ESS - TBIO - Posters apresentados em eventos científicos
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- Antimicrobial activity of food-isolated fungi extractsPublication . Ferreira, Diogo; Areal-Hermida, Lara; Baylina, Pilar; Fernandes, Rúben; Sieiro, CarmenOne major source for drug discovery are microbial metabolites. Fungi, renowned for their ability to produce an array of broad and diverse secondary metabolites, due to their extensive dispersion and diversity, offer a rich resource for drug discovery. Antibiotic resistance is a major concern. Rapid increase of resistant bacteria worldwide, dampens antibiotic efficiency, burdens healthcare services and increase morbidity and mortality. Antibiotic misuse and lack of new drug development are the main responsible for this health crisis. So, the creation of fungal libraries to find and study new compounds is essential to tackle the rising of antimicrobial resistance and continue with industrial efforts of drug discovery and production. Isolation from chestnuts, chestnut flour and sunflower seeds allowed us to obtain a collection of 165 fungal isolates. Bioactivity of fungal extracts were screened against different antibiotic resistant bacteria. Bacteria grown overnight, adjusted to 1.5 x 108 CFU/mL was exposed to fungal extracts, at a concentration of 100 μg/mL for 24 hours and inhibition rates were calculated. Several extracts showed activity against antimicrobial resistant bacteria and further studies should be made in order to find if new molecules could be responsible for our fungi antimicrobial activity.
- Avaliação do potencial antimicrobiano de fungos filamentosos: Um estudo promissor na abordagem do pé diabético e da resistência bacterianaPublication . Ferreira, D.; BAYLINA MACHADO, PILAR; Sá, S.; Areal Hermida, L.; Rocha, A. C.; Baylina, Pilar; Fernandes, R.; Sieiro, C.Prevê-se que até 2045, cerca de 700 milhões de pessoas possam ser afetadas pela Diabetes Mellitus (DM). Uma complicação frequente em indivíduos com DM é o pé diabético, que se manifesta por feridas nos pés causadas por danos nos nervos e vasos sanguíneos, resultando frequentemente na amputação dos membros inferiores. Esta situação é agravada por infeções bacterianas, causadas por estirpes resistentes, como Staphylococcus aureus resistente à meticilina (MRSA), Pseudomonas aeruginosa e Klebsiella spp. produtoras de beta-lactamases de espectro estendido (ESBL), dificultando o tratamento destas lesões. A resistência antibiótica, impulsionada pelo uso excessivo e indiscriminado de antibióticos, destaca a necessidade urgente de novos fármacos e terapias mais eficazes. Neste contexto, os fungos apresentam-se como uma fonte promissora de novos agentes antimicrobianos, devido à vasta gama e diversidade de compostos bioativos que conseguem sintetizar.
- Avaliação do potenicial antimicrobiano do extrato de Gnomoniopsis sp. contra agentes infeciosos do pé diabéticoPublication . Rocha, A. C.; Areal Hermida, L.; Baylina, Pilar; Fernandes, R.; Sieiro, C.; BAYLINA MACHADO, PILAREstima-se que até ao ano de 2045, aproximadamente 700 milhões de pessoas sofram de Diabetes mellitus (DM). O pé diabético é uma complicação comum em pacientes com DM e caracteriza-se por lesões nos pés devido a danos nos nervos e vasos sanguíneos, levando muitas vezes à amputação dos membros inferiores. A infeção causada por bactérias resistentes, como Staphyloccus aureus resistente a meticilina (MRSA), Pseudomonas aeruginosa e espécies de Klebsiella beta-lactamases de espetro estendido (ESBL), acentuam a gravidade destas lesões, tornando o seu tratamento mais complexo. A resistência a antibióticos resulta do uso exagerado e indiscriminado de antibióticos e o desenvolvimento de medicamentos inovadores e de terapias mais eficazes é urgente. Assim, os fungos, nomeadamente fungos filamentosos, surgem como um potencial reservatório para novos compostos antimicrobianos, devido à grande quantidade e diversidade de compostos bioativos produzidos por estes organismos.
- Combining natural bile acids with old basic drugs affords new triple stage antimalarial surface-active ionic liquidsPublication . Silva, Ana Teresa; Oliveira, Isabel; Duarte, Denise; Moita, Diana; Prudêncio, Miguel; Nogueira, Fátima; Ferraz, Ricardo; Marques, Eduardo F.; Gomes, PaulaIonic liquids (ILs) are special organic salts that have been gaining momentum in medicinal chemistry. Despite their simple and cost-effective synthesis, ILs offer an easy access to structures of biological interest by combining bioactive molecules with opposite polarities, e.g., via simple ionic pairing of an acid with a base. This makes ILs of special interest for treating malaria. Since this disease is prevalent mainly in low-to-middle income countries, novel chemotherapeutic strategies must be kept affordable. Malaria is caused by Plasmodium parasites, whose complex life cycle includes three developmental stages in the host: the blood stage, the liver stage, and the gametocyte stage. This complexity turns the development of new effective drugs quite difficult, which is aggravated by the fast emergence of drug-resistant strains. This fact has often led to the disuse of several antimalarials, driving the need to find new ones with multiple-stage action. In this context, we have been working on new antimalarial ILs by mixing antimalarial aminoquinolines—chloroquine and primaquine—with natural lipids. Two new families of salts derived from those antimalarial drugs and naturally-occurring bile acids were now produced by acid-base neutralization, and evaluated for their antiplasmodial action. The chloroquine-derived bile salts were found active against all the three stages of parasite development in the host. Their behavior as surface-active ionic liquids (SAILs), i.e. their interfacial and self-aggregation properties, were also investigated, as they may contribute critically to their delivery and therapeutic action.
- 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
- 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.
- Epidemiologia do cancro do colo uterino e o impacto da vacinação contra o HPV: a realidade portuguesa no contexto europeuPublication . Marques, Ana; Fernandes, Sílvia; Silva, Regina A.A atual disponibilidade de vacinas e testes de rastreio molecular para o HPV aumentaram a possibilidade de eliminar globalmente o cancro cervical e outras patologias relacionadas com o vírus. No entanto, têm surgido vários obstáculos à implementação dos programas de vacinação contra o HPV a nível mundial. Este estudo tem como objetivos compilar e analisar estatisticamente a informação acerca da vacinação contra o HPV e políticas de rastreio de cancro cervical, em Portugal e em outros países europeus; e avaliar o impacto na incidência e mortalidade de cancro cervical, ao longo dos anos.
- Ictal and interictal source connectivity during gelastic seizures in a focal cortical dysplasia of the cingulate gyrusPublication . Borges, Daniel Filipe; Dias, Daniela; Cordeiro, Helena; Leal, AlbertoGelastic seizures are rare manifestations of epilepsy often associated with hypothalamic hamartomas (HH). In the absence of HH, localization of the origin is more difficult and there are several less well-established possibilities. Non-invasive assessment is often complicated by the deep localization of the epileptogenic area and the complex dynamics of spike activity. Robust neurophysiologic methods and careful validation of intrinsically epileptogenic lesions detected by RM can improve the validity of scalp EEG analysis. To analyze the dynamics of epileptic activity in gelastic seizures associated with cortical dysplasia. A 26-year-old girl with daily refractory gelastic seizures since the age of 6 underwent a neurophysiological assessment with long-term EEG recordings (24h, 31 channels) and an MRI of the brain. She had previously been rejected in an epilepsy surgery program because no structural brain lesion had been found and the EEG information was not informative enough. A recent 3T MRI showed cortical dysplasia in the cingulate gyrus, which was highly suspicious as an epileptogenic focus. We analyzed the 31-channel EEG (ictal and interictal) using source and functional connectivity methods to obtain a solid computer model of the origin and dynamics of epileptic activity and correlate it with the anatomical information of the MRI. Source analysis of interictal activity revealed multiple sources that dominated at different time points, indicating considerable dynamics that prevented clear source identification. The ictal recordings demonstrated barely late rhythmic activity over the frontal areas, but no early focalization. Connectivity analysis, which revealed the flow of information between cortical sources, showed a consistent origin of epileptic activity in the vicinity of cortical dysplasia for both interictal and ictal activity. EEG connectivity analysis significantly improves the performance of non-invasive methods for determining intracranial dynamics of epileptic activity and correlation with structural lesions.
- 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).
- 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.