Browsing by Author "Fernandes, Rúben"
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- 3-Nitrotyrosine quantification methods: Current concepts and future challengesPublication . Teixeira, Dulce; Fernandes, Rúben; Prudêncio, Cristina; Vieira, MónicaMeasurement of 3-nitrotyrosine (3-NT) in biological samples can be used as a biomarker of nitrosative stress, since it is very stable and suitable for analysis. Increased 3-NT levels in biological samples have been associated with several physiological and pathological conditions. Different methods have been described for the detection and quantification of this molecule, such as (i) immunological methods; (ii) liquid chromatography, namely high-pressure liquid chromatography (HPLC)-based methods that use ultraviolet-visible (UV/VIS) absorption, electrochemical (ECD) and diode array (DAD) detection, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS); (iii) gas chromatography, such as gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Methods A literature review on nitrosative stress, protein nitration, as well as 3-NT quantification methods was carried out. Results This review covers the different methods for analysis of 3-NT that have been developed during the last years as well as the latest advances in this field. Overall, all methods present positive and negative aspects, although it is clear that chromatography-based methods present good sensitivity and specificity. Regarding this, GC-based methods exhibit the highest sensibility in the quantification of 3-NT, although it requires a prior time consuming derivatization step. Conversely, HPLC does not require such derivatization step, despite being not as accurate as GC. Conclusion It becomes clear that all the methods described during this literature review, although accurate for 3-NT quantification, need to be improved regarding both sensitivity and specificity. Moreover, optimization of the protocols that have been described is clearly needed.
- Adipocyte proteome and secretome influence inflammatory and hormone pathways in gliomaPublication . Almeida, Joana; Costa, J.; Coelho, Pedro; Cea, V.; Galesio, M.; Noronha, J. P.; Diniz, M. S.; Prudêncio, Cristina; Soares, R.; Sala, C.; Fernandes, RúbenGliomas represent the most common primary malignant brain tumors in adults, with an extremely poor prognosis. Among several risk factors, lifestyle was also recently identified as a major risk factor for the development of primary glioma. In the present study, we explore the relationship between obesity and glioma in a cellular model. Thus, we have study the influence of adipocytes secretome on glioma cell line GL261. Using the 3T3-L1 adipocyte cell line, and its conditioned medium (adipokines-enriched medium), we showed that adipocyte-released factors relate with glioma angiogenic, growth, hormones and metabolic behavior by MALDI-TOF-MS and proteomic array analysis. In a first view, STI1, hnRNPs and PGK1 are under expressed on CGl. Similarly, both carbonic anhydrase and aldose reductase are even suppressed in glioma cells that grown under adipokines-enriched environment. Contrariwise, RFC1, KIF5C, ANXA2, N-RAP and RACK1 are overexpressed in GL261 cell the in the presence of the adipokines-enriched medium. We further identified the factors that are released by adipocyte cells, and revealed that several pro-inflammatory and angiogenic factors, such as IL-6, IL-11, LIF, PAI-1, TNF-α, endocan, HGF, VEGF IGF-I, were secreted to the medium into a high extent, whereas TIMP-1 and SerpinE1 were under expressed on CGl. This study discloses an interesting in vitro model for the study of glioma biology under a "obesity" environment, that can be explored for the understanding of cancer cells biology, for the search of biomarkers, prognostic markers and therapeutic approaches.
- Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma CellsPublication . Moreira, Ângela; Pereira, Sofia S.; Costa, Madalena; Morais, Tiago; Pinto, Ana; Fernandes, Rúben; Monteiro, Mariana P.Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.
- Adipocyte secretome increases radioresistance of malignant melanocytes by improving cell survival and decreasing oxidative statusPublication . Coelho, Pedro; Silva, Liliana; Faria, Isabel; Vieira, Mónica; Monteiro, Armanda; Pinto, Gabriela; Prudêncio, Cristina; Fernandes, Rúben; Soares, RaquelRadiotherapy is a treatment option for the majority of malignancies. However, because melanoma is known to be radioresistant, the use of ionizing radiation as an adjuvant therapy in cutaneous melanoma patients is ineffective. Obesity has now been recognized as a risk factor for melanoma. High adiposity is generally associated with a more pro-oxidative status. Oxidative stress is a major player in radiation therapy and also a common link between obesity and cancer. Several adipocyte-released proteins are known to have a role in controlling cellular growth and pro-survival signaling. For that reason, we investigated the influence of 3T3-L1 mature adipocyte secretome in B16-F10 malignant melanocyte radiosensitivity. We evaluated B16-F10 cell survival and redox homeostasis when exposed to four daily doses of ionizing radiation (2 Gy per day) up to a total of 8 Gy in a medical linear accelerator. B16-F10 melanocytes exhibited slight alterations in survival, catalase activity, nitrative stress and total oxidant concentration after the first 2 Gy irradiation. The motility of the melanocytes was also delayed by ionizing radiation. Subsequent irradiations of the malignant melanocytes led to more prominent reductions in overall survival. Remarkably, 3T3-L1 adipocyte-secreted molecules were able to increase the viability and migration of melanocytes, as well as lessen the pro-oxidant burden induced by both the single and cumulative X-ray doses. In vitro adipocyte-released factors protected B16-F10 malignant melanocytes from both oxidative stress and loss of viability triggered by radiation, enhancing the radioresistant phenoyype of these cells with a concomitant activation of the AKT signaling pathway These results both help to elucidate how obesity influences melanoma radioresistance and support the usage of conventional medical linear accelerators as a valid model for the in vitro radiobiological study of tumor cell lines.
- Adipocyte-released factors enhance melanocyte’s proliferation and motilityPublication . Fernandes, Rúben; Coelho, Pedro; Almeida, Joana; Prudêncio, Cristina; Soares, RaquelObesity, favored by the modern lifestyle, acquired epidemic proportions nowadays. Obesity has been associated with various major causes of death and morbidity including malignant neoplasms. Cutaneous melanoma incidence rates have also been increasing uring the last four decades in several countries. Obesity involvement in melanoma etiology has been recognized, but the implicated mechanisms remain unclear. We propose to address the above relationship and investigate the mechanism interplaying between obesity and an increased risk of melanoma onset.
- Alterações morfopatológicas no rim de rato com obesidade e insulino-resistênciaPublication . Teixeira, C.; Guedes, C.; Sousa, A.; Lopes, S.; Luís, C.; Soares, R.; Baylina, Pilar; Moreira, F.; Alencastre, I.; Fernandes, RúbenA diabetes é uma das patologias com elevada taxa de comorbilidades associadas, como a nefropatia diabética que por sua vez leva a doença renal de estadio terminal (DRET). Estudos prévios demonstraram que indivíduos que desenvolveram DRET apresentavam um IMC mais elevado revelando a importância da obesidade no desenvolvimento da doença. Existe uma relação entre a atividade endócrina do tecido adiposo e a doença renal, nomeadamente o desenvolvimento de inflamação, fibrose, stress oxidativo, metabolismo lipídico anormal, uma elevada produção de insulina e maior resistência à mesma. As alterações histológicas renais mais frequentes ocorrem no glomérulo e podem ser classificadas conforme o espessamento da membrana basal glomerular, expansão mesangial ou glomerulosclerose. Avaliar as alterações histológicas renais, nomeadamente, a inflamação, fibrose e acumulação de tecido adiposo ou carboidratos. Ratos C57Bl/6J foram submetidos a dieta normal e a dieta gorda, e depois de sacrificados os tecidos (rim) foram fixados, desidratados e impregnados em parafina. Cortes histológicos foram submetidos às seguintes colorações: Hematoxilina e Eosina, Ácido Periódico de Schiff (PAS), PAS e Azul de Alcian e Tricrómio de Masson. Em ratos obesos insulino-resistentes, observou-se uma alteração de tamanho dos glomérulos e um evidente aumento do espaço capsular. Há também uma alteração da arquitetura e organização estrutural a nível tubular e uma evidente acumulação de gotículas de lípidos. Contrariamente ao esperado não foram encontradas evidências de fibrose. Conclusão: Todas as alterações esperadas foram encontradas nos cortes histológicos de tecido exceto a ausência de fibrose. Mais estudos serão necessários no sentido de perceber a relação entre este fator e a doença renal.
- Angiogenesis in Schistosoma haematobium-associated urinary bladder cancerPublication . Dematei, Anderson; Fernandes, Rúben; Soares, Raquel; Alves, Helena; Richter, Joachim; Botelho, Monica C.Schistosoma haematobium, a parasitic flatworm that infects more than 100 million people, mostly in the developing world, is the causative agent of urogenital schistosomiasis, and is associated with a high incidence of squamous cell carcinoma (SCC) of the bladder. During infection, eggs are deposited in the bladder causing an intense inflammatory reaction. Angiogenesis is defined as the formation of new blood vessels from preexisting ones and is recognized as a key event in cell proliferation and carcinogenesis and spread of malignant lesions. A growing amount of evidence points to angiogenesis playing a key role in schistosomiasis-associated bladder cancer. Thus, identifying biomarkers of this process plays an important role in the study of cancer. Here, we review recent findings on the role of angiogenesis in bladder cancer and the growth factors that induce and assist in their development, particularly SCC of the bladder associated to urogenital schistosomiasis.
- Antibacterial activity of Ionic Liquids based on ampicillin against resistant bacteriaPublication . Ferraz, Ricardo; Teixeira, Vânia; Rodrigues, Débora; Prudêncio, Cristina; Fernandes, Rúben; Noronha, João Paulo; Petrovski, Zeljko; Branco, LuísAntibacterial activity of novel Active Pharmaceutical Ingredient Ionic Liquids (API-ILs) based on ampicillin anion [Amp] have been evaluated. They showed growth inhibition and bactericidal properties on some sensitive bacteria and especially some Gram-negative resistant bacteria when compared to the [Na][Amp] and the initial bromide and chloride salts. For these studies were analysed the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBIC) against sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumonia (clinically isolated), as well as sensitive Gram positive S. Aureus ATCC 25923, Staphylococcus epidermidis and Enterococcus faecalis and completed using clinically isolated resistent strains: E. coli TEM CTX M9, E. coli CTX M2 and E. coli AmpC Mox. From the obtained MIC values of studied APIs-ILs and standard [Na][Amp] were derived RDIC values (relative decrease of inhibitory concentration). High RDIC values of [C16Pyr][Amp] especially against two resistant Gram-negative strains E. coli TEM CTX M9 (RDIC>1000) and E. coli CTX M2 (RDIC>100) point clearly to a potential promising role of APIs-ILs as antimicrobial drugs especially against resistant bacterial strains.
- Antibiotic resistance in enterobacteriaceae isolated from Portuguese deli meatsPublication . Amador, Paula; Fernandes, Rúben; Brito, Luísa; Prudêncio, CristinaThis study aimed to identify the presence of β-lactam-resistant bacteria in different types of Portuguese deli meats. The numbers of ampicillin resistant bacteria varied from negative in 25 g to 1.0 × 108colony-forming units/g. Within 78 randomly selected β-lactam-resistant bacteria, 24 different resistant phenotypes were found and 35.9% were multidrug resistant (MDR). The majority (87.2%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the blaTEM gene was detected in 23 out of 67 isolates (34.3%) and 16 of them presented MDR phenotypes. Four Klebsiella oxytoca isolates (6%) harbored a gene for the CTX-M/OXY-type enzyme. The direct sequencing of their purified amplicons confirmed the presence of three types of blaOXYgenes (blaOXY-1, blaOXY-2 and blaOXY-5). These results suggest that without good hygienic practices, deli meats may act as a vehicle of transfer of β-lactam-resistant bacteria to the gastrointestinal tract of consumers.
- Antibiotic resistance in wastewater: Occurrence and fate ofEnterobacteriaceaeproducers of Class A and Class C β-lactamasesPublication . Amador, Paula P.; Fernandes, Rúben; Prudêncio, Cristina; Barreto, Mário P.; Duarte, Isabel M.Antibiotics have been intensively used over the last decades in human and animal therapy and livestock, resulting in serious environmental and public health problems, namely due to the antibiotic residues concentration in wastewaters and to the development of antibiotic-resistant bacteria. This study aimed to access the contribution of some anthropological activities, namely urban household, hospital and a wastewater treatment plant, to the spread of antibiotic resistances in the treated wastewater released into the Mondego River, Coimbra, Portugal. Six sampling sites were selected in the wastewater network and in the river. The ampicillin-resistant Enterobacteriaceae of the water samples were enumerated, isolated and phenotypically characterized in relation to their resistance profile to 13 antibiotics. Some isolates were identified into species level and investigated for the presence of class A and class C -lactamases. Results revealed high frequency of resistance to the -lactam group, cefoxitin (53.5%), amoxicillin/clavulanic acid combination (43.5%), cefotaxime (22.7%), aztreonam (21.3) cefpirome (19.2%), ceftazidime (16.2%) and to the non--lactam group, trimethoprim/sulfamethoxazol (21.1%), tetracycline (18.2%), followed by ciprofloxacin (14.1%). The hospital effluent showed the higher rates of resistance to all antibiotic, except two (chloramphenicol and gentamicin). Similarly, higher resistance rates were detected in the wastewater treatment plant (WWTP) effluent compared with the untreated affluent. Regarding the multidrug resistance, the highest incidence was recorded in the hospital sewage and the lowest in the urban waste. The majority of the isolates altogether are potentially extended-spectrum -lactamases positive (ESBL(+)) (51.9%), followed by AmpC(+) (44.4%) and ESBL(+)/AmpC(+) (35.2%). The most prevalent genes among the potential ESBL producers were blaOXA (33.3%), blaTEM (24.1%) and blaCTX-M (5.6%) and among the AmpC producers were blaEBC (38.9%), blaFOX (1.9%) and blaCIT (1.9%). In conclusion, the hospital and the WWTP activities revealed to have the highest contribution to the spread of multidrug resistant bacteria in the study area. Such data is important for future management of the environmental and public health risk of these contaminants. This is the first embracing study in the water network of Coimbra region on the dissemination of antibiotic resistance determinants. Moreover, it is also the first report with the simultaneous detection of multiresistant bacteria producers of AmpC and ESBLs -lactamases in aquatic systems in Portugal.