Chez la brebis, les BMPs inhibent la synthèse et la libération de FSH. La détection des ARNm de certaines BMPs dans l’hypophyse et de leurs récepteurs sur les cellules gonadotropes suggère une action paracrine/autocrine des BMPs sur la production de FSH. L’objectif de cette thèse a visé à déterminer l’importance des BMPs hypophysaires, en particulier BMP-4, dans la régulation de la sécrétion de FSH chez la brebis. Nous montrons que l’expression des ARNm du système BMP-4,analysée par RT-PCR en temps réel, ne varie pas au cours du cycle oestrien ou in vitro quelque soit le temps d’incubation (6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires.Afin de déterminer si les cellules hypophysaires ovines sécrètent des BMPs, des milieux conditionnés(MC) hypophysaires ont été soumis à un test d’activité biologique reposant sur des cellules embryonnaires transfectées avec un élément de réponse BMPs couplé au gène rapporteur luciférase.Aucun effet des MC n’a été observé sur l’activité luciférase comparé au milieu non conditionnésuggérant l’absence ou la très faible activité BMP des MC, et cela quelque soit le temps d’incubation(6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires. Toutefois, nous détectons une activité inhibitrice de BMPs qui est augmentée par la GnRH et l’oestradiol suggérant l’implication d’inhibiteurs de BMPs dans la régulation de la production de FSH. En conclusion,l’ensemble de nos résultats n’est pas en faveur d’un rôle de BMP-4 hypophysaire dans la synthèse de FSH chez l’adulte. Un rôle des BMPs au niveau hypophysaire via la voie endocrine peut s’envisager puisque nous avons mis en évidence une bioactivité de type BMP dans le sérum ovin. Les inhibiteurs BMPs produits par l’hypophyse, qui restent à identifier, pourraient moduler la biodisponibilité des BMPs atteignant l’hypophyse par la voie sanguine. / BMPs inhibit FSH synthesis and release in ewe. The detection of BMP mRNAs in the pituitary as well as the colocalisation of the two types of BMP receptors on gonadotrope cells suggest that these BMPs can exert paracrine/autocrine actions on FSH production. The aim of this thesis work was to determine the importance of pituitary BMPs in the regulation of FSH production in the ewe. We showed that the level of mRNAs for BMP-4 system, analyzed by real-time RT-PCR, did not vary across the oestrous cycle or in vitro whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of pituitary cells. By using a bioactivity test based on embryonic mesenchymal cells transfected with an expression construct containing a BMP-responsive element fused to firefly luciferase reporter gene,we detected no BMP activity within conditioned media from pituitary cells whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of the pituitary cells. However, we detected a BMP inhibitory activity which is increased by GnRH and oestradiol suggesting the implication of BMP inhibitor(s) in FSH regulation. In conclusion, the results are not in favor of a role for pituitary BMP-4 in the regulation of FSH synthesis in adult ewe. An endocrine action of BMPs at pituitary level can be evoked since BMP bioactivity was detected within ovine serum. BMP inhibitors that remain to be identified can modulate the bioavailability of BMPs reaching the pituitary by the blood way.

O presente estudo foi conduzido com o objetivo de determinar se a sensibilidade do teste de Soroneutralização (SN) seria afetada quando utilizados distintos subtipos de herpesvírus bovino tipos 1 (BoHV-1) e 5 (BoHV-5). Dessa forma, soros de bovinos, coletados randomicamente (n= 287) foram testados por SN frente a três amostras de BoHV-1 (BoHV-1.1: EVI123/98 e Los Angeles (LA); BoHV-1.2a: SV265/96) e três amostras de BoHV-5 (BoHV-5a: EVI88/95; BoHV-5b: A663 e BoHV-5c: ISO 97/95), utilizando um período de incubação soro-vírus de 24 horas. A sensibilidade da SN variou significativamente dependendo da amostra viral utilizada. Esta variação foi de 77% (80/104 soros positivos) até 91% (95/104) com as amostras ISO 97/95 e LA, respectivamente, quando cada vírus foi considerado individualmente. A sensibilidade máxima (104/104) foi obtida quando os resultados positivos de uma combinação particular de quatro vírus (LA + EVI123 + SV265 + A663), algumas combinações de cinco vírus, ou ainda, todos os seis vírus foram adicionados. Estes resultados evidenciaram que quando a SN é realizada frente a uma única amostra viral, a sensibilidade pode variar significativamente, podendo comprometer programas de controle e erradicação das infecções por estes agentes. Além disso, a realização de SN frente a diferentes isolados virais mostrou aumentar significativamente a sensibilidade do teste. Com isso, a caracterização de novos isolados de campo pode favorecer futuras avaliações de diferentes subtipos de BoHV-1 e BoHV-5 e contribuir com a escolha de amostra e/ou combinação de amostras mais sensíveis. Deste modo, buscou-se caracterizar isolados de campo de BoHV-1 e BoHV-5 com base na análise molecular da região carboxi-terminal do gene que codifica a glicoproteína C (gC) e na análise com enzima de restrição (REA) do genoma viral. Para tanto, a multiplicação de 24 isolados da América do Sul foi realizada em células CRIB para posterior extração do DNA viral, PCR e sequenciamento. As seqüências foram alinhadas utilizando o programa ClustalX2 para uma inferência filogenética pelo método de Neighbor-Joining (Mega 4.0), Kimura 2-parâmetros. O alinhamento das seqüências de nucleotídeos revelou níveis de identidade variando de 70 a 99,6% entre os isolados de BoHV-1; de 66,9 a 100% entre os isolados de BoHV-5 e de 62,9 a 92,8% entre os isolados BoHV-1 e BoHV-5. A árvore filogenética mostrou o agrupamento dos vírus de acordo com o tipo (BoHV-1 e BoHV-5) e subtipo de BoHV-1 (BoHV-1.1 e BoHV-1.2). No entanto, essa técnica não permitiu a diferenciação dos isolados em diferentes subtipos de BoHV-5. Do mesmo modo, a análise por restrição enzimática não proporcionou uma clara diferenciação dos isolados em subtipos devido à presença de variações nos padrões de restrição. Somente um isolado (ISO 94/232) pode ser diferenciado como subtipo 5a. Todavia, este estudo mostrou que a análise filogenética utilizada representa uma potencial ferramenta para a diferenciação e classificação dos vírus em BoHV-1.1, BoHV-1.2 e BoHV-5. No entanto, ambas as técnicas podem ser empregadas, de maneira complementar, quando maiores informações sobre estes vírus forem requeridas. Além disso, com o presente estudo, foi possível expandir o número de amostras caracterizadas, fornecendo subsídios para estudos futuros. / This study was carried out to determine whether the sensitivity of serum neutralization (SN) tests would be affected by the use of distinct subtypes of bovine herpesvirus 1 (BoHV- 1) and 5 (BoHV-5) as test challenge viruses. Bovine sera collected from a randomized sample (n = 287) were tested in a 24 hour incubation SN against three type 1 viruses (BoHV-1.1 strains „„Los Angeles‟‟ (LA) and „„EVI 123‟‟; BoHV-1.2a strain „„SV 265‟‟) and three type 5 viruses (BoHV-5a strain „„EVI 88‟‟; BoHV-5b strain „„A 663‟‟ and BoHV-5c „„ISO 97‟‟). SN sensitivity varied greatly depending on the challenge virus used in the test, particularly when results against each virus were considered individually, where it ranged from 77% (detecting 80 out of 104 antibody-positive sera) to 91% (95/104) with ISO 97/95 and LA strain, respectively. Maximum sensitivity (104/104) was achieved when positive results to a particular combination of four of the challenge viruses (LA + EVI 123 + SV 265 + A 663) or some combinations of five viruses (or all six viruses) were added cumulatively. These results clearly shown that when SN is performed with single test challenge viruses, sensitivity could vary significantly that might compromise control or eradication efforts. Performing SN against a number of different viruses demonstrated to improve significantly the test‟s sensitivity. Thus, news field isolates characterization could aid future evaluations of different BoHV-1 and BoHV-5 subtypes and also provide the better strain and/or strains combination choice. In such case, this study aimed to characterize field isolates of BoHV-1 and BoHV-5 by molecular analyses of glycoprotein C (gC) carboxy-terminal region and viral genome restriction enzymatic analysis (REA). The 24 isolates from South America were propagated in CRIB cells for viral DNA extraction, PCR and sequencing. The sequences were aligned in ClustalX2 to perform a distance–based phylogenetic analysis by Neighbor-Joining method in MEGA 4.0 software under de Kimura 2-parameter. The nucleotide sequence alignments revealed levels of genomic similarity ranging from 70% to 99.6% between BoHV-1 isolates; from 67% to 100% among BoHV-5 isolates and from 63% to 93% between BoHV-1 and BoHV-5 isolates. The phylogenetic tree clustered the viruses according to types (BoHV-1 and BoHV-5) and BoHV-1 subtypes (BoHV-1.1, -1.2). However, this method did not allow clearly differentiated in BoHV-5 subtypes. Likewise, REA did not clear showed differentiation in subtypes due presence variation in restriction pattern. Only one isolate (ISO 94/232) could be differentiated in subtype 5a. The results suggest that the phylogenetic analysis performed could be an important tool for differentiation and classification of such viruses in BoHV-1.1, BoHV-1.2, BoHV-5. However, when adiccional informations were resquested both techniques should be performed. Furthermore, with this work it was possible to expand the number of samples characterized to support future investigation of these viruses.

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La récupération fonctionnelle des patients victimes d’un accident vasculaire cérébral (AVC) ischémique est largement sous-tendue par les propriétés plastiques du cerveau et plus précisément par sa capacité à remodeler les réseaux de neurones épargnés par l’infarctus. Les études réalisées sur différents modèles animaux d’infarctus cérébral s’accordent à montrer que ces changements plastiques sont induits par le BDNF (Brain-Derived Neurotrophic Factor). Aussi, augmenter les taux cérébraux de BDNF est considéré comme une stratégie thérapeutique prometteuse de réduction des déficiences post-AVC. Dans ce contexte, notre travail avait 2 objectifs : 1) chez le rat, identifier les cellules impliquées dans la surproduction de BDNF et évaluer la pertinence de la mesure des taux circulants de BDNF pour estimer les taux de BDNF présents dans le cerveau, 2) chez le patient victime d’un infarctus cérébral, étudier l’efficacité de la fluoxétine sur la récupération motrice à 3 mois, la fluoxétine étant un inhibiteur spécifique de la recapture de la sérotonine commercialisé comme antidépresseur et capable non seulement d’augmenter la production cérébrale de BDNF mais aussi de stimuler la plasticité post-lésionnelle.Les études précliniques ont été réalisées chez le rat soumis à l’embolisation unilatérale du cerveau par un nombre variable de microsphères (en carbone et calibrées à 50 µm) afin de reproduire le large panel de souffrance cérébrale rencontré en clinique. Le BDNF a été mesuré dans le cerveau et dans le sang (plasma et sérum par technique ELISA) avant et après (4, 24h et 8j) embolisation. Nos résultats montrent :- que la production de BDNF est plus intense et plus durable dans l’hémisphère embolisé que dans l’hémisphère non embolisé et que cette production est indépendante du degré d’embolisation, marqueur indirect de la souffrance cérébrale. - que les cellules non-neuronales deviennent une source non négligeable de BDNF en cas d’ischémie, notamment les cellules endothéliales et microgliales avant 24h et les astrocytes au temps 8j.- que les taux circulants et cérébraux de BDNF ne sont pas corrélés mais qu’il existe une corrélation entre le BDNF plasmatique mesuré au temps 4h et le degré d’embolisation.L’étude clinique correspond à un essai randomisé contrôlé en double aveugle comparant la fluoxétine (20mg/j, voie orale, pendant 3 mois et débutée entre 5 et 10j après les premiers symptômes) au placebo chez des patients présentant un déficit moteur modéré à sévère sur l’échelle motrice de Fugl-Meyer (n=59 dans chaque groupe). Nos résultats montrent que l’amélioration de la fonction motrice est meilleure sous fluoxétine que placebo. En conclusion, notre travail montre l’intérêt des médicaments capables d’augmenter le BDNF et la plasticité post-lésionnelle pour améliorer le pronostic clinique de l’AVC et identifie pour la première fois les cellules endothéliales cérébrales comme une cible potentielle de ces médicaments. Il remet également en cause l’idée largement répandue selon laquelle les taux circulants de BDNF varient dans le même sens que les taux cérébraux. / Functional recovery after ischemic stroke largely involves brain plasticity and more accurately its ability to reorganize the neuronal networks spared by the infarct. Studies conducted on animals using different ischemic stroke models have demonstrated that plastic changes are induced by BDNF (Brain-Derived Neurotrophic Factor). Hence, increasing levels of BDNF in the brain is considered a promising therapeutic strategy to reduce post-stroke impairments. In this context, our work had 2 aims: 1) In a rat model, to identify cells involved in the over-production of BDNF and to evaluate the pertinence of the measurement of circulating BDNF levels to estimate brain BDNF levels; 2) In ischemic stroke patients, to study the effectiveness of fluoxetin on 3-month motor recovery. This drug is a selective serotonin-reuptake inhibitor commercialized as an antidepressant treatment that is not only able to increase brain production of BDNF, but also to stimulate post-lesion plasticity. Animal studies were performed on rats that underwent unilateral embolization of the brain with various amounts of carbonized calibrated (50 µm) micropsheres in order to mimick the large panel of brain injury observed in humans. BDNF levels were measured in the brain and the blood (plasma and serum, ELISA method) before and after (4, 24h, and 8d) embolization. Our results show that:- The production of BDNF was more intense and longer lasting in the embolized than in the non-embolized hemisphere, and this production was independent of the degree of embolization, an indirect marker of brain injury.- Several non-neuronal cells become a non-negligible source of BDNF after ischemia, particularly endothelial cells and microglia before 24h, and astrocytes at 8d.- Brain and circulating levels of BDNF did not correlate, but a correlation between plasma BDNF at 4h and the degree of embolization was noted.Our clinical study was a randomized placebo-controlled trial that evaluated the efficacy of fluoxetine (20mg/d, oral route, over 3 months, and starting between 5 and 10d after stroke onset) in patients with moderate to severe motor impairment measured by the Fugl-Meyer motor scale (n=59 in each group). Our results showed a greater improvement in motor recovery under fluoxetin than placebo.To conclude, our work underlines the fact that treatments able to increase BDNF levels and post-lesion brain plasticity are of interest to improve the prognosis after stroke. We have shown, for the first time, that endothelial cells are a potential target for these treatments. Our study also calls into question the widespread idea according to which circulating levels of BDNF vary in the same way as levels of BDNF in the brain.

L'essor des thérapies régénératives au cours des 10 dernières années a entraîné un effort de recherche important, mais l'obtention des cellules souches humaines en quantité suffisante reste cependant encore problématique, notamment concernant les cellules souches mésenchymateuses (CSM). Ces travaux ont donc mis en œuvre une approche à la croisée de la biologie et du génie des procédés afin d'identifier les verrous limitant la croissance des CSM. L'étude des méthodes d'intensification de culture a été entreprise grâce à l'utilisation de microporteurs et d'une plateforme de minibioréacteurs de 200~mL. Puis le développement d'un milieu de culture sans sérum a été testé dans le but de maximiser la croissance cellulaire dans des conditions biochimiques contrôlées. Les CSM humaines en tant que modèle type en thérapie cellulaire ont été démontrées comme extrêmement sensibles aux phases de congélation/décongélation, aux variations de température, à un vieillissement prématuré et nécessitant un milieu de culture complexe riche en facteurs de croissance et d'adhérence. Suite à cette étude, plusieurs écueils pourront être évités lors de la montée en échelle d'un procédé de culture de CSM afin d'intégrer leurs paramètres biologiques intrinsèques aux paramètres d'ingénierie des bioréacteurs (transfert de chaleur, contraintes hydrodynamiques, surface d'adhérence) / Progress in regenerative medicines over the past ten years have led to an important research mobilisation, but obtaining a sufficient amount of human stem cells remains nonetheless problematic, especially for mesenchymal stem cells (MSC). Hence, this work developed an approach coupling biology and process engineering to identify barriers limiting MSC growth. The study of scaled-up amplification methods was performed using microcarriers and a 200~mL minibioreactors platform. In order to maximise MSC growth in a biochemically controlled environment, a serum free medium development was tested as well. Human MSC as model cell type for cellular therapies have thus been demonstrated as extremely sensitive to freeze/thaw cycles, temperature variations, subject to premature aging and needing a complex medium enriched in multiple growth and adherence factors. Following this study, several pitfalls might be avoided during MSC process scale-up by integrating the cells biology into the bioreactors' process engineering parameters (heat transfer, hydrodamic stress, adhesion surface)

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Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03 · 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.

Este estudo visou observar os efeitos plásticos induzidos pelo exercício a curto prazo em regiões motoras do encéfalo de ratos. Observou-se a expressão das subunidades GluR1 e GluR2/3. Os animais foram divididos em grupos de: 3 dias(COR3), 7 dias(COR7) e 15 dias(COR15); e um grupo controle(CONT). Empregaram-se as técnicas de imuno-histoquímica e immunoblotting. A expressão de GluR1 no cerebelo, demonstrou um decréscimo em COR3. No hipocampo houve uma queda na expressão em COR3(40%), retornando aos níveis basais em COR7. No córtex cerebral observou-se uma queda da expressão com máxima queda em COR7(52%), retornando à expressão basal em COR15. O estriado não sofreu alterações na expressão de GluR1 ao longo dos primeiros 7 dias, tendo um aumento em COR15(90%). A expressão de GluR2/3 não foi alterada, exceto no cerebelo, onde houve um decréscimo em dois momentos distintos, COR3(55%) e COR15(25%), retornando à expressão basal em COR7. Os nossos dados revelam que o exercício físico a curto prazo foi capaz de promover alterações plásticas ao longo do treinamento. / This study aimed at analyzing the plastic effects of the short-term exercise upon the rat motor area. We check the expression of GluR1 and GluR2/3. We divided into 3 experimental groups based on duration of exercise: 3 days(COR3), 7 days(COR7), and 15 days(COR15); and a control group(CONT). The experimental animals were subjected to a treadmill exercise protocol. The brains were subjected to the techniques of immunohistochemistry and immunoblotting. In the cerebellum, there was a decrease for COR3(17%). In the hippocampus, there was a decrease of the GluR1 expression for COR3 (40%). In the cerebral cortex there was a drop of GluR1 expression for COR3 and COR7(52%). In the striatum, there was no change of GluR1 expression during the first seven days, with a increase for COR15(90%). The GluR2/3 expression did not change in any brain structure analyzed, except in the cerebellum, where there was a significant decrease for two distinct groups, COR3(55%) and COR15(25%). Our data show that short-term physical exercise was able to promote plastic changes during training.

Submitted by Tony Rogerio de Lima Dadamos null (tonyrlrl@yahoo.com.br) on 2018-02-20T16:25:35Z No. of bitstreams: 1 Tese_AU_TRLD_2018.pdf: 2071412 bytes, checksum: ba59227c90cba94171f8548d07346f1a (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2018-02-21T19:00:37Z (GMT) No. of bitstreams: 1 dadamos_trl_dr_sjrp.pdf: 2071412 bytes, checksum: ba59227c90cba94171f8548d07346f1a (MD5) / Made available in DSpace on 2018-02-21T19:00:37Z (GMT). No. of bitstreams: 1 dadamos_trl_dr_sjrp.pdf: 2071412 bytes, checksum: ba59227c90cba94171f8548d07346f1a (MD5) Previous issue date: 2018-01-24 / Este trabalho apresenta metodologias de desenvolvimento, otimização, e validação de procedimentos de medição eletroquímica de parâmetros bioquímicos em fluidos biológicos. As ferramentas desenvolvidas foram aplicadas à medição voltamétrica, simples e de baixo custo de ácido úrico (AU) em soro humano que se apresenta como uma alternativa confiável às medições realizadas pelo método de referência espectrofométrico. Foi estudada a medição de AU no soro visto a ser utilizada no diagnóstico de várias doenças, tais como gota, doenças cardiovasculares e neoplasias. O eletrodo de trabalho modificado desenvolvido e otimizado é composto por 25 % de lignina, 60 % de nanografite, 15 % de óleo mineral e cobre metálico eletrodepositado. As medições foram realizadas por um método de adição cumulativa de padrão (MAP-C), recorrendo a um novo tratamento estatístico dos dados de calibração, que permite a calibração da instrumentação num pequeno volume de soro. A incerteza da medição foi estimada recorrendo a diversas abordagens bottom-up desenvolvidas e usando abordagens pragmáticas top-down apresentadas na bibliografia. As componentes de incerteza foram combinadas usando a Lei de Propagação de Incertezas ou os métodos numéricos de Kragten e Monte Carlo. Procedeu-se igualmente a uma avaliação bayesiana da incerteza da medição que envolve a atualização de dados da prevalência de ácido úrico na população com o resultado da medição do soro específico analisado. Os modelos metrológicos desenvolvidos foram implementados em folhas de cálculo MSExcel de fácil utilização pelo analista. As avaliações bottom-up da incerteza da medição recorreram a estimativas da incerteza de extrapolação produzidas pelo modelo de regressão ou usando simulações de Monte Carlo aplicáveis quando os pressupostos do modelo de regressão não são cumpridos. O método de adição cumulativa de padrão foi aplicado com sucesso à análise de soro humano pela adição de 1,0, 3,0, 5,0, 7,0 e 9,0 mg dL-1 de AU a 5 mL de soro. A adequação da qualidade das medições eletroquímicas foi avaliada comparando sua incerteza com um valor alvo de 0,56 mg dL-1 (uma oitava parte das faixas de AU de indivíduos saudáveis) e avaliado a compatibilidades das medições com determinações realizadas pelo procedimento de referência. As medições realizadas revelaram-se válidas e com menor custo do que as produzidas pelo método de referência. As ferramentas desenvolvidas para a construção e otimização de eletrodos de trabalho são aplicáveis à medição de outras espécies de análise e em outras matrizes. O método de adição cumulativa de padrão desenvolvido, bem como os modelos de medição respectivos desenvolvidos, são aplicáveis a qualquer tipo de medição instrumental não destrutiva de soluções. / This work presents methodologies for the development, optimization and validation of procedures for the electrochemical measurement of biochemical parameters in biological fluids. The developed tools were applied to voltammetric, simple and low-cost measurements of uric acid (AU) in human serum, which proved to be a reliable alternative to the spectrophotometric reference method. It was studied the measurement of AU in serum since it is used in the diagnosis of various diseases, such as gout, cardiovascular diseases and neoplasms. The developed and optimized modified working electrode is composed of 25 % lignin, 60 % nanocarbon, 15 % mineral oil and electrodeposited copper. The measurements were performed using a cumulative standard addition method (MAP-C), using a new statistical treatment of the calibration data, which allows the calibration of the instrumentation in a small volume of serum. The measurement uncertainty was estimated using developed bottom-up approaches and using pragmatic top-down approaches presented in the literature. The uncertainty components were combined using the Uncertainty Propagation Law or the numerical Kragten and Monte Carlo methods. A bayesian evaluation of the measurement uncertainty was performed, involving the updating of uric acid prevalence in the population with the result of the measurement of AU in the specific serum sample. The developed metrological models were implemented in user-friendly MS-Excel spreadsheets. The bottom-up assessments of measurements uncertainty involve the estimation of the extrapolation uncertainty from the regression model or using Monte Carlo simulations applicable to when the assumptions of the regression model are not valid. The cumulative standard addition method was successfully applied to the analysis of human serum by adding 1.0, 3.0, 5.0, 7.0 and 9.0 mg dL-1 of AU to 5 mL of serum. The adequacy of the electrochemical measurements was assessed by comparing their uncertainty with a target value of 0.56 mg dL-1 (one-eighth of the range of AU in healthy individuals) and by evaluating the compatibility of measurements with determinations performed by the reference procedure. The performed measurements are valid and significantly cheaper than those produced by the reference method. The tools developed for the construction and optimization of working electrodes are applicable to the measurement of other analytes and matrices. The developed cumulative standard addition method and respective measurement models, are applicable to any kind of non-destructive chemical measurement of a solution.

Made available in DSpace on 2015-04-17T15:02:51Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 677279 bytes, checksum: 5d3ab2a60a6e82e4a31371e12aa51b6d (MD5) Previous issue date: 2011-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The senior population represents today a significant share in most countries, being susceptible to not transferable chronic diseases that may be associated with vitamin A status and the presence of inflammation. The aim of this study was therefore to verify the association of serum retinol, C-reactive protein and total fiber intake with hypertension in the elderly in the city of Joao Pessoa. This is a cross-sectional epidemiological research, population-based, with 212 individuals between 60 to 90 years old, both sexes, with or without chronic diseases and use of antihypertensive medication or not. The dietary survey was conducted by the Quantitative Food Frequency Questionnaire, retinol serum by liquid chromatography high resolution and the CRP ultrasensitive (hs-CRP) by particle agglutination by latex; for statistical analysis was applied Fisher Exact Test, which was adopted a significance level of 5% to reject the null hypothesis. The prevalence of hypertension alone was 48.08% and other morbidities associated with hypertension was 51.92%, 97.85% of elderly hypertensives were using hypotensive and had adequate levels of serum retinol (&#8805; 1.05 mmol / L), 89.13% had adequate levels of CRP (<8.5 mg / dL for females and <7.9 mg / dL for males) and 96.77% had inadequate intake of fiber (&#8805; 30 g / day for men and &#8805; 21g/dia for females). There was no significant relationship between serum retinol and hypertension (p = 0.1661) or between CRP and hypertension (p = 0.557). There was a significant association between inadequate intake of fiber and hypertension (p = 0.0035), suggesting for the first time in the elderly of different socioeconomic levels, based on the literature, that this group of hypertensive patients should be informed about the positive function of adequated consumption of fiber and it may helps the treatment of hypertension and thus reduce the amount of hypotensive drugs used by this population. We conclude that, even considering the inadequate consumption of fiber, most of the elderly hypertensive population showed serum retinol and CRP-us appropriate, probably, these values did not rise because it is the elderly hypertensive patients without acute inflammation or other chronic decompensated diseases. / A população idosa representa hoje uma parcela significativa na maioria dos países, sendo suscetível às doenças crônicas não transmissíveis que pode estar associada ao estado vitamínico A e à presença de inflamação. O objetivo deste estudo foi, portanto, verificar a associação do retinol sérico, proteína C-reativa e consumo de fibras totais com hipertensão arterial, em idosos do município de João Pessoa. Trata-se de uma pesquisa epidemiológica transversal, de base populacional, com 212 indivíduos entre 60 a 90 anos de idade, de ambos os sexos, portadores ou não de doenças crônicas e em uso ou não de medicação hipotensora. O inquérito alimentar foi realizado por meio do Questionário Quantitativo de Frequência do Consumo Alimentar, o retinol sérico por cromatografia líquida de alta resolução e a proteína C reativa ultra-sensível (PCR-us) por aglutinação de partículas por látex; para a análise estatística foi aplicado o Teste Exato de Fisher, na qual adotou-se nível de significância de 5% para rejeição da hipótese de nulidade. A prevalência de hipertensão isolada foi de 48,08% e hipertensão associada a outras morbidades 51,92%; 97,85% dos idosos hipertensos e que faziam uso de hipotensores, apresentavam níveis adequados de retinol sérico (&#8805;1,05 &#956;mol/L), 89,13% apresentavam valores de PCR adequada (< 8,5 mg/dL para o sexo feminino e < 7,9 mg/dL para o sexo masculino) e 96,77% apresentavam consumo inadequado de fibras (&#8805;30g/dia para sexo masculino e &#8805; 21g/dia para sexo feminino). Não houve relação significativa entre valores séricos de retinol e hipertensão (p=0,1661) ou entre valores de PCR e hipertensão (p=0,557). Houve associação significativa entre o consumo inadequado de fibras e hipertensão (p=0,0035), sugerindo pela primeira vez em idosos de diferentes níveis socioeconômicos, com base na literatura consultada, que esse grupo de hipertensos deveria ser esclarecido quanto à função positiva do consumo adequado de fibras, o que auxiliaria o tratamento da hipertensão e consequentemente reduziria a quantidade de hipotensores utilizados por esta população. Conclui-se que mesmo considerando o consumo inadequado de fibras, a população idosa hipertensa apresentou, em sua maioria, valores séricos de retinol e de PCR-us adequados, provavelmente, esses valores não se elevaram por tratar-se de idosos hipertensos sem quadro agudo de inflamação ou de outras doenças crônicas descompensadas.