Browsing by Author "Gomes, Anita Q."
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- Análise do efeito biológico de extratos de folhas de Carica papaya na viabilidade e na proliferação de células K562Publication . Canteiro, Beatriz; Mendes, Maria; Delgadinho, Mariana; Oliveira, Ketlyn; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Introdução – A anemia falciforme é uma doença monogénica causada por mutações no gene da β-globina que afeta a estrutura da hemoglobina, sendo associada a diversas complicações clínicas com elevadas taxas de morbilidade e mortalidade. A reativação farmacológica da hemoglobina fetal (HbF) por compostos como a hidroxiureia (HU) é um dos tratamentos atualmente disponíveis; contudo, o seu perfil de segurança e o elevado custo em países subdesenvolvidos limitam a sua utilização. Nesse contexto é essencial estudar novos compostos indutores da HbF com baixa citotoxicidade e que possam estar amplamente disponíveis, como é o caso de extratos de folhas da Carica papaya (CP), uma planta medicinal com propriedades antioxidantes e anti-inflamatórias. Objetivos – Este estudo pretende avaliar o efeito do extrato metanólico das folhas de CP (EMFCP) em parâmetros biológicos como a proliferação e a viabilidade celular em células K562. Método – As células K562 foram expostas durante 72h ao EMFCP a 500 µg/mL e durante 24 horas ao EMFCP (0,5; 50; 100 µg/mL) e à HU (25 μg/mL). A proliferação e viabilidade celular foram analisadas através da quantificação celular pelo método de exclusão do azul de tripano. Resultados – Os resultados demonstram que a proliferação e a viabilidade celular foram afetadas pelo EMFCP apenas na concentração de 500 µg/mL, não se tendo verificado alteração nestes parâmetros nas restantes concentrações utilizadas. Conclusão – Os resultados mostraram que os EMFCP não são citotóxicos quando incubados em células K562 em concentrações inferiores ou iguais a 100 μg/mL, permitindo assim explorar este composto na avaliação do seu potencial terapêutico no contexto da anemia falciforme.
- Aspergillus flavus contamination in two Portuguese wastewater treatment plantsPublication . Viegas, Carla; Dias, R.; Gomes, Anita Q.; Meneses, Márcia; Sabino, Raquel; Viegas, SusanaFilamentous fungi from genus Aspergillus were previously detected in wastewater treatment plants (WWTP) as being Aspergillus flavus (A. flavus), an important toxigenic fungus producing aflatoxins. This study aimed to determine occupational exposure adverse effects due to fungal contamination produced by A. flavus complex in two Portuguese WWTP using conventional and molecular methodologies. Air samples from two WWTP were collected at 1 m height through impaction method. Surface samples were collected by swabbing surfaces of the same indoor sites. After counting A. flavus and identification, detection of aflatoxin production was ensured through inoculation of seven inoculates in coconut-milk agar. Plates were examined under long-wave ultraviolet (UV; 365 nm) illumination to search for the presence of fluorescence in the growing colonies. To apply molecular methods, air samples were also collected using the impinger method. Samples were collected and collection liquid was subsequently used for DNA extraction. Molecular identification of A. flavus was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR detection system (Corbett). Among the Aspergillus genus, the species that were more abundant in air samples from both WWTP were Aspergillus versicolor (38%), Aspergillus candidus (29.1%), and Aspergillus sydowii (12.7%). However, the most commonly species found on surfaces were A. flavus (47.3%), Aspergillus fumigatus (34.4%), and Aspergillus sydowii (10.8%). Aspergillus flavus isolates that were inoculated in coconut agar medium were not identified as toxigenic strains and were not detected by RT-PCR in any of the analyzed samples from both plants. Data in this study indicate the need for monitoring fungal contamination in this setting. Although toxigenic strains were not detected from A. flavus complex, one cannot disregard the eventual presence and potential toxicity of aflatoxins.
- Assessment of children’s potential exposure to bioburden in indoor environmentsPublication . Viegas, Carla; Almeida, Beatriz; Dias, Marta; Aranha Caetano, Liliana; Carolino, Elisabete; Gomes, Anita Q.; Faria, Tiago; Martins, Vânia; Almeida, Susana MartaExposure to particles and bioaerosols has been associated with the increase in health effects in children. The objective of this study was to assess the indoor exposure to bioburden in the indoor microenvironments more frequented by children. Air particulate matter (PM) and settled dust were sampled in 33 dwellings and four schools with a medium volume sampler and with a passive method using electrostatic dust collectors (EDC), respectively. Settled dust collected by EDC was analyzed by culture-based methods (including azole resistance profile) and using qPCR. Results showed that the PM2.5 and PM10 concentrations in classrooms (31.15 μg/m3 and 57.83 μg/m3, respectively) were higher than in homes (15.26 μg/m3 and 18.95 μg/m3, respectively) and highly exceeded the limit values established by the Portuguese legislation for indoor air quality. The fungal species most commonly found in bedrooms was Penicillium sp. (91.79%), whereas, in living rooms, it was Rhizopus sp. (37.95%). Aspergillus sections with toxigenic potential were found in bedrooms and living rooms and were able to grow on VOR. Although not correlated with PM, EDC provided information regarding the bioburden. Future studies, applying EDC coupled with PM assessment, should be implemented to allow for a long-term integrated sample of organic dust.
- Assessment of fungal contamination in waste sorting and incineration: case study in PortugalPublication . Viegas, Carla; Gomes, Anita Q.; Abegão, João; Sabino, Raquel; Graça, Tiago; Viegas, SusanaOrganic waste is a rich substrate for microbial growth, and because of that, workers from waste industry are at higher risk of exposure to bioaerosols. This study aimed to assess fungal contamination in two plants handling solid waste management. Air samples from the two plants were collected through an impaction method. Surface samples were also collected by swabbing surfaces of the same indoor sites. All collected samples were incubated at 27◦C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. Air samples were also subjected to molecular methods by real-time polymerase chain reaction (RT PCR) using an impinger method to measure DNA of Aspergillus flavus complex and Stachybotrys chartarum. Assessment of particulate matter (PM) was also conducted with portable direct-reading equipment. Particles concentration measurement was performed at five different sizes (PM0.5; PM1; PM2.5; PM5; PM10). With respect to the waste sorting plant, three species more frequently isolated in air and surfaces were A. niger (73.9%; 66.1%), A. fumigatus (16%; 13.8%), and A. flavus (8.7%; 14.2%). In the incineration plant, the most prevalent species detected in air samples were Penicillium sp. (62.9%), A. fumigatus (18%), and A. flavus (6%), while the most frequently isolated in surface samples were Penicillium sp. (57.5%), A. fumigatus (22.3%) and A. niger (12.8%). Stachybotrys chartarum and other toxinogenic strains from A. flavus complex were not detected. The most common PM sizes obtained were the PM10 and PM5 (inhalable fraction). Since waste is the main internal fungal source in the analyzed settings, preventive and protective measures need to be maintained to avoid worker exposure to fungi and their metabolites.
- Bioburden assessment by passive methods on a clinical pathology service in one central hospital from Lisbon: what can it tell us regarding patients and staff exposure?Publication . Viegas, Carla; Twarużek, Magdalena; Lourenço, Raquel; Dias, Marta; Almeida, Beatriz; Aranha Caetano, Liliana; Carolino, Elisabete; Gomes, Anita Q.; Kosicki, Robert; Soszczyńska, Ewelina; Viegas, SusanaThe assessment and control of microbial contamination in healthcare facilities is presently a mandatory and vital part of strategies to prevent and control hospital-acquired infections. This study aims to assess the bioburden with two passive sampling methods (30 ventilations grids swabs and 16 electrostatic dust collectors (EDCs)) at Clinical Pathology Services. The fungal burden was characterized through molecular tools, antifungal resistance, and the mycotoxins and cytotoxicity profile. Total bacteria presented the highest prevalence in both matrixes, whereas Gram-bacteria presented the lowest. Swabs presented a higher prevalence (27.6%) for fungal burden. Chrysonilia sitophila presented the highest prevalence in swabs, whereas for EDCs, C. sitophila and Mucor sp. were the most prevalent. Concerning Aspergillus genera on swabs, section Flavi was the one with the highest prevalence (58.02%), whereas, for EDCs, section Versicolores was the only section observed (100%). Aspergillus section Fumigati was detected in 10 swabs and 7 EDC samples and Aspergillus section Versicolores were detected in one EDC sample. Fungal growth on azole-supplemented media was observed in eight EDC samples. No mycotoxins were detected in any of the samples. A low cytotoxic effect was observed in two sites upon incubation of collected samples with A549 and SK cells and in two other sites upon incubation of collected samples with SK cells only. A medium cytotoxic effect was observed with one EDC sample upon incubation with A549 cells. This study reinforces the need for determination of the azole resistance profile for fungal species and allowed a preliminary risk characterization regarding the cytotoxicity. An intervention including the use of ultraviolet with a wavelength between 200 nm and 280 nm (UVC)—emitting device and increased maintenance and cleaning of the central heating, ventilation, and air conditioning (HVAC) systems should be ensured to promote the reduction of microbial contamination.
- Bioburden in sleeping environments from Portuguese dwellingsPublication . Viegas, Carla; Dias, Marta; Monteiro, Ana; Faria, Tiago; Lage, Joana; Carolino, Elisabete; Caetano, Liliana Aranha; Gomes, Anita Q.; Almeida, Susana Marta; Verde, Sandra Cabo; Belo, Joana; Canha, NunoA wider characterization of indoor air quality during sleep is still lacking in the literature. This study intends to assess bioburden before and after sleeping periods in Portuguese dwellings through active methods (air sampling) coupled with passive methods, such as electrostatic dust cloths (EDC); and investigate associations between before and after sleeping and bioburden. In addition, and driven by the lack of information regarding fungi azole-resistance in Portuguese dwellings, a screening with supplemented media was also performed. The most prevalent genera of airborne bacteria identified in the indoor air of the bedrooms were Micrococcus (41%), Staphylococcus (15%) and Neisseria (9%). The major indoor bacterial species isolated in all ten studied bedrooms were Micrococcus luteus (30%), Staphylococcus aureus (13%) and Micrococcus varians (11%). Our results highlight that our bodies are the source of the majority of the bacteria found in the indoor air of our homes. Regarding air fungal contamination, Chrysosporium spp. presented the highest prevalence both in after the sleeping period (40.8%) and before the sleeping period (28.8%) followed by Penicillium spp. (23.47% morning; 23.6% night) and Chrysonilia spp. (12.4% morning; 20.3% night). Several Aspergillus sections were identified in air and EDC samples. However, none of the fungal species/strains (Aspergillus sections Fumigati, Flavi, Nidulantes and Circumdati) were amplified by qPCR in the analyzed EDC. The correlations observed suggest reduced susceptibility to antifungal drugs of some fungal species found in sleeping environments. Toxigenic fungal species and indicators of harmful fungal contamination were observed in sleeping environments.
- Cell-specific regulation of acetylcholinesterase expression under inflammatory conditionsPublication . Oliveira, P. de; Gomes, Anita Q.; Pacheco, T. R.; Vitorino de Almeida, V.; Saldanha, C.; Calado, A.Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were aurprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.
- Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cellsPublication . Barroso, M.; Rocha, M. S.; Esse, R.; Gonçalves, I. Jr; Gomes, Anita Q.; Teerlink, T.; Jakobs, C.; Blom, H. J.; Loscalzo, J.; Rivera, I.; de Almeida, I. T.; Castro, R.Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.
- Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cellsPublication . Moutinho, Miguel; Nunes, Maria João; Gomes, Anita Q.; Gama, Maria João; Cedazo-Minguez, Angel; Rodrigues, C. M.; Björkhem, Ingemar; Rodrigues, ElsaThe neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.
- Commercial green tea from Portugal: comprehensive microbiologic analysesPublication . Viegas, Carla; Sá, Flávio; Mateus, Margarida; Santos, Patrícia; Almeida, Beatriz; Aranha Caetano, Liliana; Gomes, Anita Q.; Viegas, SusanaIn recent times green tea (GT) consumption has increased, due to the numerous studies that indicate a wide variety of health benefits following its regular consumption. The aim of this study was to assess the bioburden (bacteria and fungi) of bulk and bags of GT marketed in Lisbon and to obtain a more refined fungal burden characterization, including azole resistance profile. The bacteriota in tea bags before boiling ranged from lower than the detection limit to 1770 CFU.g−1, whereas in brew samples ranged from lower than the detection limit to 54.55 CFU.mL−1. In bulk samples before boiling ranged from lower than the detection limit to 2636 CFU.g−1, while after boiling ranged from lower than the detection limit to 72.73 CFU.mL−1. Fungal contamination on teabags before boiling ranged from lower than the detection limit to 66.67 CFU.g−1 and after boiling, all samples presented results lower than the detection limit. Concerning bulk samples before boiling ranged from lower than the detection limit to 96.97 CFU.g−1, whereas after boiling ranged from lower the detection limit to 30.3 CFU.mL−1. Before boiling, the most common fungal species in the bagged tea (90.91 CFU.g−1; 45.45%) and bulk samples (66.67 CFU.g−1; 91.67%) was Aspergillus section Nigri. Fungal diversity was higher on bulk samples than in tea bags. Aspergillus section Nigri and Rhizopus sp. growth was observed mostly on itraconazole-supplemented Sabouraud dextrose agar media, which require further investigation. Aspergillus sections Fumigati and Nidulantes were detected by using real-time PCR, but not in the GT samples in which they were identified through culture-based methods. A significant reduction of bacterial contamination after boiling was observed, however fungal contamination with toxigenic potential was observed before and after boiling. Future research work needs to characterize in detail the mycotoxins contamination to allow a risk-benefit assessment to estimate the human health benefits and risks following tea consumption and to support policy actions, if and when needed. The results also suggest that the conditions of how tea is packed can influence the fungal diversity and this variable should be further investigated.