Le cancer colorectal (CCR) reste encore aujourd'hui une cause majeure de mortalité dans le monde, avec une incidence annuelle d'environ 1 million de cas et une mortalité d'environ 700 000 personnes. La stratégie de dépistage du CCR se base sur la détection de sang occulte dans les selles par le test iFOBT. Malgré une spécificité acceptable, la sensibilité de détection reste toujours insuffisante dans une population asymptomatique (27% de sensibilité pour un adénome, 65% pour un CCR). Aujourd'hui, il existe un réel besoin de trouver de nouveaux biomarqueurs spécifiques du CCR ainsi que de développer des tests de diagnostic faciles à mettre en oeuvre dans les laboratoires de biologie médicale. Les progrès récents de l'analyse protéomique à haut débit permettent aujourd'hui d'identifier et de quantifier un nombre toujours plus grand de protéines. C'est pourquoi, nous avons développé des stratégies en protéomique quantitative pour la recherche et la validation de biomarqueurs du cancer colorectal. L'approche iTRAQ et Label-free ont permis l'identification d'environ 800 protéines et la quantification de 214 protéines. Nous avons entrepris avec succès, la validation de 4 protéines par des tests ELISA et de nouvelles technologie en protéomique quantitative ciblée (LC-SRM). Au Final, nous proposons un test multiplex de 3 protéines qui permettra d'affirmer ou d'infirmer les résultats obtenus par le test iFOBT. L'ajout du test pourrait réduire de manière significative le nombre de coloscopies inutiles, potentiellement dangereuses pour les patients et onéreuses pour les autorités de santé. / Colorectal cancer (CRC) remains a major cause of cancer mortality throughout the world with an annual incidence of approximately 1 million cases and an annual mortality around 700 000. CRC outcome is highly dependent upon the stage of detection. The 5-year survival rate of patients with metastases is less than 20% while patients with localized adenomatous polyps have an excellent outcome with 90% survival rate. Current screening methods of CRC include the iFOBT and colonoscopy. The iFOBT has an acceptable 95% specificity but always an unsatisfactory sensitivity of detection in the asymptomatic population (27% sensitivity for adenoma, 65% for carcinoma). That is why, there is a real need to find new biomarkers and develop new diagnostic test, specific and easy to implement in clinical research. Recent advances in the analysis of biological fluids by high throughput mass spectrometry allow us now to identify and quantify a large number of proteins. Due to these improvements of proteomic strategies, it seems to be a promising way to find new potential proteins markers in the CRC disease. The quantitative iTRAQ & Label-free approaches allowed the identification of 800 proteins and the quantification of 200 proteins. Therefore, we have successfully validated four proteins by targeted proteomics using mass spectrometry (LC-SRM) and ELISA assays. Finally, we suggest a multiplex test of 3 proteins which confirm or contradict the FOBT outcome. The addition of the test could significantly reduce the number of unnecessary colonoscopies which are potentially dangerous for patients and expensive for public health authorities.

Processos metabólicos naturais no organismo humano levam à formação de substâncias como compostos orgânicos voláteis (VOCs), de modo que, em um quadro patológico, processos diferenciados podem ocorrer nas células, fazendo com que um conjunto diferente de compostos seja produzido. Com esta hipótese, VOCs podem ser analisados em amostras biológicas com a intenção de se verificar alterações em seus perfis que sejam indicativos de certas patologias. No presente estudo, foi selecionado o suor como matriz, amostra de coleta simples e não-invasiva, de composição menos complexa e relacionada aos níveis sanguíneos e emanações da pele. Adicionalmente, foram também analisadas amostras de urina, para obtenção de dados comparativos. O presente estudo compreendeu amostras de voluntários saudáveis (grupo controle-C) e com diagnóstico de câncer confirmado (grupo positivo- P). As amostras de suor foram coletadas com o dispositivo PharmChek®, após, o patch foi removido, inserido em frasco e os VOCs isolados com emprego de técnica otimizada de Headspace estático (HS). Para as amostras de urina, estas foram analisadas com e sem tratamento por ?-glucuronidase. Perfis de VOCs foram obtidos por análise por cromatografia gasosa acoplada a espectrometria de massas (GC-MS) para todas amostras. A tentativa de identificação dos compostos detectados foi feita por busca na biblioteca NIST08 e uso do software AMDIS32. Diferenças qualitativas (teste chi-quadrado, p<<0,01) e quantitativas (teste U de Mann-Whitney, p<<0,01) foram avaliadas entre os perfis do grupo controle e positivo. Para o suor foram selecionados os potenciais biomarcadores pentanal, hexanal, heptanal, octanal, limoneno, 2-etil-1-hexanol, 1-undeceno, fenol, 2,6-dimetil-7-octen-2-ol (DMOL), nonanal, decanal e tridecano; para a urina, fenol e DMOL, hidrólise-dependentes, foram selecionados. Método HS-GC-FID (acoplado a detector por ionização de chama) foi desenvolvido e validado segundo a RDC 27/2012 da ANVISA, para ambas amostras. No suor, os analitos apresentaram limites inferiores de quantificação (LIQ) de 1 ng/adesivo, 5 ng/adesivo para o fenol; na urina foram de 2 ng mL-1 para o DMOL e 10 ng mL-1 para o fenol. Linearidade foi observada para faixa de 2 a 150 ng/adesivo e, 2 e 5 a 400 ng mL-1 na urina. No suor, a precisão variou de 0,08 a 12,35% e os analitos foram demonstrados estáveis para os ensaios realizados. Curvas ROC (Receiver Operating Characteristic) foram avaliadas e áreas sob a curva foram todas próximas a 1, com valores cut-off de 1,71 a 35,44 ng/adesivo no suor e 8,71 e 52,86 ng mL-1 na urina. 2-etil-1-hexanol se demonstrou negativamente correlacionado com o estágio clínico em adenocarcinomas (rô= -0,527) e DMOL, no suor, e aldeídos C5-C8 positivamente relacionados ao estágio do câncer de próstata (rô= 0,779 e 0,684, respectivamente). Conclui-se, portanto, que o método apresentado se mostrou eficiente, contudo, prático e de baixo custo, e os resultados obtidos corroboram para ideia da determinação de VOCs como promissora ferramenta auxiliar de diagnóstico no câncer. / Natural metabolic processes in the human body lead to the formation of substances such as volatile organic compounds (VOCs), so that, in a pathological context, differentiated processes can occur in the cells, causing a different set of compounds to be produced. With this hypothesis, VOCs can be analyzed in biological samples with the intention to verify changes in their profiles that are indicative of certain pathologies. In the present study, sweat was selected as the matrix, due simple and non-invasive collection, with lower complexity composition and related to blood levels and skin emanations. In addition, urine samples were also analyzed to obtain comparative data. The present study comprised samples from healthy volunteers (control-C group) and individuals with confirmed cancer diagnosis (positive-P group). The sweat samples were collected with PharmChek® device, next, the patch was removed, inserted in a vial and the VOCs were isolated using an optimized Static Headspace (HS) technique. For urine samples, these were analyzed with and without ?-glucuronidase treatment. VOC profiles were obtained by gas chromatography coupled to mass spectrometry (GC-MS) for all samples. The attempt to identify the detected compounds was made by searching the NIST08 library and using the AMDIS32 software. Qualitative differences (chi-square test, p << 0.01) and quantitative tests (Mann-Whitney U test, p << 0.01) were evaluated between the profiles of the control and positive groups. For the sweat, the potential biomarkers pentanal, hexanal, heptanal, octanal, limonene, 2-ethyl-1-hexanol, 1-undecene, phenol, 2,6-dimethyl-7-octen-2-ol (DMOL), nonanal, decanal and tridecane; for urine, phenol and DMOL, both hydrolysis-dependent, were selected. HS-GC-FID (coupled to flame ionization detector) method was developed and validated according to RDC 27/2012- ANVISA, for both samples. In sweat, the analytes presented limits of quantification (LOQ) of 1 ng/patch, 5 ng/patch for phenol; in urine were 2 ng mL-1 for DMOL and 10 ng mL-1 for phenol. Linearity was observed for the range of 2 to 150 ng/patch and, 2 and 5 to 400 ng mL-1 in urine. In sweat, the precision ranged from 0.08 to 12.35% and the analytes were shown to be stable for the assays performed. Receiver Operating Characteristic (ROC) curves were evaluated and areas under the curve were all near to 1, with cut-off values of 1.71 to 35.44 ng/patch in sweat and 8.71 and 52.86 ng mL-1 in urine. 2-ethyl-1-hexanol was shown to be negatively correlated with the clinical stage in adenocarcinomas (rho= -0.527) and DMOL, in sweat, and C5-C8 aldehydes sum, positively related to the stage of prostate cancer (rho= 0.779 and 0.684, respectively). It was concluded, therefore, that the method presented proved to be efficient, however, practical and low cost, and the results corroborate to the idea of VOCs determination as a promising diagnostic tool for cancer diagnosis.

Role of blood biomarkers in spontaneous intracerebral hemorrhage Background: The study of blood biomarkers can offer new possibilities in diagnostics, prognostication, determination of etiology, and management of spontaneous intracerebral hemorrhage. The aim of our study was to assess the relationship between a panel of selected blood biomarkers and clinical and radiodiagnostic parameters in patients with spontaneous intracerebral hemorrhage. Primarily, the aim was to find a prognostic biomarker which could help in deciding on the optimal categorization of treatment. Patients and methods: A total of 70 patients were prospectively included in this study. The following blood biomarkers were determined: glial fibrillary acidic protein, S100B protein, matrix metalloproteinase 9, interleukin 6, interleukin 10, 25-hydroxyvitamin D, 1,25- dihydroxyvitamin D, total cholesterol, leukocyte counts, blood glucose and C-reactive protein. These were then correlated with selected clinical and radiodiagnostic parameters. Results: Relative to hematoma volume a statistically significant positive correlation was found for S100B, interleukin 10, interleukin 6 and blood glucose (S100B: ρ= 0,54, p< 0,001, IL-10: ρ= 0,43, p< 0,001, IL-6: ρ= 0,26, p= 0,027, blood glucose: ρ= 0,24, p= 0,045). Using multivariate analysis, a...

L’étiologie des troubles du spectre autistique (TSA) est multifactorielle mettant en jeu une forte composante génétique ainsi que des facteurs environnementaux. A ce jour, il n’existe aucun biomarqueur facilitant un diagnostic précoce des TSA, ce qui permettrait une meilleure prise en charge des patients. De plus, les mécanismes physiopathologiques sont encore mal connus. La majorité des études sur les TSA ont été réalisées sur des populations occidentales. Ainsi, nous avons voulu étudier un groupe de patients libanais atteints de TSA. Nos objectifs s’articulent autour de trois volets : 1) L’identification de facteurs de risques environnementaux dans le but de mieux comprendre la pathologie. Les résultats de cette étude ont montré que plusieurs facteurs tels que la consanguinité, le stress au cours de la grossesse et la prématurité semblent être des facteurs de risques. 2) L’identification des variations structurales dans le génome des enfants atteints de TSA afin d'évaluer la contribution de CNV par l'approche de CGH array à haute résolution. Cette étude nous a permis d’identifier de nouveaux gènes dans les TSA ainsi de confirmer la présence des régions et des gènes déjà cités dans des études précédentes sur les TSA. 3) L’identification des métabolites urinaires et des voies métaboliques associés afin de proposer des biomarqueurs aidant à un diagnostic précoce et à une meilleure compréhension de la physiopathologie de la maladie. De nouveaux métabolites ont été identifiés, comme nous avons confirmé des variations anomalies déjà citées dans la littérature concernant certains métabolites qui pourraient représenter des marqueurs robustes de la maladie. / The etiology of autism spectrum disorders (ASD) is multifactorial involving a strong genetic component as well as environmental factors. To date, there is no biomarker facilitating early diagnosis of ASD to improve patient management and outcomes. In addition, the physiopathological mechanisms are still poorly understood. Most studies on ASD have been conducted in Western populations. Thus, we wanted to study a group of Lebanese patients with ASD. Our objectives focus around three aspects: 1) The identification of environmental risk factors in order to better understand the pathology. The results of this study showed that several factors such as consanguinity, stress during pregnancy and prematurity appear to be risk factors. 2) The identification of structural variations in the genome of children with ASD in order to evaluate the contribution of CNV by high resolution CGH array approach. This study allowed us to identify new genes in ASD as well as to confirm the presence of regions and genes already cited in previous ASD studies. 3) The identification of metabolites and associated metabolic pathways to provide biomarkers for early diagnosis and to better understanding of the pathophysiology of the disease. New metabolites have been identified and we have confirmed variations already mentioned in the literature concerning certain metabolites that could represent robust markers of the disease.

INTRODUCTION: The techniques currently in practice to diagnose and assess interval disease activity in patients with inflammatory bowel disease (IBD) are costly and invasive. Physicians typically rely on information derived from a combination of endoscopic, radiologic, and histologic studies to diagnose and determine the extent and severity of the two most common forms of IBD, Crohn disease (CD) and ulcerative colitis (UC). The development of noninvasive methods of assessing response to therapy is of increasing importance to pediatric healthcare providers. Previous studies have demonstrated that serum and fecal biomarkers are reliable measures of inflammation in the gastrointestinal tract. However, existing biomarkers are non-specific and their levels can be elevated in the context of either acute and chronic inflammation (IBD) or infection. As such, further studies are required to develop newer and novel biomarkers that have greater specificity for use in the diagnosis and interval assessment in children and adults with IBD. OBJECTIVES: The goal of this study is to further assess the relationship between biomarkers in the stool and serum of patients with IBD that are being treated with the anti-TNF therapy, infliximab (Remicade). To accomplish this, we will assess the changes in serum and fecal biomarker levels over the course of treatment and correlate the changes in fecal and serum biomarker levels with clinical, biochemical, and endoscopic outcome variables. METHODS: We conducted a prospective longitudinal cohort study in pediatric patients with IBD receiving long-term immunosuppressive therapy with Remicade. Pediatric patients diagnosed with either CD or UC who receive Remicade at Boston Children’s Hospital were recruited. Patients were drawn from subsets of patients that were either naïve to Remicade, had received Remicade for less than 6 months, or had received Remicade for more than one year at the time of enrollment. We collected longitudinal data over the course of their first 6 consecutive infusions following enrollment, including blood and stool samples, disease activity indexes, as well as a patient-reported outcome measure (IMPACT-III Questionnaire) at each infusion session. RESULTS: A total of 33 patients with IBD who fit our eligibility criteria and provided informed consent were enrolled to date. Of these, 20 had a CD diagnosis and 13 had a UC diagnosis. We collected baseline serum, fecal, and IMPACT-III score data and followed enrolled patients over the course of subsequent infusions. Mean baseline fecal ASCA levels from 8 CD and 6 UC patients were 0.08±0.021 OD and 0.02±0.0015 OD, respectively. At baseline, serum lactoferrin (p<0.10), calprotectin (p<0.10), ESR (p<0.05), and CRP (p<0.10) were significantly higher among CD patients. CONCLUSION: Our data demonstrate the potential for serum and fecal biomarkers to evaluate therapeutic response to Remicade. Completion of study enrollment and data collection will be necessary to determine if individual or combinations of fecal and serum biomarkers yield the most robust measures for use in the diagnosis and interval assessment of children and adults with IBD.

INTRODUÇÃO: A endometriose é doença ginecológica prevalente, com intervalo de tempo longo entre o início dos sintomas e o diagnóstico definitivo. A endometriose profunda intestinal se apresenta com sintomas mais severos, e maior morbidade cirúrgica. Um método minimamente invasivo para o diagnóstico precoce se faz necessário, e impediria a progressão para as formas mais profundas da doença. Estudos de biomarcadores com técnicas em proteômica podem trazer melhor entendimento das bases moleculares da doença, possibilidade de diagnóstico precoce, e do desenvolvimento de terapias-alvo. OBJETIVO: Comparar o perfil de expressão de proteínas do endométrio tópico entre pacientes saudáveis e com endometriose profunda intestinal, por técnica de espectrometria de massas shotgun. MÉTODOS: Estudo caso-controle exploratório, com total de 24 pacientes divididas em dois grupos: pacientes com endometriose profunda intestinal com comprovação cirúrgica, e pacientes do grupo controle saúdavel submetidas a cirurgia de laqueadura tubárea. Amostras de endométrio tópico foram obtidas por meio de auxílio de catéter de aspiração. As amostras foram submetidas a extração, digestão, dessalinização peptídica, e analisadas seguindo uma abordagem de espectrometria de massas shotgun, label-free de DDA (aquisição dependente de dados), utilizando o instrumento Orbitrap Elite (Thermo Fisher, EUA). RESULTADOS: a análise proteômica dos resultados foi realizada através do software Progenesis QI (Nonlinear Dynamics, Newcastle upon Tyne, UK), e resultou em um painel de 595 proteínas diferencialmente expressas entre os grupos controle e endometriose. Os resultados revelaram diferentes moléculas de acordo com a fase do ciclo menstrual, e o estádio da doença. As principais proteínas com expressão aumentada no grupo de pacientes com endometriose incluíram SETSIP, SRRM2, CAPS, H2AFV e SAFB, enquanto moléculas reguladas negativamente incluíram BPIFB1, FAM184A, SLPI, PIGR, JCHAÍNA, HLA-A e LTF. As proteínas diferenciais entre os grupos apresentaram o câncer como principal doença associada (p - valor de 4,14E-02 - 3,91E-08), de acordo com as análises realizadas pelo software IPA (Ingenuity Pathway Analysis System; Ingenuity Systems, Redwood City, CA, USA). As principais proteínas com expressão aumentada nos estádios mais avançados da doença foram CHMP4B, DES, EIF3I, CDH13, LIMS1, HSPA4, HSP90AB1, TUBB, NPM1, MYL6, e foram descritas relacionadas a metástases e pior prognóstico de diversos tipos de cânceres. CONCLUSÃO: CHMP4B, DES, EIF3I, CDH13, LIMS1, HSPA4, HSP90AB1, TUBB, NPM1, MYL6 são proteínas com expressão aumentada no endométrio das pacientes portadoras de endometriose profunda intestinal em estádios avançados. O presente estudo revelou proteínas diferencialmente expressas na endometriose em associação com diversos tipos de cânceres. Os achados constituem possíveis marcadores para o desenvolvimento de técnicas para o diagnóstico minimamente invasivo, e potenciais alvos terapêuticos para a doença / INTRODUCTION: Endometriosis is a prevalent gynecological disease, with a long time elapsed from onset of symptoms to definitive diagnosis. Deep intestinal endometriosis presents with more severe symptoms, and greater surgical morbidity. A minimally invasive method for early diagnosis becomes necessary and would prevent progression to the deeper presentations of the disease. Studies of biomarkers with proteomics techniques can provide a better understanding of the molecular basis of the disease, the possibility of early diagnosis, and the development of targeted therapies. OBJECTIVE: To compare the expression of eutopic endometrial proteins between healthy and deep intestinal endometriosis patients, using a shotgun mass spectrometry technique. METHODS: An exploratory case-control study with a total of 24 patients divided in two groups: patients with intestinal deep endometriosis with surgical confirmation, and patients in the healthy control group submitted to tubal ligation surgery. Eutopic endometrial samples were obtained by aspiration catheter. The samples were submitted to extraction, digestion, peptide desalting and analyzed using a DDA (data-dependent acquisition) label-free mass spectrometry approach by the Orbitrap Elite instrument (Thermo Fisher, USA). RESULTS: Proteomic analysis was performed using the Progenesis QI software (Nonlinear Dynamics, Newcastle upon Tyne, UK) to profile 595 differentially expressed proteins between the control and endometriosis groups. The results revealed different molecules according to the phase of the menstrual cycle and stage of disease. Top upregulated molecules included SETSIP, SRRM2, CAPS, H2AFV, and SAFB, whereas downregulated molecules included BPIFB1, FAM184A, SLPI, PIGR, JCHAIN, HLA-A, and LTF. Among top diseases associated molecules between groups were related to cancer (p - value of 4.14E-02 - 3.91E-08), according to the analysis performed by IPA software (Ingenuity Pathway Analysis System, Ingenuity Systems, Redwood City, CA, USA). Top upregulated proteins among patientes with advanced stage of endomeriosis were CHMP4B, DES, EIF3I, CDH13, LIMS1, HSPA4, HSP90AB1, TUBB, NPM1, MYL6. Those proteins were related to metastasis and poor survival of several cancer types. CONCLUSION: CHMP4B, DES, EIF3I, CDH13, LIMS1, HSPA4, HSP90AB1, TUBB, NPM1, MYL6 are among the top upregulated proteins in patients with advanced stage of deep intestinal endomeriosis. The present study revealed proteins differentially expressed in endometriosis as potential biomarkers for the development of techniques for minimally invasive diagnosis, and potential therapeutic targets for the disease

Orientador: Maria Lucia Gomes Lourenço / Resumo: Ambulatorial electrocardiographic monitoring, or Holter method, has been shown to be an effective tool in veterinary medicine to detect early heart lesions, not only to monitor the electrical activity of the heart, but also to control the autonomic nervous system of this organ. It is also known that the main infectious diseases of dogs, such as canine distemper and canine monocytic ehrlichiosis (CME) cause considerable lesions in the heart, proven by histopathological examination. It has already been proven the occurrence of myocarditis in the CME, leading to frequent presence of changes in the generation and conduction of the cardiac electrical impulse. The present study analyzed the electrical activity of the heart during 24 hours, focusing on the prevalence of arrhythmias, heart rate variability study and the biomarkers concentration of dogs with chronic CME (sick group) compared to healthy animals (control group). Forty-five percent of the animals in the diseased group had a high frequency of arrhythmias during the study. The mean concentration of cardiac troponin I and creatinokinase MB (CK-MB) was significant (0.24 ng / mL ± 0.5; 229 ± 205 IU / mL) compared to the control group (0.042 ± 0.07 ng / ML, 126 ± 46.12 IU / mL). The standard deviation of the mean of all NN (SDNN) intervals and the percentage of adjacent RR intervals with a duration difference greater than 50 milliseconds (pnn50%) were also extremely significant (83 ± 65 and 14.56 ± 20) when compared to Healthy... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

(in English) Air pollution is a serious worldwide problem associated with the risk of cancer. The negative effect of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), including benzo[a]pyrene (B[a]P), on human health is analyzed using specific biomarkers. Among them biomarkers of early effect play an important role. This work summarizes the results of cytogenetic analyses performed by fluorescence in situ hybridization (FISH) (whole chromosome painting of chromosomes #1 and #4) and automated image analysis of micronuclei (MN). During the analyses a total set of 1304 samples was analyzed by the FISH method and 885 samples by the automated image analysis of MN. Studied groups including city policemen, garage men, bus drivers, administrative workers, mothers, newborns, healthy children and children with bronchial asthma and laboratory workers were from Prague, Ostrava and Ceske Budejovice. The locations significantly differed in levels of air pollutants and the type of air pollution. The exposure of participants of the study was assessed by personal and stationary monitoring. The impact of other factors including age, smoking or intake of vitamins was also evaluated in these studies. The results obtained by the FISH method in Prague showed the impact of seasonal variability of concentrations of...

Ce manuscrit est consacré au développement de méthodes de dérivation intra-capillaire ultrasensible et de méthodes de séparation électrocinétique hautement résolutives en vue d’une intégration dans un outil microfluidique de diagnostic de la maladie d’Alzheimer. La partie bibliographique situe d'abord le contexte épidémiologique et neurophysiopathologique de la maladie d’Alzheimer ainsi que les outils de diagnostic actuellement utilisés. Ensuite, l'état de l'art sur l'utilisation de méthodes de séparation électrocinétique, ainsi que les outils analytiques disponibles pour améliorer l’analyse d’échantillon biologique sont présentés. La partie expérimentale a porté tout d'abord sur le développement de méthodes de dérivation intra-capillaire permettant une détection ultrasensible de biomarqueurs de la maladie d’Alzheimer, en particulier l’ubiquitine, dans le liquide céphalo-rachidien. Le travail expérimental s’est ensuite prolongé par le développement d’une méthode de séparations hautement résolutives en puce en verre de différentes formes de peptides beta-amyloïdes, biomarqueurs pertinents en vue d’un diagnostic de la maladie d’Alzheimer. Pour terminer, l’apport de deux nouveaux substrats polymériques, le COC et le thiolène, pour la microfabrication de puce d’électrophorèse capillaire a été démontré. / This work is dedicated to the development of highly sensitive intra-capillary derivatization methods and highly resolutive electrokinetic separation methods, with the aim to integrate them in a microfluidic diagnostic tool of Alzheimer’s disease. The bibliographic chapter explains the epidemiologic and neurophysiopathologic background of Alzheimer’s disease and describes the diagnostic tools currently used. Then, a state of the art related to the use of electrokinetic separation methods, as well as the analytical techniques available to improve the analyses of biological samples, is presented. The first experimental part focused on the development of intra-capillary derivatization methods allowing highly sensitive detection of Alzheimer’s disease biomarkers, especially ubiquitine, in the cerebrospinal liquid. The second part was dedicated to the development of a capillary electrophoresis method, in a glass chip, capable of separating with a high resolution different forms of beta-amyloid peptides, which are relevant biomarkers of Alzheimer’s disease. Finally, the usefulness of new polymeric materials, such as COC and thiolene, for the manufacturing of microchip device was demonstrated. A careful study of their physico-chemical properties led to the development of relevant strategies for surface treatment, so as to minimize the adsorption of proteins. For the first time, a thiolene chip covered with a new copolymer allowed an electrokinetic separation of proteins.

Os corantes são utilizados pelo homem há mais de 20 mil anos, com emprego nas indústrias têxteis, farmacêuticas, alimentícias, cosméticas, fotográficas, entre outras. Contudo, apesar de sua importância, os corantes podem ser tóxicos, mutagênicos e resistentes a muitos processos de degradação utilizados em estações de tratamento de águas residuais. Dentro deste contexto, o objetivo desta pesquisa foi avaliar os efeitos ecotoxicológicos dos corantes têxteis Disperse Red 60 (DR 60), Disperse Red 73 (DR 73) e Disperse Red 78 (DR 78) por meio de testes de toxicidade em três organismos de diferentes níveis tróficos: a bactéria Vibrio fischeri, o microcrustáceo Daphnia similis e o peixe Danio rerio (zebrafish), além da mutagenicidade na bactéria Salmonella typhimurium. Nossos resultados mostraram que os corantes DR 73 e DR 78 induzem toxicidade aguda em Vibrio fischeri e Daphnia similis, reduzindo a luminescência da bactéria e causando imobilidade no microcrustáceo, ao passo que o corante Disperse Red 60 não altera esses endpoints. No entanto, todos os corantes causam efeitos tóxicos em embriões e larvas de Danio rerio. O corante DR 60 ocasionou edemas oculares, alteração no comportamento e indução de efeitos pró-oxidantes em zebrafish. O corante DR 73 reduziu a inflação da bexiga natatória, induziu edemas do pericárdio, promoveu escoliose e alteração no saco vitelínico. Além disso, os corantes DR 73 e DR 78 alteraram o comportamento e balanço energético, induziram efeitos pró-oxidantes e neurotoxicidade. Quanto à mutagenicidade, o corante DR 73 induziu deslocamento do quadro de leitura e substituição de pares de base, enquanto o corante DR 78 causou apenas deslocamento do quadro de leitura e o corante DR 60 não induziu mutagenicidade. Em conclusão, os corantes Disperse Red 73 e Disperse Red 78 foram os mais tóxicos para os organismos avaliados, uma vez que alteraram maior número de endpoints, assim como causaram efeitos em menores concentrações, a partir de 1 ?g/L. Este estudo também demonstra a importância do uso de organismos testes de diferentes níveis tróficos, bem como a necessidade de uma maior atenção quanto ao registro e liberação para uso de corantes, visando prevenir danos ao ambiente e à saúde humana / Dyes have been used for more than twenty thousand years in the textile, pharmaceutical, food, cosmetic, and photographic industry, among others. Despite their importance in these applications, dyes can be toxic, mutagenic and resistant to many degradation processes used in wastewater treatment plants. In this context, the aim of this research was to evaluate the ecotoxicological effects of the Disperse Red 60 (DR 60), Disperse Red 73 (DR 73) and Disperse Red 78 (DR 78) textile dyes by means of toxicity tests on three organisms of different trophic levels: the bacterium Vibrio fischeri, the microcrustacean Daphnia similis and the fish Danio rerio (zebrafish). In addition, the mutagenicity of these dyes to the bacterium Salmonella typhimurium was evaluated. Our results demonstrate that the dyes DR 73 and DR 78 induce acute toxicity to V. fischeri (luminescence reduction) and D. similis (immobility), whereas the DR 60 did not alter these endpoints at the concentrations tested. However, all dyes caused toxic effects on embryos and larvae of D. rerio. DR 60 caused ocular edema, altered behavior and induced pro-oxidant effects in zebrafish. DR 73 reduced swim bladder inflation, induced pericardial edema, promoted scoliosis and changes in yolk sacs. In addition, DR 73 and DR 78 altered the zebrafish juvenile behavior and energy balance, and induced pro-oxidant effects and neurotoxicity. As for the mutagenicity test, DR 73 induced frameshift and base pair substitution, whereas DR 78 caused only frameshift and DR 60 did not induce any mutagenic effects. In conclusion, DR 73 and DR 78 were the most toxic for the organisms tested since they altered a greater number of endpoints and caused toxic effects at lower concentrations, i.e. from 1 ?g/L onwards. These results indicate the importance of using a battery of test organisms from different trophic levels when evaluating the environmental risks of dyes, as well as the need for greater attention regarding the Registration and authorization of the use of dyes in order to prevent unacceptable risks to the environment and human health.