Browsing by Author "Rocha, Ana Catarina"
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- Cannabidiol and terpene formulation reducing SARS-CoV-2 infectivity tackling a therapeutic strategyPublication . Santos, Susana; Barata, Pedro; Charmier, Adilia; Lehmann, Inês; Rodrigues, Suzilaine; Melosini, Matteo M.; Pais, Patrick J.; Sousa, André P.; Teixeira, Catarina; Santos, Inês; Rocha, Ana Catarina; Baylina, Pilar; Fernandes, RubenIn late 2019, COVID-19 emerged in Wuhan, China. Currently, it is an ongoing global health threat stressing the need for therapeutic compounds. Linking the virus life cycle and its interaction with cell receptors and internal cellular machinery is key to developing therapies based on the control of infectivity and inflammation. In this framework, we evaluate the combination of cannabidiol (CBD), as an anti-inflammatory molecule, and terpenes, by their anti-microbiological properties, in reducing SARS-CoV-2 infectivity. Our group settled six formulations combining CBD and terpenes purified from Cannabis sativa L, Origanum vulgare, and Thymus mastichina. The formulations were analyzed by HPLC and GC-MS and evaluated for virucide and antiviral potential by in vitro studies in alveolar basal epithelial, colon, kidney, and keratinocyte human cell lines.
- Creation of a fungal library and screening of antimicrobial and anticancer activityPublication . Ferreira, Diogo; Hermida, Lara Areal; Rocha, Ana Catarina; Baylina, Pilar; Sieiro, Carmen; Fernandes, Rúben; BAYLINA MACHADO, PILARAccording to the World Health Organization, cancer and infectious diseases are two of the most problematic diseases nowadays. Cancer kills 10 million people every year and the emergence of resistance to antitumoral drugs is an important medical challenge. At the same time, antimicrobial resistance (AMR) is also a serious threat to human and environmental health. Besides mortality, AMR burdens healthcare services and dampens medical procedures such as surgeries, cancer treatments and other invasive procedures. The development of new drug therapies to fight drug resistance is essential to contest the rising of resistant bacteria and reduction of the effectiveness of antitumoral drugs. Microorganisms have been a major source for natural compounds throughout the years. Fungi, renowned for their ability to produce an array of broad and diverse secondary metabolites, offer a rich resource for drug discovery. We built a collection of fungal species, isolated from chestnuts, sunflower seeds, and chestnut flour, and explored their extracts for potential antimicrobial and anticancer activity. Fungi cultures for secondary metabolite biosynthesis were done in submerged fermentation in Malt Extract broth for 15 days at 26 °C. Liquid-liquid extraction techniques, with ethyl acetate as a solvent, were applied to obtain crude secondary metabolite extracts. Clinical resistant bacteria, yeasts, and prostate cell lines (human prostate epithelial cells – HpepiC; human caucasian prostate adenocarcinoma cells - PC3) were exposed to fungal extracts at a single concentration of 100 µg/mL. Our results so far show several extracts with antimicrobial and/or anticancer activity without decreasing cell viability of non-tumoral cells, showing their potential as therapeutic drugs without possible secondary effects. Although, more studies should be done, and pending fungal identification will allow us to select which extracts will be further investigated to find if the displayed bioactivity could be happening due to unknown natural compounds
- Enhanced 3T3-L1 differentiation into adipocytes by pioglitazone pharmacological activation of peroxisome proliferator activated receptor-gamma (PPAR-gamma)Publication . Teixeira, Catarina; Sousa, André P.; Santos, Inês; Rocha, Ana Catarina; Alencastre, Inês; Pereira, Ana Cláudia; Martins-Mendes, Daniela; Barata, Pedro; Baylina, Pilar; Fernandes, RúbenDespite the primary function of pioglitazone in antidiabetic treatment, this drug is a potent inducer of PPAR-γ, a crucial receptor that is involved in adipocyte differentiation. In this work, we propose an optimized methodology to enhance the differentiation of 3T3-L1 fibroblasts into adipocytes. This process is crucial for adipocyte secretome release, which is fundamental for understanding the molecular mechanisms that are involved in obesity for in vitro studies. To achieve this, a pioglitazone dose-response assay was determined over a range varying from 0 to 10 µM. Lipid accumulation was evaluated using Oil-Red-O. The results showed that 10 µM pioglitazone enhanced differentiation and increased secretome production. This secretome was then added into two cell lines: PC3 and RAW264.7. In the PC3 cells, an increase of aggressiveness was observed in terms of viability and proliferation, with the increase of anti-inflammatory cytokines. Conversely, in RAW264.7 cells, a reduction of viability and proliferation was observed, with a decrease in the overexpression of pro-inflammatory cytokines. Overall, the present work constitutes an improved method for adipocyte secretome production that is suitable for experimental biology studies and that could help with our understanding of the molecular mechanisms underlying adiposity influence in other cells.
- Ensaios in vitro de avaliação do efeito antitumoral de inibidores glicolíticosPublication . Cunha, Andrea; Rocha, Ana Catarina; Silva, Daniela; Barbosa, Flávia; Sarmento, Bruno; Queirós, OdíliaA quimioterapia é particularmente tóxica para as células tumorais; porém, não é específica, sendo responsável por importantes efeitos adversos associados ao tratamento. O "Efeito Warburg" é uma característica do cancro, que consiste numa alteração metabólica na produção de energia da fosforilação oxidativa para a glicólise, mesmo na presença de O2. A ativação contínua da glicólise conduz a um rápido aumento de energia e de lactato, promovendo a proliferação, a invasão e a resistência à quimioterapia. O 3-bromopiruvato (3BP), o dicloroacetato (DCA) e a 2-desoxiglucose (2DG) são agentes anti-glicolíticos que inibem o metabolismo energético da célula tumoral, levando à depleção do ATP celular.
- Harvesting the power of green synthesis: gold nanoparticles tailored for prostate cancer therapyPublication . Oliveira, Marco; Sousa, André; Sá, Sara; Soares, Sílvia; Pereira, Ana Cláudia; Rocha, Ana Catarina; Pais, Patrick; Ferreira, Diogo; Almeida, Cátia; Luís, Carla; Lima, Cláudio; Almeida, Fábio; Gestoso, Álvaro; Duarte, Miguel-Correa; Barata, Pedro; Martins-Mendes, Daniela; Baylina, Pilar; Pereira, Carla F.; Fernandes, RúbenBiosynthetic gold nanoparticles (bAuNPs) present a promising avenue for enhancing biocompatibility and offering an economically and environmentally responsible alternative to traditional production methods, achieved through a reduction in the use of hazardous chemicals. While the potential of bAuNPs as anticancer agents has been explored, there is a limited body of research focusing on the crucial physicochemical conditions influencing bAuNP production. In this study, we aim to identify the optimal growth phase of Pseudomonas aeruginosa cultures that maximizes the redox potential and coordinates the formation of bAuNPs with increased efficiency. The investigation employs 2,6-dichlorophenolindophenol (DCIP) as a redox indicator. Simultaneously, we explore the impact of temperature, pH, and incubation duration on the biosynthesis of bAuNPs, with a specific emphasis on their potential application as antitumor agents. Characterization of the resulting bAuNPs is conducted using ATR-FT-IR, TEM, and UV-Vis spectroscopy. To gain insights into the anticancer potential of bAuNPs, an experimental model is employed, utilizing both non-neoplastic (HPEpiC) and neoplastic (PC3) epithelial cell lines. Notably, P. aeruginosa cultures at 9 h/OD600 = 1, combined with biosynthesis at pH 9.0 for 24 h at 58 ◦C, produce bAuNPs that exhibit smaller, more spherical, and less aggregated characteristics. Crucially, these nanoparticles demonstrate negligible effects on HPEpiC cells while significantly impacting PC3 cells, resulting in reduced viability, migration, and lower IL-6 levels. This research lays the groundwork for the development of more specialized, economical, and ecologically friendly treatment modalities.
- Potential anticancer activity from food-isolated fungi extractsPublication . Ferreira, Diogo; Rocha, Ana Catarina; Baylina, Pilar; Sieiro, Carmen; Fernandes, RúbenFungal species have demonstrated great potential to produce a wide range of metabolites, including enzymes, antibiotics, and other bioactive compounds with therapeutic interest. Prostate cancer (PCa) is one of the most frequent cancers in men. This type of tumors have high levels of heterogeneity, leading to therapeutic failures and increasing resistance against chemotherapeutic drugs. Hence, is essential to research new therapeutic agents against PCa. Exploring the rich reservoir of fungal diversity, this study aims to uncover bioactive compounds that may serve as valuable candidates for developing novel therapeutics against prostate cancer. Isolation from chestnuts, chestnut flour and sunflower seeds led to the creation of a fungal collection of 165 isolates. Fungi isolates grew in flask cultures for 15 days, and culture broths were extracted with ethyl acetate. Human prostate epithelial cells (HPepiC) and the human prostate cancer cell line (PC3) were exposed to the fungal extracts at a concentration 100 μg/mL, and cell viability was evaluated by MTT assay. Results show that several fungal extracts significantly reduce the viability of tumor cells, with some showing little to no effect on healthy human cells, however, species identification is essential to carry on our studies.
- Virulence-linked mutations in rubredoxin reductase and glutaredoxin: impact on antibiotic susceptibility and phage therapy in Pseudomonas aeruginosaPublication . Sá, Sara; Silva, Carina; Dias, Maria Clara; Veiga, Marlene; Lopes, Sofia; Fernandes, Ruben; Rocha, Ana Catarina; Pais, Patrick J.; Oliveira, Marco; Mendes, João; Novais, Gonçalo; Luís, Carla; Gestoso, Álvaro; Macedo, José Mário; Martins-Mendes, Daniela; Pereira, Ana Cláudia; Baylina, PilarPseudomonas aeruginosa (PAO1) is an opportunistic pathogen, lethal in immunocompromised individuals. The clinical management of PAO1 infections still depends deeply on antibiotic therapy. However, this therapy has been alarmingly overpowered by growing bacterial resistance mechanisms over the years. One of these bacterial mechanisms is quorum sensing (QS). QS is involved in the production of biofilm, rhamnolipids and pyocyanin, among other factors. The present study aimed to study the effect of the mutations in the genes of rubredoxin (Rub A1 and Rub A2) and glutaredoxin (GLRx) in the production of virulence traits and susceptibility of PAO1 to the antibiotic ciprofloxacin (CIP) and to infection by a phage cocktail. Rub A1, Rub A2, and GLRx showed a decrease in the expression of genes lasI, lasR, mvfR, and rpsL when compared to the wild type, PAO1. Rub A1 and Rub A2 also showed a decrease in the expression of the gene pqsA, while the mutant GLRx showed an increase of over 200% in expression compared to PAO1. The biofilm produced by the mutants Rub A1, Rub A2, and GLRx increased more than 1.5 times in comparison to PAO1, with statistical significance (p < 0.0001). In the viability assay, the mutant strain Rub A2 was the most susceptible to ciprofloxacin in both concentrations tested (p < 0.0001). The production of proteases increased in the mutant strains when compared to PAO1 (p < 0.05). However, there was a decrease in the production of rhamnolipids and pyocyanins in the mutant strains. In the phage assay, we could perceive a reduction in the growth of the mutant strains when compared to PAO1. Additionally, after the addition of the phages, all the strains showed susceptibility to the phage assay (p < 0.0001), observed in the decrease in the absorbance values. These results may highlight the relevance of the genes Rub A1, Rub A2, and GLRX in the proliferation and treatment of infections with PAO1. Overall, this study gives preliminary insights into how gene expression may be helpful in strategies to overcome antibiotic resistance.