Nutritional composition of low protein and phenylalanine-restricted dishes prepared for phenylketonuric patients

Nutritional management is essential for Phenylketonuria (PKU) treatment, consisting in a semi-synthetic and low phenylalanine (Phe) diet, which includes strictly controlled amounts of low protein natural foods (essentially fruits and vegetables) supplemented with Phe-free protein substitutes and dietetic low-protein products. PKU diet has to be carefully planned, providing the best ingredient combinations, so that patients can achieve good metabolic control and an adequate nutritional status. Hereupon, it is mandatory to know the detailed composition of natural and/or cooked foodstuffs prepared specifically for these patients. We intended to evaluate sixteen dishes specifically prepared for PKU patients, regarding the nutritional composition, Phe and tyrosine (Tyr) contents, fatty acids profile, and vitamins E and B12 amounts. The nutritional composition of the cooked samples was 15.5–92.0 g/100 g, for moisture; 0.7–3.2 g/100 g, for protein; 0.1–25.0 g/100 g, for total fat; and 5.0–62.0 g/100 g, for total carbohydrates. Fatty acids profile and vitamin E amount reflected the type of fat used. All samples were poor in vitamin B12 (0.3–0.8 μg/100 g). Boiled rice presented the highest Phe content: 50.3 mg/g of protein. These data allow a more accurate calculation of the diet portions to be ingested by the patients according to their individual tolerance.

Organic versus conventional tomatoes: Influence on physicochemical parameters, bioactive compounds and sensorial attributes

The effect of organic and conventional agricultural systems on the physicochemical parameters, bioactive compounds content, and sensorial attributes of tomatoes (‘‘Redondo’’ cultivar) was studied. The influence on phytochemicals distribution among peel, pulp and seeds was also accessed. Organic tomatoes were richer in lycopene (+20%), vitamin C (+30%), total phenolics (+24%) and flavonoids (+21%) and had higher (+6%) in vitro antioxidant activity. In the conventional fruits, lycopene was mainly concentrated in the pulp, whereas in the organic ones, the peel and seeds contained high levels of bioactive compounds. Only the phenolic compounds had a similar distribution among the different fractions of both types of tomatoes. Furthermore, a sensorial analysis indicated that organic farming improved the gustative properties of this tomato cultivar.

Optimization of antioxidants extraction from coffee silverskin, aroasting by-product, having in view a sustainable process

Coffee silverskin is a major roasting by-product that could be valued as a source of antioxidant compounds. The effect of the major variables (solvent polarity, temperature and extraction time) affecting the extraction yields of bioactive compounds and antioxidant activity of silverskin extracts was evaluated. The extracts composition varied significantly with the extraction conditions used. A factorial experimental design showed that the use of a hydroalcoholic solvent (50%:50%) at 40 °C for 60 min is a sustainable option to maximize the extraction yield of bioactive compounds and the antioxidant capacity of extracts. Using this set of conditions it was possible to obtain extracts containing total phenolics (302.5 ± 7.1 mg GAE/L), tannins (0.43 ± 0.06 mg TAE/L), and flavonoids (83.0 ± 1.4 mg ECE/L), exhibiting DPPHradical dot scavenging activity (326.0 ± 5.7 mg TE/L) and ferric reducing antioxidant power (1791.9 ± 126.3 mg SFE/L). These conditions allowed, in comparison with other “more effective” for some individual parameters, a cost reduction, saving time and energy.

Construção de Biossensores de ADN para a Avaliação da Capacidade Antioxidante

Mestrado em Engenharia da Computação e Instrumentação Médica

PEC e a vertente empresarial: impacto na gestão de tesouraria das PME

Dissertação de Mestrado apresentada ao Instituto de Contabilidade e Administração do Porto para a obtenção do grau de Mestre em Contabilidade e Finanças, sob orientação de Cláudia Maria Pereira Esta versão contém as críticas e sugestões dos elementos do júri.

Impacto da Radioterapia no stress oxidativo em modelos celulares radiorresistentes em contexto de obesidade

Introdução: Estudos anteriores em modelos tumorais de glioma e melanoma, tumores radiorresistentes, indicaram que a obesidade pode estar relacionada com um aumento do status oxidativo e com a diminuição da resistência à radiação. Como a Radioterapia é o tratamento frequentemente utilizado para esta patologia, propomo-nos, desta forma, a explorar a influência da obesidade em células de glioma, as BC3H1, e melanoma, B16F10, submetidas a Radioterapia, na presença de agentes oxidantes e antioxidantes, para o estudo da sua influência ao nível da viabilidade celular e do impacto do stress oxidativo. Métodos: As células BC3H1 e B16F10 foram tratadas com t-BOOH (150μM e 50 μM, respetivamente), TUDCA (25μM e 1μM, respetivamente) e com a mistura de t-BOOH+TUDCA em meio DMEM sem soro e meio condicionado (CM), a partir de adipócitos 3T3-L1. Em seguida, parte das células foram irradiadas com uma dose total de 2Gy. Posteriormente avaliou-se a viabilidade celular (teste MTT) e o stress oxidativo (teste TBARS, atividade da catalase, concentração da GSH, e status antioxidante total), às 4h e 12h. Resultados: Observou-se um aumento da capacidade antioxidante total das células irradiadas, comparativamente com as células não irradiadas. O meio condicionado reduziu o stress oxidativo nas BC3H1, ao mesmo tempo que reduziu a sua viabilidade celular. O TUDCA nas células incubadas com MC e submetidas a radioterapia, tendencialmente diminuiu a viabilidade celular, nas concertações em estudo. Discussão/Conclusão: O meio condicionado e a radioterapia, por si só, aumentam a resposta antioxidante total na célula, às 4h e às 12h. O TUDCA nas células incubadas com meio condicionado e submetidas a radioterapia, teve um comportamento citotóxico para as BC3H1, nas concentrações testadas. Revelando a necessidade de aprofundar os estudos da ação deste composto como agente radiossensibilizador, neste e noutros modelos celulares de carcinogénese.

Intake of milk, but not total dairy, yogurt, or cheese, is negatively associated with the clustering of cardiometabolic risk factors in adolescents

Epidemiologic studies have reported an inverse association between dairy product consumption and cardiometabolic risk factors in adults, but this relation is relatively unexplored in adolescents. We hypothesized that a higher dairy product intake is associated with lower cardiometabolic risk factor clustering in adolescents. To test this hypothesis, a cross-sectional study was conducted with 494 adolescents aged 15 to 18 years from the Azorean Archipelago, Portugal. We measured fasting glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, systolic blood pressure, body fat, and cardiorespiratory fitness. We also calculated homeostatic model assessment and total cholesterol/high-density lipoprotein cholesterol ratio. For each one of these variables, a z score was computed using age and sex. A cardiometabolic risk score (CMRS) was constructed by summing up the z scores of all individual risk factors. High risk was considered to exist when an individual had at least 1 SD from this score. Diet was evaluated using a food frequency questionnaire, and the intake of total dairy (included milk, yogurt, and cheese), milk, yogurt, and cheese was categorized as low (equal to or below the median of the total sample) or “appropriate” (above the median of the total sample).The association between dairy product intake and CMRS was evaluated using separate logistic regression, and the results were adjusted for confounders. Adolescents with high milk intake had lower CMRS, compared with those with low intake (10.6% vs 18.1%, P = .018). Adolescents with appropriate milk intake were less likely to have high CMRS than those with low milk intake (odds ratio, 0.531; 95% confidence interval, 0.302-0.931). No association was found between CMRS and total dairy, yogurt, and cheese intake. Only milk intake seems to be inversely related to CMRS in adolescents.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Disposable immunosensor with simple antibody orientation for label-free real-time detection of a cancer biomarker

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2’-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The Anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented proposal favored Ab/Ag affinity. The immunosensor design was evaluated by Quartz-Crystal microbalance with Dissipation, Atomic Force Microscopy, Electrochemical Impedance Spectroscopy (EIS) and Square-Wave Voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charged transfer resistance across the electrochemical sep-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from Glucose, Urea and Creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Label-free human chorionic gonadotropin detection at picogram levels using oriented antibodies bound to graphene screen-printed electrodes

Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for cancers in the prostate, ovaries and bladder and therefore of great importance in diagnosis. For this purpose, a new immunosensor of screen-printed electrodes (SPEs) is presented here. The device was fabricated by introducing a polyaniline (PANI) conductive layer, via in situ electropolymerization of aniline, onto a screen-printed graphene support. The PANI-coated graphene acts as the working electrode of a three terminal electrochemical sensor. The working electrode is functionalised with anti-hCG, by means of a simple process that enabled oriented antibody binding to the PANI layer. The antibody was attached to PANI following activation of the –COOH group at the Fc terminal. Functionalisation of the electrode was analysed and optimized using Electrochemical Impedance Spectroscopy (EIS). Chemical modification of the surface was characterised using Fourier transform infrared, and Raman spectroscopy with confocal microscopy. The graphene–SPE–PANI devices displayed linear responses to hCG in EIS assays from 0.001 to 50 ng mL−1 in real urine, with a detection limit of 0.286 pg mL−1. High selectivity was observed with respect to the presence of the constituent components of urine (urea, creatinine, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, ammonium chloride, potassium sulphate and sodium chloride) at their normal levels, with a negligible sensor response to these chemicals. Successful detection of hCG was also achieved in spiked samples of real urine from a pregnant woman. The immunosensor developed is a promising tool for point-of-care detection of hCG, due to its excellent detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Specific label-free and real-time detection of oxidized low density lipoprotein (oxLDL) using an immunosensor with three monoclonal antibodies

Increased levels of plasma oxLDL, which is the oxidized fraction of Low Density Lipoprotein (LDL), are associated with atherosclerosis, an inflammatory disease, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL in serum. A new immunosensor employing three monoclonal antibodies (mAbs) against oxLDL is proposed in this work as a quick and effective way to monitor oxLDL. The oxLDL was first employed to produce anti-oxLDL monoclonal antibodies by hybridoma cells that were previously obtained. The immunosensor was set-up by selfassembling cysteamine (Cyst) on a gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Three mAbs were allowed to react with N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDAC), and subsequently incubated in the Au/Cys. Albumin from bovine serum (BSA) was immobilized further to ensure that other molecules apart from oxLDL could not bind to the electrode surface. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The analytical operation of the immunosensor was obtained by incubating the sensing layer of the device in oxLDL for 15 minutes, prior to EIS and SWV. This was done by using standard oxLDL solutions prepared in foetal calf serum, in order to simulate patient's plasma with circulating oxLDL. A sensitive response was observed from 0.5 to 18.0 mg mL 1 . The device was successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. The use of multiple monoclonal antibodies on a biosensing platform seemed to be a successful approach to produce a specific response towards a complex multi-analyte target, correlating well with the level of oxLDL within atherosclerosis disease, in a simple, fast and cheap way.

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Label-free Detection of Microcystin-LR in Waters Using Real-Time Potentiometric Biosensors Based on Single-Walled Carbon Nanotubes Imprinted Polymers

Microcystin-LR (MC-LR) is a dangerous toxin found in environmental waters, quantified by high performance liquid chromatography and/or enzyme-linked immunosorbent assays. Quick, low cost and on-site analysis is thus required to ensure human safety and wide screening programs. This work proposes label-free potentiometric sensors made of solid-contact electrodes coated with a surface imprinted polymer on the surface of Multi-Walled Carbon NanoTubes (CNTs) incorporated in a polyvinyl chloride membrane. The imprinting effect was checked by using non-imprinted materials. The MC-LR sensitive sensors were evaluated, characterized and applied successfully in spiked environmental waters. The presented method offered the advantages of low cost, portability, easy operation and suitability for adaptation to flow methods.

Assessing and comparing the total antioxidant capacity of commercial beverages: application to beers, wines, waters and soft drinks using TRAP, TEAC and FRAP methods.

This work measures and tries to compare the Antioxidant Capacity (AC) of 50 commercial beverages of different kinds: 6 wines, 12 beers, 18 soft drinks and 14 flavoured waters. Because there is no reference procedure established for this purpose, three different optical methods were used to analyse these samples: Total Radical trapping Antioxidant Parameter (TRAP), Trolox Equivalent Antioxidant Capacity (TEAC) and Ferric ion Reducing Antioxidant Parameter (FRAP). These methods differ on the chemical background and nature of redox system. The TRAP method involves the transfer of hydrogen atoms while TEAC and FRAP involves electron transfer reactions. The AC was also assessed against three antioxidants of reference, Ascorbic acid (AA), Gallic acid (GA) and 6-hydroxy-2,5,7,8-tetramethyl- 2-carboxylic acid (Trolox). The results obtained were analyzed statistically. Anova one-way tests were applied to all results and suggested that methods and standards exhibited significant statistical differences. The possible effect of sample features in the AC, such as gas, flavours, food colouring, sweeteners, acidity regulators, preservatives, stabilizers, vitamins, juice percentage, alcohol percentage, antioxidants and the colour was also investigated. The AC levels seemed to change with brand, kind of antioxidants added, and kind of flavour, depending on the sample. In general, higher ACs were obtained for FRAP as method, and beer for kind of sample, and the standard expressing the smaller AC values was GA.