Early detection and monitoring of metabolic progression of type 2 diabetes

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Desenvolvimento de um biossensor eletroquímico para monitorização da radioterapia no melanoma

Publication . Cerqueira, Sofia Manuela Vasconcelos; Fernandes, Ruben Miguel Pereira

A incidência do cancro da pele em Portugal continua a ser preocupante em relação à média europeia. No que diz respeito ao melanoma, apesar de representar apenas 4% de todos os cancros da pele, representa a forma mais agressiva, devido ao seu elevado potencial de metastização, sendo responsável por cerca de 75% das mortes que estão associadas às neoplasias cutâneas. Alguns estudos demonstraram que a expressão da galactina-3 (Gal-3) estava associada à progressão do melanoma, pelo que poderá ser usada como um marcador da monitorização da eficácia terapêutica, como por exemplo da radioterapia. Adicionalmente, a Gal-3 tem revelado uma enorme versatilidade diagnóstica, tendo sido demonstrada a sua importância como biomarcador em diversos tipos de cancro, em doenças cardíacas e mais recentemente em distúrbios metabólicos como a diabetes. Neste sentido, este trabalho tem como objetivo o desenvolvimento e a otimização de um biossensor eletroquímico com base em polímeros de impressão molecular (do inglês, molecularly-imprinted polymer, MIP) para a monitorização do efeito da radioterapia no melanoma, através da deteção da Gal-3. O reconhecimento da molécula alvo foi feita através de um material MIP, criado na superfície do elétrodo por eletropolimerização da mistura do analito (Gal-3) com o monómero (2-Aminofenol). Este processo formou então um polímero não condutor com locais de reconhecimento, demonstrando afinidade para a Gal-3. Posteriormente, a proteína foi removida por um tratamento com ácido oxálico. O controlo da modificação da superfície foi feito por espetroscopia de RAMAN e por técnicas eletroanalíticas, nomeadamente a espetroscopia de impedância eletroquímica (do inglês, electrochemical impedance spectroscopy, EIS) e a voltametria cíclica (do inglês, cyclic voltammetry, CV). A avaliação do desempenho analítico do sensor foi feita por EIS seguindo a resposta obtida com soluções padrão entre os 0,5 ng/mL e 5 ng/mL de Gal-3. A resposta obtida foi de interesse analítico, considerando que indivíduos saudáveis apresentam um nível fisiológico normal de 30 ng/mL. De uma forma geral, o biossensor apresentou um bom desempenho analítico geral em termos de limite de deteção, tempo de resposta e reprodutibilidade. As características de desempenho do material MIP em soro foram também adequadas, considerando possíveis aplicações posteriores em amostras reais, revelando um reduzido impacto dos vários componentes deste fluído na resposta do biossensor. Face ao exposto, o biossensor desenvolvido com base em anticorpos plásticos (MIP´s) como elemento de biorreconhecimento, mostrou características relevantes em termos de simplicidade e sensibilidade, demonstrando um grande potencial para a aplicação no local em pesquisa médica e diagnóstico clínico.

2019Master thesis

Open access

Vitamin A Enhances Macrophages Activity Against B16-F10 Malignant Melanocytes: A New Player for Cancer Immunotherapy?

Publication . Oliveira, Sofia; Costa, José; Faria, Isabel; Guerreiro, Susana G; Fernandes, Rúben

Background and objectives: The incidence of cutaneous melanoma has been increasing. Melanoma is an aggressive form of skin cancer irresponsive to radiation and chemotherapy, rendering this cancer a disease with poor prognosis: In order to surpass some of the limitations addressed to melanoma treatment, alternatives like vitamins have been investigated. In the present study, we address this relationship and investigate the possible role of vitamin A. Materials and Methods: We perform a co-culture assay using a macrophage cell model and RAW 264.7 from mouse, and also a murine melanoma cell line B16-F10. Macrophages were stimulated with both Escherichia coli lipopolysaccharides (LPS) as control, and also with LPS plus vitamin A. Results: Using B16-F10 and RAW 264.7 cell lines, we were able to demonstrate that low concentrations of vitamin A increase cytotoxic activity of macrophages, whereas higher concentrations have the opposite effect. Conclusion: These findings can constitute a new point of view related to immunostimulation by nutrients, which may be considered one major preventive strategy by enhancing the natural defense system of the body.

2019-09-18Journal article

Open access

Plastic Antibody of Polypyrrole/Multiwall Carbon Nanotubes on Screen-Printed Electrodes for Cystatin C Detection

Publication . Gomes, Rui S.; Gomez-Rodríguez, Blanca Azucena; Fernandes, Ruben; Sales, Goreti; Moreira, Felismina; Dutra, Rosa F.

This work reports the design of a novel plastic antibody for cystatin C (Cys-C), an acute kidney injury biomarker, and its application in point-of-care (PoC) testing. The synthetic antibody was obtained by tailoring a molecularly imprinted polymer (MIP) on a carbon screen-printed electrode (SPE). The MIP was obtained by electropolymerizing pyrrole (Py) with carboxylated Py (Py-COOH) in the presence of Cys-C and multiwall carbon nanotubes (MWCNTs). Cys-C was removed from the molecularly imprinted poly(Py) matrix (MPPy) by urea treatment. As a control, a non-imprinted poly(Py) matrix (NPPy) was obtained by the same procedure, but without Cys-C. The assembly of the MIP material was evaluated in situ by Raman spectroscopy and the binding ability of Cys-C was evaluated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. The MIP sensor responses were measured by the DPV anodic peaks obtained in the presence of ferro/ferricyanide. The peak currents decreased linearly from 0.5 to 20.0 ng/mL of Cys-C at each 20 min successive incubation and a limit of detection below 0.5 ng/mL was obtained at pH 6.0. The MPPy/SPE was used to analyze Cys-C in spiked serum samples, showing recoveries <3%. This device showed promising features in terms of simplicity, cost and sensitivity for acute kidney injury diagnosis at the point of care.

2021Journal article

Open access

Warburg Effect Inversion: Adiposity shifts central primary metabolism in MCF-7 breast cancer cells

Publication . Luís, Carla; Duarte, Fernanda; Faria, Isabel; Jarak, Ivana; Oliveira, Pedro F.; Alves, Marco G.; Soares, Raquel; Fernandes, Rúben

Obesity is a complex health disorder and a trigger to many diseases like Diabetes mellitus (DM) and breast cancer (BrCa), both leading causes of morbidity and mortality worldwide. Also evidence demonstrates that abnormal glucose metabolism termed ‘the Warburg effect’ in cancer cell is closely associated with malignant phenotypes and promote the aggressiveness of several types of cancer, including BrCa. In this study, we evaluated the breast cancer cell metabolism in normoglycemia, hyperglycemia and in an obesity condition in order to clarify the potential underlined mechanisms that link these disorders. Materials and methods MCF-7 cells were exposed to low and high glucose levels, the latter either in the presence of 3T3-L1 adipocyte conditioned medium (CM), thus mimicking the adiposity observed in obese patients. Cell viability, migration, proliferation, cytotoxicity and cell death assays were performed under the different culture conditions. Hormonal and lipid profile were also characterized by biochemical assays and primary metabolism was determined by Nuclear Magnetic Resonance (NMR)-based metabolomics. Results Our results show an increased aggressiveness in the condition mimicking diabetogenic obesity with an altered energy/lipid metabolism. Interestingly in the experimental obesity-mimicking status, lipids and amino acids were expended while glucose was produced by tumor cells from lactate. These findings reveal a shift on tumor cells metabolism that is opposite to ‘the Warburg effect’. Conclusions Overall, this experimentally obesity-mimicking condition not only revealed an increased tumor proliferation and aggressiveness but also disclosed a new mechanism of cancer metabolism, the ‘Warburg Effect Inversion’.

2019Journal article

Open access