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- Ceftaroline Resistance in MRSA- identification of cell wall modificationsPublication . Máximo, Carolina da Fonseca; Sobral, Rita Gonçalves; Dias, JorgeMethicillin-Resistant Staphylococcus aureus (MRSA) is a critical global health issue that causes major therapeutic challenges. Its resistance to β-lactam antibiotics results from the acquisition of mecA gene, which encodes penicillin-binding protein 2a (PBP2a); consequently, new therapeutic options are essential to fight MRSA resistance. With these therapeutic challenges emerged Ceftaroline (CPT), a fifth-generation cephalosporin with strong anti-MRSA activity due to its allosteric binding. Importantly, MRSA infections are prevalent in patients with Cystic Fibrosis (CF); since CPT is also used for the treatment of patients with acute pulmonary exacerbations of CF, and cases of CPT-resistant strains have been reported in these patients, it is crucial to understand the mechanisms behind resistance. Studies also suggested that TrfA, an adaptor involved in cell wall biosynthesis, is an important regulatory amplifier of CPT resistance in MRSA; therefore, a better understanding of its influence is needed. The aim of this project was to determine the biochemical and molecular mechanisms underlying the formation of this muropeptide associated with CPT resistance in MRSA. Considering this, studies were also conducted on ΔmecA knockouts to elucidate the molecular origin of this muropeptide, and on ΔtrfA knockouts to further comprehend how auxiliary factors may influence the formation of this cyclization. Studies on CPT-intermediate strains (4519 and 5006) were also conducted to understand the biochemical requirements of this peptide. This analysis showed that the mecA gene is essential for the muropeptide biosynthesis and that the Y446N substitution in the PBP2a transpeptidase domain may be required to stabilize it. To further understand how alterations in the cell wall influence physiological changes in S. aureus, cytochrome-binding assays and autolysis assays were performed in the clinical strains 3957 and 5007. Results exhibit a more electron-negative charge in the 3957 strain, even in the presence of CPT, and these results may also influence the autolysin pattern exhibited. To understand how mutations in the allosteric and active sites of PBP2a are important for the phenotype in clinical isolates, revertant mutants are also being studied.
- Avaliação de fármacos para reposicionamento como inibidores de biofilmes maduros em Staphylococcus aureus e Staphylococcus epidermidisPublication . Nunes, Matilde Batista; Costa, Sofia; Pinto, Sandra; Couto, IsabelA resistência aos antimicrobianos constitui um dos maiores desafios de saúde pública da atualidade, especialmente nas infeções causadas por Staphylococcus aureus e Staphylococcus epidermidis. Estas espécies destacam-se pela elevada capacidade de formar biofilmes, estruturas complexas que conferem proteção acrescida contra antibióticos e respostas imunitárias, contribuindo para a persistência e recidiva de infeções. Dada a escassez de novas opções terapêuticas, o reposicionamento de fármacos tem surgido como uma estratégia promissora para identificar fármacos com potencial antibiofilme. Neste trabalho, procurou-se avaliar a capacidade de seis fármacos candidatos, amlodipina, imipramina, doxepina, desipramina, fluoxetina e sunitinib, previamente identificados como inibidores de efluxo e da formação de biofilme, em reduzir ou inibir biofilmes maduros de S. aureus e S. epidermidis, explorando a potencial relação efluxo-biofilme. Para isso, foram utilizados dois pares de estirpes isogénicas, um de S. aureus e um de S. epidermidis, compostos por uma estirpe parental e respetiva estirpe adaptada com sobreexpressão do gene norA, que codifica para a bomba de efluxo NorA. A atividade dos fármacos candidatos foi analisada através de ensaios MBEC. Nestes, a imipramina e doxepina demonstraram atividade relevante de redução de biofilmes maduros de S. aureus, particularmente na estirpe adaptada com atividade de efluxo aumentada, para a qual estes fármacos promoveram a erradicação completa do biofilme. A amlodipina revelou apenas um efeito inibitório moderado, enquanto a fluoxetina, desipramina e sunitinib não apresentaram atividade significativa contra os biofilmes maduros testados. Em S. epidermidis, nenhum dos fármacos avaliados apresentou efeito. A análise por microscopia confirmou o efeito da imipramina, evidenciado pela redução acentuada de células viáveis, mas não revelou diferenças estruturais dos biofilmes relevantes entre estirpes parentais e adaptadas. Estes resultados reforçam a capacidade da imipramina, doxepina e amlodipina, como potenciais fármacos para reposicionamento e uma nova estratégia promissora no combate a infeções associadas a biofilmes maduros, particularmente em S. aureus.
- ESTABLISHING MICRONUTRIENT DEFICIENCIES IMPACT ON METABOLIC DRUGS-GUT MICROBIOME INTERACTIONSPublication . Santos, Mafalda Barata dos; Santamarina, SarelaAtherosclerotic cardiovascular disease is the leading cause of mortality worldwide, and statin therapy is recommended for its prevention and management. By inhibiting 3-hydroxy-3- methylglutaryl-CoA reductase in the liver, statins lower Low-Density Lipoprotein (LDL) cholesterol levels. However, their efficacy varies between individuals. Because statins are absorbed in the gut, bidirectional interactions with the microbiota may contribute to this variability: drugs can alter microbial composition, while the microbiota can modulate drug bioavailability. Yet, the microbial targets of statins remain unclear. Recent work showed that high simvastatin concentrations upregulate genes encoding iron– sulfur cluster (Fe-S) proteins in Gram-positive gut bacteria. We hypothesized that statins might be toxic to gut bacteria by disrupting iron-related pathways. To test this, bacteria were cultured under iron-deficient conditions with statins; only simvastatin demonstrated synergy. Iron-binding assays and xanthine oxidase tests indicated no direct interaction with iron. Cell viability assays showed that Coprococcus comes, a common gut bacterium, became more sensitive to a membrane-disrupting agent under high simvastatin, suggesting Fe–S protein upregulation may be a collateral response to membrane disruption. These findings are preliminary and require further validation.
- Molecular and cellular alterations caused by environmental genotoxicants on the regenerative ability of marine speciesPublication . Abreu, Mariana Guerreiro; Costa, Pedro; Diniz, MárioIn recent years, regenerative medicine has emerged as a rapidly advancing field within biomedicine, aiming to develop innovative treatments to overcome the limitations of conventional surgical and therapeutic approaches. While humans possess limited regenerative capacities, other species have the ability to regenerate their whole body, like planarians and sea anemones. Several internal and external factors influence the regeneration process, including chemical stressors, such as carcinogenic com- pounds. Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants that represent a threat to both environment and human health, with benzo[α]pyrene, being a well-known carcinogenic model of this group. The main objective of this thesis was to evaluate whether pollution can have a significant impact on the regeneration process, and to translate this knowledge into new perspectives for humans health. Upon exposure to 100ng/L of B[α]P, planarians (Leptoplana tremellaris) exhibited high mortality, how- ever, a greater regeneration success in the head fragment regenerating the tail was observed. In sea anemones (Actinia equina) exposed to two concentrations of B[α]P (10 and 100 ng/L of B[α]P), exhib- ited histopathological alterations. Metabolic activation B[α]P led to a slight increase in the activity of biochemical stress markers (GST, SOD) in the group exposed to the higher concentration. The exposure to B[α]P resulted in DNA damage, which in turn activated a P53 response, consequently leading to cell death. Evidences of DNA damage and cell death were observed through comet assay and histopatho- logical analysis of the tentacles. The present work showed that pollutants that interfere with cell cycle and differentiation can hinder regeneration. However, the relatively high sensitivity to B[α]P, reduced genomic resources and knowledge gaps in the physiology of marine invertebrates must the addressed.
- Biological role of carboxyhemoglobin (COHb) against hemorrhagic stroke: anti-inflammatory and anti-oxidant effectsPublication . Rodrigues, Beatriz Faria; Vieira, HelenaHemorrhagic stroke is one of the leading causes of mortality and disability worldwide. Its pathophysiology involves processes such as neuroinflammation, oxidative stress, cell death, and disruption of the blood-brain barrier. These phenomena result primarily from the rupture of blood vessels and the subsequent release of erythrocytes. Hemolysis promotes the release of large amounts of hemoglobin (Hb), which comes into contact with brain cells. Free Hb rapidly undergoes autooxidation, forming its oxidized form, methemoglobin (MetHb), a pro-oxidant that intensifies the aforementioned dam- age. Carbon monoxide (CO) is an endogenous gas produced primarily by the degradation of the heme group mediated by heme oxygenase enzymes. Although toxic in high concentrations, causing hypoxia and inhibiting mitochondrial respiration, CO has demonstrated anti-inflamma- tory, anti-oxidant, anti-apoptotic, and cytoprotective properties at low concentrations. CO binds with high affinity to Hb, forming carboxyhemoglobin (COHb). For many years, COHb was associ- ated with CO toxicity, functioning as a measure of toxicity. However, recently, there has been growing interest in its potential protective role. The present study aimed to investigate the biological role of COHb in the context of hem- orrhagic stroke. Specifically, its anti-inflammatory and anti-oxidant effects on microglia relative to MetHb were explored, as well as the impact of intracellular COHb formation in erythrocytes, evaluating changes in their redox state and secretome, and how these can modulate microglial function. The results suggest that COHb exerts anti-inflammatory effects by reducing nitric oxide (NO) production and anti-oxidant effects by increasing catalase activity in microglia. Further- more, it has been shown to reduce the production of reactive oxygen species (ROS) in erythro- cytes and increase the levels of glutathione-associated metabolites, reinforcing its protective role. Although preliminary, these findings point to COHb as a potential therapeutic candidate for modulating inflammation and oxidative stress in hemorrhagic stroke.
- Interplay of Ageing and metabolism: Impact on osteogenic differentiationPublication . Manco, João Palmela; Deus, Claúdia; Baptista, PedroBone is a mineralized, dynamic, and metabolically active connective tissue composed of organic and inorganic matrices. Bone homeostasis relies on three main cell types, among which osteoblasts play a key role by driving osteogenesis. Ageing significantly impairs this process, resulting in increased bone resorption. This impairment is associated with cellular senescence, accumulation of reactive oxygen species and functional decline of osteoblasts and osteocytes, ultimately contributing to bone-related pathologies. However, the specific impact of cellular senescence on osteogenic differentiation remains unclear. This work aimed to investigate how cellular senescence influences osteogenic differentiation and its related metabolism To address this, Warton-Jelly-derived mesenchymal stem cells (WJ-MSC) with different senes- cence ratios were characterized and used as an ageing model. Different senescence rates with and without dexamethasone (Dex), an osteogenic stimulator, were tested. The activity of early marker of bone formation, alkaline phosphatase (ALP) and the expression of osteogenic and metabolism-related genes was assessed by standard methods Our results demonstrated that increasing senescence correlated with higher ALP activity. ALP gene expression analysis confirmed this trend, indicating enhanced osteogenic differentiation in senescent cells. Interestingly, RUNX-2 gene expression was virtually absent, suggesting that differentiation occurred through RUNX-2-independent mechanisms. In parallel, the expression of metabolic-related genes such as AMPK, NDUFA9, and ATP5G1 showed only minor changes in senescent cells, whereas NRF2 gene expression remained unaltered. Acute/transient inflammatory responses triggered by senescence cells may explain some of our results on osteogenic differentiation. Further experiments are needed to confirm the presence of inflammatory mediators and to perform long-term osteogenic differentiation analyses. Overall, our results highlight the complex influence of cellular senescence on early bone formation and suggest potential implications for the development of novel regenerative strategies, including biomaterial scaffolds, to treat age-related bone disorders.
- Generation of an HFpEF in vitro model using hiPSC-derived cardiac organoidsPublication . Araújo, Beatriz da Conceição; Belo , José; Videira, PaulaHeart failure with preserved ejection fraction (HFpEF) affects over 32 million people worldwide, with fibrosis representing a key underlying mechanism. Effective therapies remain lacking due to the challenge of establishing HFpEF models. A recent study from our laboratory identified miRNA-mRNA interactions potentially relevant to HFpEF and validated them in 2D cardiomyocyte cultures: Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) was downregulated by hsa-miR-25-3p and hsa-miR-26a-5p, while Natriuretic Peptide B (NPPB) was downregulated by hsa-miR-26a-5p. Although human induced pluripotent stem cell (hiPSC)- based cardiac models are useful tools for studying cardiac disease, 2D systems do not replicate the complexity of in vivo (patho)physiology. To overcome this, hiPSC-derived cardiac organ- oids have emerged as a promising alternative. hiPSC-derived human cardioids that recapitulate essential structural and functional features of the native heart were generated in the frame of this work. Here, I aimed to advance the establishment of a reliable HFpEF in vitro model by addressing the potential role of hsa-miR-25-3p and hsa-miR-26a-5p in myocardial stiffness, using hiPSC-derived cardioids. The suitability of cardioids for studying fibrosis-related mechanisms was demonstrated by cardiac fibroblast (CF) activation, evidenced by increased α-smooth muscle actin (α-SMA) expression, as well as by extracellular matrix (ECM) accumulation, evidenced by a statistically significant increase in collagen deposits following Transforming Growth Factor β (TGF-β) treatment. miRNA delivery into cardioids was optimized, with a liposomal-based approach using 1 μM miRNA and serum supplementation after complex formation as the most effective condition, ensuring distribution throughout the inner regions of the cardioid for six days. Repeated delivery of hsa-miR-25-3p and hsa-miR-26a-5p reduced HAPLN1 expression in one cardioid batch, although no significant overall reduction was observed for either HAPLN1 or NPPB. Under the same conditions, CF activation was promoted, although not significantly. ECM deposition did not increase significantly following the delivery of the miRNAs. Altogether, this work demonstrates that our cardioid model effectively recapitulates features of the human heart and is a suitable platform to investigate fibrosis-associated mechanisms, supporting its potential as an HFpEF model. Moreover, we established a protocol for miRNA delivery into 3D cardiac systems. Further studies are required to confirm the down- regulation of HAPLN1 and NPPB by hsa-miR-25-3p and hsa-miR-26a-5p in cardioids, as well as to clarify their role in modulating CF activation and ECM deposition, processes that contribute to myocardial stiffness in HFpEF.
- Functional and structural characterization of higher-order oligomers of phenylalanine hydroxylasePublication . Sarmento, Licínia Isabel Martins; Leandro, Ana; Baptista, PedroPhenylalanine hydroxylase (PAH), which catalyses the conversion of L-phenylalanine (L-Phe) into L-tyrosine (L-Tyr), biologically assembles as homotetramers (TM) but in solution is consistently found in higher-order oligomeric forms such as octamers (OM). Mutations in the PAH gene causes phenylke- tonuria (PKU), the most common inborn metabolic disorder (IMD) of amino acid catabolism, associated with over 3400 pathogenic variants. PKU is characterized as a conformational disorder, resulting from impaired folding and reduced thermodynamic stability of variant PAH proteins, leading to intracellular premature degradation. PKU results in a neurotoxic accumulation of L-Phe in the brain, which can cause severe intellectual and motor impairment. It has been described that most PAH gene missense mutations promote PAH aggregation and assembly into higher-order oligomers with fibrillar structures described for the p.G46S variant, suggesting a link between disturbed oligomerization and PKU pathogenesis. Recently, supra-assembly of metabolic enzymes has emerged as a novel mechanism of enzyme activity regulation. This study aimed to isolate and characterize OM forms of human PAH to determine whether they represent the starting point for aggregation or if they could represent supra-assembled structures with a biological role. PAH was overexpressed and purified using three prokaryotic expression systems (pTrc, pET-28a+ and pMAL) and both TM and OM, tagged and untagged, were purified. Functional characterization was performed through enzymatic activity assays, while structural properties were eval- uated using far-ultraviolet circular dichroism (Far-UV CD), limited proteolysis by trypsin, differential scanning fluorimetry (DSF) and dynamic light scattering (DLS). The obtained results showed that OM are enzymatically active and maintain secondary structure profiles comparable to TM. Structural evi- dence suggests that OM assembly involves interactions predominantly through the catalytic domain (CD) rather than the regulatory domain (RD), while preserving ligand responsiveness and increased stability. OM revealed higher resistance to aggregation, strongly suggesting these species are not the starting point for aggregation.
- Developing targeted chronotherapy protocols in T-cell acute lymphoblastic leukemiaPublication . Marques, Tomás Francisco da Silva; Barata, João; Baptista, PedroCircadian rhythms are an essential mechanism to the proper adaptation of organisms to their environment and to the periodical changes which occur inherent to life on Earth. The disruption of circadian rhythms contributes to oncogenesis by promoting the onset of all can- cer hallmarks. Furthermore, a link exists between the Circadian Molecular Clock, which regu- lates circadian oscillations in humans, and the PI3K/AKT/mTOR pathway, which is hyperac- tivated in T-ALL patients. This link resides in the activity of CK2, a kinase that contributes towards the activation of PI3K signaling by inhibiting PTEN, and towards the establishment of rhythmicity by phosphorylating BMAL1, allowing its nuclear localization. There are a lot of unknowns regarding the persistence of circadian rhythms in T-ALL, and thus, in this work, we set out to characterize circadian rhythms in leukemic cells and to compare them to healthy counterparts. First, we found a distinct lack of BMAL1 in most leukemic samples we analyzed. However, some rhythmicity was still present in T-ALL cells, even if slightly less pronounced than that displayed by healthy thymocytes. Out of all the genes analyzed, IL7R appeared as the most consistently rhythmic. The data presented considerable heterogeneity. Thus, syn- chronization was employed to improve consistency. To truly assess the effect of BMAL1 ab- sence, we performed qPCRs on CMC genes and checked for survival differences in mice with different levels of BMAL expression. The results suggested that absence of BMAL1 expression correlates negatively with rhythmicity and survival. Finally, we successfully tested a lucifer- ase-based PI3K signaling activity readout reporter, that should facilitate the analysis of PI3K pathway oscillations. Coupled with the knowledge we are acquiring about circadian oscilla- tions, this novel tool will be of key importance in defining the best points for the development of targeted chronotherapeutic protocols for T-ALL in the future.
- Unveiling the Mechanism of Action of Spiro-β-Lactam Antivirals: A Single-Cell Transcriptomics ApproachPublication . Lopes, Inês dos Santos Pais Bernardo; Taveira, Nuno; Bártolo, Inês; Sobral, RitaO HIV, ou vírus da imunodeficiência humana, continua a constituir um desafio de saúde pública de grandes proporções, agravado pela emergência de estirpes multirresistentes. Os compostos espiro- β-lactâmicos, incluindo o BSS-730A, demonstraram uma potente atividade contra o HIV e outros vírus RNA, mas o seu mecanismo de ação permanece pouco compreendido. O presente estudo teve como objetivo investigar o mecanismo de ação antiviral do BSS-730A, recorrendo a uma análise transcriptómica de células únicas (scRNA-seq) de linfócitos do sangue periférico. O tratamento com BSS-730A induziu uma forte regulação positiva dos genes da ferritina (FTL e FTH1) e SQSTM1 (p62), um regulador chave das respostas ao stress celular, enquanto regulou negativamente genes que podem facilitar a replicação do HIV. A inibição da ferroptose com Ferrostatina-1 (Fer-1) aumentou a replicação viral, indicando que a ferroptose da célula hospedeira pode restringir o HIV. Embora o BSS-730A tenha antagonizado a atividade da Fer-1, um análogo estruturalmente relacionado sem atividade antiviral (CP9A) também o fez, sugerindo que a ferroptose não é o principal mecanismo de ação antiviral. Estas descobertas suportam um modelo no qual o BSS-730A suprime a replicação do HIV, sobretudo através da sequestração do ferro intracelular, resultando na redução da atividade da transcriptase reversa, com contribuições adicionais das vias de resposta ao stress. Os possíveis papéis da ferritinofagia e da ferroptose ainda carecem de esclarecimento. Estudos adicionais são necessários para validar estes mecanismos de ação no HIV e avaliar se eles se estendem a outros vírus de RNA, contribuindo para o desenvolvimento de derivados de espiro- β-lactâmicos como antivirais de amplo espetro.