Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.

Construção de Biossensores de ADN para a Avaliação da Capacidade Antioxidante

Mestrado em Engenharia da Computação e Instrumentação Médica

Estudo eletroquímico do dano oxidativo promovido por RNOS e PAH e o efeito protetor de antioxidantes no ADN

No dia-a-dia, os organismos vivos estão sujeitos a vários tipos de agressões de origem endógena e exógena. A produção endógena exagerada de agentes oxidantes que ocorre nos processos metabólicos dos seres vivos está intimamente associada ao aparecimento e desenvolvimento de várias patologias. Por outro lado, e devido às atividades antropogénicas, muitos agentes oxidantes de origem ambiental e alimentar entram por via exógena no organismo dos seres vivos provocando igualmente danos a nível celular. De modo a protegerem-se dos efeitos pejorativos provocados por estes compostos, os organismos vivos desenvolveram mecanismos complexos de defesa antioxidante. Este trabalho consistiu no estudo eletroquímico do dano oxidativo induzido por agentes oxidantes (PAH (hidrocarbonetos aromáticos policíclicos), H2O2, NO• e HClO) e do efeito protetor, ao dano oxidativo, promovido por antioxidantes no material baseado no ADN recorrendo à utilização de um biossensor de bases púricas, adenina-EPC (elétrodo pasta de carbono) e dA20-EPC, utilizando a voltametria de onda quadrada (VOQ) como técnica de deteção. A aplicação da eletroquímica apresenta várias vantagens para a quantificação da capacidade antioxidante total (CAT) pois, permite a redução da quantidade de reagentes e amostra em análise, elimina a etapa de remoção de cor (a cor é um interferente nos métodos óticos) e não requer equipamentos dispendiosos. Foram seguidas diferentes abordagens para a construção dos biossensores. A primeira consistiu na construção de um adenina-EPC em três etapas: i) condicionamento do EPC, ii) eletrodeposição da adenina no EPC e iii) leitura do sinal eletroquímico. Assim, foram otimizados diversos parâmetros: concentração de adenina (150,0 mg/L), potencial de condicionamento (Ec) (+ 1,80 V), potencial de deposição (Ed) (+ 0,40 V), tempo de condicionamento (tc) (180 s) e tempo de deposição (td) (240 s). Foi aplicado o adenina-EPC no estudo do dano oxidativo provocado por PAH (benzo (g,h,i) perileno) e constatou-se que era necessário transformar o benzo (g,h,i) perileno num radical para se possível observar danos oxidativos induzidos no biossensor. A nova estratégia consistiu na construção de um dA20-EPC, através da adsorção física de uma gota de dA20 na superfície do EPC, com posterior secagem e leitura do sinal eletroquímico. Neste procedimento foi otimizada a concentração de dA20 (100,0 mg/L). O dano oxidativo provocado pelo H2O2, NO• e HClO foi estudado sobre o dA20-EPC e verificou-se que os três agentes oxidantes induziam dano oxidativo no dA20-EPC. Confirmou-se a capacidade do ácido ascórbico (AA) em proteger o dA20-EPC do dano oxidativo induzido por H2O2 e NO•. O biossensor desenvolvido (dA20-EPC) foi aplicado na avaliação da CAT de diferentes amostras reais (café, sumo de laranja e água aromatizada de laranja) usando-se como agentes oxidantes o H2O2 e NO•. Todas as amostras analisadas apresentaram ter capacidade antioxidante. Quando se utilizou o dA20-EPC na presença de H2O2, verificou-se que as amostras de café apresentam valores mais elevados de CAT (1130-1488 mg AAE/L) do que as amostras de bebidas (110 mg AAE/L em água aromatizada e 775 mg AAE/L em sumo). Os valores de CAT obtidos para amostras de sumo e água aromatizada na presença de NO• indicam que a amostra de sumo possui maior teor de CAT (871 mg AAE/L) conforme era esperado, do que a amostra de água aromatizada (172 mg AAE/L). Na presença de HClO, o valor de CAT mais elevado pertence a uma amostra de sumo (513 mg AAE/L) mas, o valor de CAT da amostra de sumo natural é muito mais baixa do que o esperado (17 mg AAE/L). Foram estudados outros antioxidantes para além do AA (ácido cumárico, ácido gálico e ácido cafeico), e constatou-se que cada um deles promove proteção ao dA20-EPC na presença de cada um dos diferentes contaminantes (H2O2, NO• e HClO).

Estudo eletroquímico do dano oxidativo promovido por RNS e PAH e o efeito protetor de antioxidantes no ADN

No dia-a-dia, os organismos vivos estão sujeitos a vários tipos de agressões de origem endógena e exógena. A produção endógena exagerada de contaminantes que ocorre nos processos metabólicos dos seres vivos está intimamente associada ao aparecimento e desenvolvimento de várias patologias. Por outro lado, e devido às atividades antropogénicas, muitos contaminantes de origem ambiental e alimentar entram por via exógena no organismo dos seres vivos provocando igualmente danos a nível celular. De modo a protegerem-se dos efeitos pejorativos provocados por estes compostos, os organismos vivos desenvolveram mecanismos complexos de defesa antioxidante. Este trabalho consistiu no estudo eletroquímico do dano oxidativo induzido por contaminantes (PAH (hidrocarbonetos aromáticos policíclicos), H2O2, NO• e HClO) e do efeito protetor, ao dano oxidativo, promovido por antioxidantes no material baseado no ADN recorrendo à utilização de um biossensor de bases púricas, adenina-EPC (elétrodo pasta de carbono) e dA20-EPC, utilizando a voltametria de onda quadrada (VOQ) como técnica de deteção. A aplicação da eletroquímica apresenta várias vantagens para a quantificação da capacidade antioxidante total (CAT) pois, permite a redução da quantidade de reagentes e amostra em análise, elimina a etapa de remoção de cor (a cor é um interferente nos métodos óticos) e não requer equipamentos dispendiosos. Foram seguidas diferentes abordagens para a construção dos biossensores. A primeira consistiu na construção de um adenina-EPC em três etapas: i) condicionamento do EPC, ii) eletrodeposição da adenina no EPC e iii) leitura do sinal eletroquímico. Assim, foram otimizados diversos parâmetros: concentração de adenina (150,0 mg/L), potencial de condicionamento (Ec) (+ 1,80 V), potencial de deposição (Ed) (+ 0,40 V), tempo de condicionamento (tc) (180 s) e tempo de deposição (td) (240 s). Foi aplicado o adenina-EPC no estudo do dano oxidativo provocado por PAH (benzo (g,h,i) perileno) e constatou-se que era necessário transformar o benzo (g,h,i) perileno num radical para se possível observar danos oxidativos induzidos no biossensor. A nova estratégia consistiu na construção de um dA20-EPC, através da adsorção física de uma gota de dA20 na superfície do EPC, com posterior secagem e leitura do sinal eletroquímico. Neste procedimento foi otimizada a concentração de dA20 (100,0 mg/L). O dano oxidativo provocado pelo H2O2, NO• e HClO foi estudado sobre o dA20-EPC e verificou-se que os três contaminantes induziam dano oxidativo no dA20-EPC. Confirmou-se a capacidade do ácido ascórbico (AA) em proteger o dA20-EPC do dano oxidativo induzido por H2O2 e NO•. O biossensor desenvolvido (dA20-EPC) foi aplicado na avaliação da CAT de diferentes amostras reais (café, sumo de laranja e água aromatizada de laranja) usando-se como contaminantes o H2O2 e NO•. Todas as amostras analisadas apresentaram ter capacidade antioxidante. Quando se usou o dA20-EPC na presença de H2O2, verificou-se que as amostras de café apresentam valores mais elevados de CAT (1130-1488 mg AAE/L) do que as amostras de bebidas (110 mg AAE/L em água aromatizada e 775 mg AAE/L em sumo). Os valores de CAT obtidos para amostras de sumo e água aromatizada na presença de NO• indicam que a amostra de sumo possui maior teor de CAT (526 mg AAE/L) conforme era esperado, do que a amostra de água aromatizada (172 mg AAE/L).

A biosensor based on immobilization of lactate oxidase in a PB-CTAB film for FIA determination of lactate in beer samples

An amperometric lactate biosensor with lactate oxidase immobilized into a Prussian Blue (PB) modified electrode was fabricated. The advantage of using cetyltrimethylammonium bromide (CTAB) in the electrodeposition step of PB films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of the measurements. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 µmol L-1 lactate with a limit of detection of 0.84 mmol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The usefulness of the method was demonstrated by determining lactate in beer samples and the results were in good agreement with those obtained by using a reference spectrophotometric enzyme method.

Determination of parathion and carbaryl pesticides in water and food samples using a self assembled monolayer/acetylcholinesterase electrochemical biosensor

An acetylcholinesterase (AchE) based amperometric biosensor was developed by immobilisation of the enzyme onto a self assembled modified gold electrode. Cyclic voltammetric experiments performed with the SAM-AchE biosensor in phosphate buffer solutions ( pH = 7.2) containing acetylthiocholine confirmed the formation of thiocholine and its electrochemical oxidation at E-p = 0.28 V vs Ag/AgCl. An indirect methodology involving the inhibition effect of parathion and carbaryl on the enzymatic reaction was developed and employed to measure both pesticides in spiked natural water and food samples without pre-treatment or pre-concentration steps. Values higher than 91-98.0% in recovery experiments indicated the feasibility of the proposed electroanalytical methodology to quantify both pesticides in water or food samples. HPLC measurements were also performed for comparison and confirmed the values measured amperometrically.

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

Electrochemical evaluation of total antioxidant capacity of beverages using a purine-biosensor

In this paper, it was evaluated the total antioxidant capacity (TAC) of beverages using an electrochemical biosensor. The biosensor consisted on the purine base (guanine or adenine) electro-immobilization on a glassy carbon electrode surface (GCE). Purine base damage was induced by the hydroxyl radical generated by Fenton-type reaction. Five antioxidants were applied to counteract the deleterious effects of the hydroxyl radical. The antioxidants used were ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol. These antioxidants have the ability to scavenger the hydroxyl radical and protect the guanine and adenine immobilized on the GCE surface. The interaction carried out between the purinebase immobilized and the free radical in the absence and presence of antioxidants was evaluated by means of changes in the guanine and adenine anodic peak obtained by square wave voltammetry (SWV). The results demonstrated that the purine-biosensors are suitable for rapid assessment of TAC in beverages.

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH• radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADHmeasured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50nMof AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi- walled carbon nanotubes(MWCNTs)paste electrode modified by dispersion of laccase(3%,w/w) within the optimum composite matrix(60:40%,w/w,MWCNTs and paraffin binder)showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate4- aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90 ×10- 7 to 1.15 ×10- 5 molL 1 using 4- aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%).The limit of detection obtained was 1.8 × 10-7 molL 1 (0.04 mgkg 1 on a fresh weight vegetable basis).The high activity and catalytic properties of the laccase- based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0±0.1% to 101.0 ± 0.3% were attained. No significant effects in the pirimicarb electro- analysis were observed by the presence of pro-vitamin A, vitamins B1 and C,and glucose in the vegetable extracts. The proposed biosensor- based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

Molinate quantification in environmental water by a glutathione-S-transferase based biosensor

A glutathione-S-transferase (GST)based biosensor was developed to quantify the thiocarbamate herbicide molinate in environmental water.The biosensor construction was based on GST immobilization onto a glassy carbon electrode via aminosilane–glutaraldehyde covalent attachment. The principle supporting the use of this biosensor consists of the GST inhibition process promoted by molinate. Differential pulse voltammetry was used to obtain a calibration curve for molinate concentration, ranging from 0.19 to 7.9 mgL -1 and presenting a detection limit of 0.064 mgL- 1. The developed biosensor is stable,and reusable during 15 days.The GST-based biosensor was successfully applied to quantify molinate in rice paddy field floodwater samples. The results achieved with the developed biosensor were in accordance with those obtained by high performance liquid chromatography. The proposed device is suitable for screening environmental water analysis and, since no sample preparation is required, it can be used in situ and in real-time measurements.

Transferência de electrão em bases de ADN por colisões átomo-molécula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Engenharia Biomédica

Simple laccase-based biosensor for formetanate hydrochloride quantification in fruits

This work describes the development of an electrochemical enzymatic biosensor for quantification of the pesticide formetanate hydrochloride (FMT). It is based on a gold electrode modified with electrodeposited gold nanoparticles and laccase. The principle behind its development relies on FMT's capacity to inhibit the laccase catalytic reaction that occurs in the presence of phenolic substrates. The optimum values for the relevant experimental variables such as gold nanoparticles electrochemical deposition (at − 0.2 V for 100 s), laccase immobilization (via glutaraldehyde cross-linking), laccase concentration (12.4 mg/mL), substrate selection and concentration (5.83×10−5 M of aminophenol), pH (5.0), buffer (Britton–Robinson), and square-wave voltammetric parameters were determined. The developed biosensor was successfully applied to FMT determination in mango and grapes. The attained limit of detection was 9.5×10−8 ± 9.5×10−10 M (0.02 ± 2.6×10−4 mg/kg on a fresh fruit weight basis). Recoveries for the five tested spiking levels ranged from 95.5 ± 2.9 (grapes) to 108.6 ± 2.5% (mango). The results indicated that the proposed device presents suitable characteristics in terms of sensitivity (20.58 ± 0.49 A/μM), linearity (9.43×10−7 to 1.13×10−5 M), accuracy, repeatability (RSD of 1.4%), reproducibility (RSD of 1.8%) and stability (19 days) for testing of compliance with established maximum residue limits of FMT in fruits and vegetables.

Sensitive bi-enzymatic biosensor based on polyphenoloxidases–gold nanoparticles–chitosan hybrid film–graphene doped carbon paste electrode for carbamates detection

A bi-enzymatic biosensor (LACC–TYR–AuNPs–CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC–TYR–AuNPs–CS/GPE exhibited an improved Michaelis–Menten kinetic constant (26.9 ± 0.5 M) when compared with LACC–AuNPs–CS/GPE (37.8 ± 0.2 M) and TYR–AuNPs–CS/GPE (52.3 ± 0.4 M). Using 4-aminophenol as substrate at pH 5.5, the device presented wide linear ranges, low detection limits (1.68×10− 9 ± 1.18×10− 10 – 2.15×10− 7 ± 3.41×10− 9 M), high accuracy, sensitivity (1.13×106 ± 8.11×104 – 2.19×108 ± 2.51×107 %inhibition M− 1), repeatability (1.2–5.8% RSD), reproducibility (3.2–6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8 ± 0.3% (lemon) to 97.8 ± 0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.

Terapêuticas antigénio-especificas no tratamento das doenças desmielinizantes: estudos sobre a vacinação com ADN e a descoberta de um novo alvo antigénico

RESUMO A Esclerose Múltipla (EM) é uma doença desmielinizante crónica do Sistema Nervoso Central (SNC), provocada, em grande parte, por um ataque imuno-mediado contra diversos elementos da bainha de mielina. Dentro dos alvos antigénicos desta resposta autoimune, vários componentes proteicos e lipídicos da mielina têm vindo a ser identificados ao longo dos anos, entre os quais se destacam a proteína básica de mielina(MBP), glicoproteína ligodendrocitária da mielina (MOG), proteína proteolipídica (PLP) e glicoproteína associada à mielina (MAG). Com o desenvolvimento do modelo animal de Encefalomielite Autoimune Experimental (EAE), diversas terapias antigénio-específicas foram desenhadas, baseadas na modificação benéfica da resposta autoimune contra a mielina, tais como a administração de mielina ou seus componentes, os copolímeros terapêuticos, os ligandos peptídeos alterados e, recentemente, a vacinação com ácido desoxirribonucleico (ADN) codificador de proteínas de mielina, integrado em plasmídeos e purificado para administração parentérica. Neste trabalho, apresentamos os resultados de um extenso conjunto de experiências, subordinadas a dois temas fundamentais: 1) avaliação do potencial terapêutico, e dos mecanismos de acção, da vacinação tolerizadora com ADN codificador de proteínas de mielina (MBP, MOG, PLP, MAG) na EAE, e da associação desta vacinação com a administração de ADN de citocinas Th2, ou de oligonucleótidos imunomoduladores; 2) identificação e caracterização da resposta imune contra um novo componente da mielina com potencial antigénico, a proteína inibidora do recrescimento axonal, Nogo-A. No que respeita à vacinação com ADN, os nossos resultados comprovam a eficácia desta terapêutica antigénio-específica na prevenção e tratamento da EAE. Os seus mecanismos de acção incluem, entre outros, a supressão anérgica da proliferação antigénioespecífica dos linfócitos T anti-mielina (no modo de prevenção da doença), o enviesamento Th2 da resposta imune (quando co-administrada com a vacina de ADN codificadora da citocina IL-4, funcionando como terapia génica local), e a redução da diversificação de epítopos da resposta humoral anti-mielina, avaliada através de myelin spotted arrays. A associação das vacinas de ADN com oligonucleótidos imunomoduladores GpG, desenvolvidos para contrariar as sequências CpG imunoestimuladoras presentes no vector de vacinação, levou à melhoria da sua eficácia terapêutica, devida, provavelmente, ao efeito estimulador preferencial dos oligonucleótidos GpG sobre linfócitos Th2 e sobre células reguladoras NK-T. Com base nestes resultados a vacinação com ADN foi desenvolvida para o tratamento da EM em humanos, com ensaios clínicos a decorrerem neste momento. Em relação à proteína Nogo-A, estudos de estrutura primária e de previsão de antigenicidade identificaram a região Nogo-66 como alvo antigénico potencial para a EAE. Nas estirpes de ratinho SJL/J e C57BL/6, fomos capazes de induzir sinais clínicos e histológicos de EAE após imunização com os epítopos encefalitogénicos Nogo1-22, Nogo23- 44 e Nogo45-66, utilizando protocolos de quebra de tolerância imune. Ao mesmo tempo, identificámos e caracterizámos uma resposta linfocitária T específica contra os antigénios contidos na região Nogo-66, e uma resposta linfocitária B com diversificação intra e intermolecular a vários determinantes presentes noutras proteínas da mielina. A transferência adoptiva de linhas celulares Th2 anti-Nogo45-66, levou à melhoria clínica e histológica da EAE em animais recipientes induzidos com outros antigénios de mielina, após migração destas células para o SNC. Estes dados comprovam a importância da Nogo-66 como antigénio na EAE, e a eficácia de terapias antigénio-específicas nela baseadas. No seu conjunto, os nossos resultados confirmam o potencial terapêutico das vacinas de ADN codificadoras de proteínas de mielina, bem como a importância dos encefalitogénios contidos na proteína Nogo-A para a fisiopatologia da EAE e da EM, com eventual relevância para o desenvolvimento de novas terapias antigénio-específicas. O aperfeiçoamento futuro destas terapias poderá levar, eventualmente, a uma capacidade de manipulação da resposta imune que permita o tratamento eficaz das doenças inflamatórias desmielinizantes, como a Esclerose Múltipla. ABSTRACT Multiple Sclerosis (MS) is a chronic demyelinating disease of the Central Nervous System (CNS), caused, mainly, by an immune-mediated attack against several elements of the myelin sheath. Among the antigenic targets for this autoimmune response, several proteic and lipidic myelin components have been identified throughout the years, of which myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipidic protein (PLP), and myelin associated glycoprotein (MAG) are the best characterized. With the development of the animal model for MS, Experimental Autoimmune Encephalomyelitis (EAE), several antigen-specific therapies have been designed, based on beneficial modifications of the autoimmune response against myelin. These have included myelin and myelin component administration, therapeutic copolymers, altered peptide ligands and, more recently, vaccination with myelin-protein encoding deoxyribonucleic acid (DNA), integrated into plasmids and purified for parenteral administration. In this work we present the results of an extensive series of experiments, subordinate to two fundamental areas: 1) evaluating the therapeutic potential, and mechanisms of action, of tolerizing myelin protein (MBP, MOG, PLP, MAG) DNA vaccination in EAE, alone and in association with Th2 cytokine DNA administration, or immunomodulatory oligonucleotides; 2) identifying and characterizing the immuneresponse against a new myelin component with antigenic potential, the axonal regrowth inhibitor Nogo-A. Regarding DNA vaccination, our results prove the efficacy of this antigen-specific therapy for the prevention and treatment of EAE. Its mechanisms of action include, among others, anergic suppression of antigen-specific T-cell proliferation against myelin (in prevention mode), Th2 biasing of the immune response (when co-administered with the IL- 4 codifying DNA vaccine, acting as local gene therapy), and reduction of epitope spreading of the anti-myelin antibody response, assessed by myelin spotted arrays. The combination of myelin DNA vaccination with the administration of GpG immunomodulatory oligonucleotides, designed to counteract immunostimulatory CpG motifs present in the vaccination vector, led to an improvement in therapeutic efficacy, probably due to the preferential stimulatory effect of GpG oligonucleotides on Th2 lymphocytes and on regulatory NK-T cells. Based on these results, tolerizing DNA vaccination is being developed for human use, with ongoing clinical trials. As concerns the Nogo-A protein, based on studies of primary structure and prediction of antigenicity, we identified the Nogo-66 region (responsible for the most of the inhibitory capacity of this protein) as a potential antigenic target for EAE. In the SJL/Jand C57BL/6 mouse strains, we were able to induce clinical and histological signs of EAE,after immunization with the encefalitogenic epitopes Nogo1-22, Nogo23-44 and Nogo45-66,using a tolerance breakdown protocol. Concomitantly, we identified and characterized a specific T cell response against these antigens, together with a B cell response which showed extensive intra and intermolecular epitope spread to several determinants present in other myelin proteins. Adoptive transfer of nti-Nogo45-66 Th2 cell lines resulted in clinical and histological improvement of EAE in recipient animals induced with other myelin antigens, after intraparenchymal CNS migration of anti-Nogo cells. These data confirm the relevance of Nogo-66 as an antigen in EAE, as well as the efficacy of antigenspecific therapies based on the response against this protein.In conclusion, our results substantiate the therapeutic potential of myelin-encoding DNA vaccination, as well as the importance of encefalitogenic epitopes present in the Nogo-A protein for the pathophysiology of EAE and MS, with potential relevance for the creation of new antigen specific-therapies. The future development of these therapies may eventually lead to a degree of manipulation of the immune response that allows the effective treatment of autoimmune, inflammatory, demyelinating diseases, such as Multiple Sclerosis.