ESS - TBIO - Posters apresentados em eventos científicos
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Browsing ESS - TBIO - Posters apresentados em eventos científicos by Author "Coelho, Pedro"
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- Development of a stable melanoma dual reporter cell line expressing Luciferase and GFPPublication . Aguiar, Gonçalo; Torres, Sílvia; Prudêncio, Cristina; Soares, Raquel; Coelho, Pedro; Prudêncio, Cristina; Coelho, PedroMelanoma is the most aggressive and lethal form of skin cancer, with a high risk of metastatic spread. Obesity is recognized as a risk factor for various types of cancer. However, regarding melanoma, this association remains controversial. Obesity might act as a double-edged sword in melanoma, promoting primary tumour growth but at the same time limiting metastatic spread - the "obesity paradox”. Herein, we aimed to create a stable murine B16F10 melanoma cell line expressing both firefly luciferase (Luc) and green fluorescent protein (GFP), which will later be engrafted into diet induced-obesity animal model for future in vivo studies. B16F10-Luc-GFP cells were generated by transfection with premade lentiviral particles, featuring a construct with Luc and GFP under a cytomegalovirus promoter and mediated by a F2A element. The antibiotic selection marker (puromycin) is expressed under a Rous sarcoma virus promoter. Afterwards, the transfected cells were selected with 1 μg/ml of puromycin. The clones with the highest levels of GFP-positive cells and GFP fluorescence were purified by two rounds of cell sorting and submitted to fluorescence and bioluminescence quantification, morphology, injury, BrdU incorporation, 7-AAD, and PI cell cycle assays and compared to the parental cell line. B16F10-Luc-GFP were successfully generated, and both GFP fluorescence and D-luciferin bioluminescence are present and proportional to cell density. As expected, the parental cell line didn’t display GFP or Luc activities. Moreover, transduced cells exhibit similar morphology, motility, proliferation, viability, and cell cycle progression as B16F10 cells. Conclusions: Altogether, the future engraftment of B16F10-LucGFP in obese mice, will improve melanoma research models, enabling the in vivo and ex vivo visualization of primary tumours and metastasis, providing a better understanding of the underlying molecular mechanisms, to clarify the “obesity paradox” in melanoma.
- In vitro evaluation of peptides with potential antioxidant, anti-inflammatory and antihypertensive activitiesPublication . Borges, Thais; Ferraz, Ricardo; Coelho, Pedro; Prudêncio, Cristina; Gomes, Ana; Gomes, PaulaHypertension develops from genetic and environmental factors, and is exacerbated by disorders that increase systemic vascular resistance, like oxidative stress, inflammation and immune system dysfunction1,2. The search for natural compounds as an alternative or a complement to the drugs used to treat hypertension and other chronic diseases has gained momentum in recent years1 . One example is that of food-derived peptides as nutraceuticals3,4,5. In this context, we are exploring the antioxidant, anti-inflammatory, and antihypertensive potential of synthetic peptides derived from proteins found in milk (lactoferrins from different species of mammals) and in other food sources (e.g., jumbo squid - Dosidicus gigas). Antioxidant activity in vitro was determined by both the DPPH radical scavenging activity and the Ferric Reducing Antioxidant Power (FRAP) assays. While none of the squid peptides was active, lactoferrin (LF) ones showed antioxidant potential: aLF-17-31, from donkey (Equus africanus asinus) LF, displayed the stronger radical scavenging activity (IC50 3.53 mol/mol DPPH), and nhLF268-284, from human (Homo sapiens) LF, showed the stronger reducing power (1.26 ± 0.86 mM Fe2+ equivalents). The ABTS radical scavenging activity of LF peptides was further assessed, with bLF-1-11 from bovine (Bos taurus) LF standing out with a Total Antioxidant Status (TAS) of 5.68 ± 9.23 mM. The peptides’ ability to inhibit the angiotensin converting enzyme 1 (ACE1) was also tested in vitro, as an indication of their antihypertensive potential; squid peptide RC7 inhibited ACE1 with an IC50 of 908.6 µM. Relevantly, none of the peptides was cytotoxic (MTT assay on macrophages), as only bLF1-11 showed some toxicity (IC50 417.6 µg/mL). In conclusion, new non-toxic food-derived peptides with ntioxidant/antihypertensive activity were found. Ongoing studies will assess their anti-inflammatory activity (Griess method) as well as their effect of on the TAS in macrophages (superoxide anion production).