of 94%, highlighting the valuable role of citizen science in microbiome research. Future studies involving larger and more geographically diverse cohorts will be essential to clarify the observed alpha diversity variations and further explore the functional dynamics of the gut microbiome
- Understanding cell response to AuNP- mediated hyperthermiaPublication . Cruz, Suzana Filomena Vicente Luz de Sousa; Baptista, Pedro; Fernandes, Maria AlexandraColorectal cancer is a significant public health issue, with a growing incidence and mortality rate. To address this increasing incidence, alternative approaches such as early de- tection, lifestyle intervention, and targeted treatments have been in progress. Among these targeted treatments, the use of gold nanoparticles (AuNPs)-mediated photothermal therapy has been a new approach to selectively kill cancer cells. AuNPs have physical and optical characteristics that allow them to be used in photothermal therapy. Previous studies have demonstrated that AuNP-mediated photothermal therapy induced by different green laser light sources promotes different types of cellular responses. Thus, this thesis has the objective of controlling different green laser light intensities in function of cellular response in HCT116 cell lines. AuNP@100%PEG was synthesized and characterized with success before heat gen- eration studies and in vitro evaluation. Heat generation studies confirmed that the hyper- thermia range was reached with 2.33 and 2.74 W/cm2 laser power. AuNP-mediated hyper- thermia effect in HCT116 showed that a combination of 0.33nM AuNPs and 2.74W/cm 2 pro- moted a decrease of cell viability associated with increased oxidative stress. Thermotolerance studies, associated with heat shock protein expression did not show prominent outcomes in any condition, however, modifications in the experimental design are required.
- A multi-omics approach to identify novel players in the pathogenesis of metabolic dysfunction-associated steatotic liver disease-associated liver cancerPublication . Simão, André Daniel Lopes; Grosso, Ana Rita; Rodrigues , PedroMetabolic dysfunction-associated steatotic liver disease (MASLD), the most common chronic liver disease worldwide, significantly increases the risk of hepatocellular carcinoma (HCC). Despite its rising incidence, effective strategies to halt MASLD progression towards HCC are lacking. In this sense, microRNAs (miRNAs/miRs) are key regulators of MASLD pathogenesis and progression. The aim of this project is to provide an in-depth study of MASLD and MASLD-associated HCC by conducting an integrative multi-omics analysis of distinct MASLD experimental models, focusing on identifying miRNA-associated pathways as novel therapeutic targets. Our analysis revealed differentially expressed miRNAs, protein and metabolites consistently dysregulated across the models. Among these, miR-146b-5p, miR-582-5p, and miR-34a-5p were upregulated in at least 6/8 experimental groups. Further correlation analyses between identified miRNAs and protein dataset, combined with miRNA-binding predictions, suggested potential direct regulatory interactions. Interestingly, CES2, potentially targeted by miR-146b-5p/miR-582-5p, correlated negatively with levels of some triglycerides and oleic acid, positively correlating with phosphocholines. Of note, miR-146b-5p was also increased in HCC tumors compared with adjacent non-tumorous liver tissue, highlighting its potential role as a driver of carcinogenesis in MASLD. Overall, our results provide a deeper understanding into miRNA-related networks crucial for the development of specific epigenetic-based signatures for disease progression.
- Single-Cell Profiling in Zebrafish Xenografts: Insights into Cancer ResistancePublication . Matos, João Lourenço Pereira de; Grosso, Ana Rita; Póvoa, VandaO cancro da mama triplo negativo (TNBC) é um subtipo agressivo caracterizado por elevada heterogeneidade, ausência de terapias alvo e remodelação extensa do microambiente tumoral (TME). Agentes antiangiogénicos como o bevacizumab podem reduzir a expansão tumoral, mas os mecanismos de resistência e modulação imunitária permanecem pouco esclarecidos. Xenógrafos de peixe-zebra constituem um modelo in vivo versátil para investigar estes processos em resolução celular. Para garantir análises fiáveis de sequenciação de RNA de célula única (scRNA-seq), foram comparadas estratégias de dissociação e fixação (células vivas, metanol, ACME) em xenógrafos da linha de TNBC Hs578T. Comparativamente ao protocolo com células vivas, a fixação por ACME revelou uma maior recuperação celular e preservação da integridade transcriptómica, enquanto o metanol manteve igualmente padrões de expressão génica e constitui uma alternativa válida. A scRNA-seq de xenógrafos a um e quatro dias pós-injeção, sob tratamento com PBS ou bevacizumab, identificou três estados tumorais principais: proliferativo, quiescente e inflamatório. O bevacizumab reduziu a população proliferativa e promoveu um programa inflamatório enriquecido em vias TNF, NF-κB e IL-17. A análise de comunicação célula–célula a um dia pós-injeção revelou aumento da sinalização de tumor para macrófagos através de ligandos da matriz extracelular que ativam o sindecano-4 (SDC4) em macrófagos associados ao tumor (TAMs). Estes resultados suportam um modelo em que o bloqueio de VEGF-A induz reprogramação inflamatória e remodelação da matriz, potenciando a retenção e polarização dos TAMs mediada por SDC4. No seu conjunto, este trabalho estabelece protocolos otimizados de scRNA-seq em xenógrafos em peixe-zebra e fornece nova perspetiva mecanística sobre a resistência induzida por bevacizumab no TNBC.
- Advancing CRISPR Therapies for lung genetic diseasesPublication . Branco, Mariana Morgado; Santos, Lúcia; Barreto, VascoCystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene, affecting mainly the lungs. Effective treatments for all CFTR variants remain a significant challenge. CRISPR-based technologies, particularly base editing, offer precise correction of single-nucleotide mutations. However, base editing components must be transiently expressed in target cells to minimize off-target effects. Delivering these components as ribonucleoproteins (RNPs) via engineered virus-like particles (eVLPs) presents a potentially safer option due to their limited lifespan within cells. This study aimed to evaluate the capacity of eVLPs pseudotyped with different viral envelope glycoproteins (Vesicular Stomatitis, SARS-CoV-2, and NipaH Virus) to target lung cells. As proof-of-concept, we developed a lung epithelial cell line (BCi-NS1.1) stably expressing a mutated version of the Green Fluorescent Protein with abolished fluorescence (dGFP) to evaluate the delivery capacity of the pseudotyped eVLPs. Our findings demonstrate that eVLPs can deliver base editing components to cells, thereby enabling the precise correction of the GFP mutation and restoring fluorescence. These results underscore the promise of base editing as a precise gene-correcting approach for CF, particularly for nonsense mutations unresponsive to current therapies. This strategy presents a potential pathway for restoring CFTR function and mitigating disease progression, warranting further refinement.
- Implementação e validação do novo kit de quantificação liofilizado investigator quantiplex pro flx, no laboratório de polícia científicaPublication . Grilo, Daniela Sofia da Silva; Ferreira, Paulo Miguel; Baptista, PedroNa atualidade forense a análise de vestígios que contenham frações suficientes de conteúdo biológico, do qual seja possível extrair quantidades significativas de ADN, trata-se do primeiro obstáculo enfrentado nesta área. Para as ciências forenses a obtenção de uma amostragem rica em DNA nem sempre é a realidade observada, ainda que na maioria das vezes por onde quer que passamos possamos através do toque ou da perda de um simples cabelo, deixar para trás informação identificativa. Não obstante a existência de material biológico, distintos fatores como a degradação da amostra ou a presença de inibidores poderão colocar em causa a obtenção de resultados com valor identificativo. A quantificação do ADN é desta forma um procedimento essencial para a otimização do processo de obtenção de um perfil genético com valor identificativo. Através da técnica de quantificação de material genético qPCR, surge o novo kit Investigator Quantiplex Pro FLX (QIAGEN) com a principal vantagem de conter os seus reagentes liofilizados, quantificar ínfimas concentrações de ADN humano e especial desempenho na quantificação de ADN masculino. Por conseguinte, o principal objetivo do presente estudo consistiu na implementação e validação deste kit no setor de biologia do Laboratório de Polícia Científica (LPC). Tendo sido utilizados dois principais grupos de amostras: amostras controlo (de modo a efetuar a validação) e amostras work case (com a finalidade de fundamentar a implementação), ambos quantificados com kit em estudo e com o kit em vigor no LPC. Em suma, a performance dos kits em estudo não foi ao encontro dos resultados expectados. E embora o kit Investigator Quantiplex Pro FLX demonstre elevada sensibilidade na deteção de ADN masculino, não foram constatadas diferenças significativas entre este kit e o kit atualmente em uso no LPC, pelo que, a aquisição do kit Investigator Quantiplex Pro FLX não representa uma vantagem significativa.
- Testing new glycosyltransferase inhibitors for cancer therapyPublication . Carrilho, João Pedro Nunes; Videira, Paula AlexandraSialyl Lewis x (sLex) is a fucosylated glycan epitope that mediates cell–cell interactions through binding to selectins on endothelial cells, facilitating leukocyte trafficking under physiological conditions. Its biosynthesis depends on α1-3 fucosyltransferases (FUTs), with FUT7 being the key enzyme catalyzing the terminal fucosylation step. FUT7 expression strongly correlates with sLex abundance on the cell surface. In pathological settings such as cancer, aberrant FUT7 expression and altered sLex levels promote tumour cell adhesion and metastasis. Because of this, both sLex and FUT7 are considered promising therapeutic targets. To date, there are no therapies directly targeting FUT7, only strategies affecting general fucosylation or other FUTs. Unpublished results suggested that new mimetic compounds able to specifically bind to FUT7 could specifically inhibit the activity of this enzyme. Their efficacy was tested by exofucosylation, which means extracellularly by incubating live cells with FUT7. Yet the effect on cell fucosylation machinery remains unknown. This thesis aimed to validate the in vitro activity of the most promising inhibitor. Gene expression analysis, combined with flow cytometry to analyse cell-surface glycans, was used to explore the molecular mode of action of this compound in three cell models (THP-1, KG-1a, and COLO205). The results did not directly confirm the inhibitor’s ability to reduce sLex expression in any cell line. However, in COLO205 cells, preliminary data suggested increased dimeric-sLex levels after treatment, likely due to substrate redirection to other FUTs following FUT7 inhibition, generating dimeric-sLex. In THP-1 cells, no compensatory FUT overexpression was detected. Additionally, MAL-I lectin staining in THP-1 revealed changes in Siaα2-3Galβ1-4GlcNAc structures. Furthermore, low DMSO concentrations also reduced sLex, suggesting solvent interference in biosynthesis. In conclusion, although direct validation of inhibitor efficacy was not achieved, this work highlights cell- and condition-specific regulation of sLex and the overlooked impact of solvents such as DMSO in therapeutic studies.
- A Pilot Study on the Genetic and Functional Characterization of RNases in Legionella pneumophilaPublication . David, Inês de Sousa Tavares; Chasqueira, Maria de Jesus; Matos, RuteLegionella, the etiological agent of Legionnaires’ disease, is ubiquitous in man-made water systems. In the environment, Legionella is constantly facing and surviving to stress conditions. RNases, enzymes involved in RNA metabolism, have an important role in bacterial adaptation to stress but are still poorly studied in Legionella. In this work we aim to comprehend the role of RNases in Legionella. The work was divided in three parts. We started by doing growth curves at 37 ºC (the optimal temperature of Legionella) and 15 ºC (a stress temperature) for all L. pneumophila clinical isolates from different serogroups. The results showed that all isolates grow slower at 15 ºC. Moreover, while at 37 ºC the growth curves are very similar to all the isolates under study, the growth at 15 ºC revealed differences in the adaptation of the isolates at this temperature. We also analysed the genetic variability of the genes encoding two RNases in all strains under study: rnc for RNase III, and rnr for RNase R. The sequencing results showed the presence of mutations that resulted in amino acid changes. Most of these changes are located in the catalytic domains of both enzymes, suggesting that they may impact their activity. Finally, we expressed and purified Legionella RNases, and performed activities assays with PNPase at 37 ºC and 15 ºC. The results showed that PNPase is active at both temperatures, although its activity was reduced at 15 ºC. As a pilot study, this works allowed us to obtain preliminary results about the role of RNases in Legionella and its adaptation to the environmental conditions. The study of new regulatory targets of Legionella such as RNases allow for a diversification of strategies to control its survival, which could be important to improve the prevention measures and reduce the number of infection cases.
- The sensor and effector arms of the Clostridioides difficile response to β-lactamsPublication . Novais, Maria Inês Teixeira Duarte de Sousa; Henriques, Adriano José; Miranda, Mónica PaulaClostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhea. Treating C. difficile infection is challenging because antibiotics are both the primary therapy and the major risk factor for the disease. Most strains of C. difficile have four penicillin-binding proteins (PBPs). Clinical isolates of ribotype 017, however, which often display resistance to carbapenems such as imipenem (IMI), carry an additional PBP, termed PBP5. PBP5 is a structural homologue of PBP2a, which confers broad β-lactam resistance in Staphylococcus aureus and its production is induced by β-lactams. While previous work showed that PBP5 alone is insufficient to confer IMI resistance, its precise contribution remained unclear. Here, we aimed to determine whether PBP5 contributes to IMI resistance and how its expression is coordinated with that of the blaI/R and blaX/CDD operons, the latter coding for the BlaCDD β-lactamase, and thought to shape the response of C. difficile to β-lactams. In other organisms, BlaI represses gene transcription by binding to target promoters, whereas BlaR, a transmembrane β-lactam sensor, becomes activated upon antibiotic exposure and cleaves BlaI, thereby relieving repression. PBP5 is extremely sensitive to ampicillin (AMP) and amoxicillin (AMX). We show that PBP5 is required for IMI resistance as its chemical inhibition with AMP or AMX restores IMI susceptibility to resistant strains. Using transcriptional SNAPCd fusions, we show that like pbp5 the blaX/CDD and blaI/R operons are repressed under non-inducing conditions, and that exposure to IMI relieves this repression in a BlaI/R-dependent manner. In the absence of BlaI, these genes are constitutively expressed, confirming that BlaI acts as a transcriptional repressor. Finally, electrophoretic mobility shift assays (EMSAs) revealed that BlaI binds directly to all three promoter regions, and quantitative analysis allowed us to estimate dissociation constants (Kd) for these interactions.Together, these findings establish PBP5 as a key determinant of IMI resistance and uncover the BlaI/R-mediated regulatory mechanism coordinating β-lactam resistance in C. difficile.