Les travaux de cette thèse sont menés dans le cadre d’un projet porté par le centre de recherche LGI2P, (IMT Mines Ales). L’objectif final de ce projet vise à permettre le retour à l’emploi et à améliorer le quotidien des personnes malvoyantes atteintes de rétinite pigmentaire et de glaucome. Le dispositif final est conçu pour aider les personnes dans leurs activités en mobilité : détection d’obstacles, recherche d’indices visuels, en adressant les problèmes liés à l’éblouissement et à l’héméralopie dont souffrent ces utilisateurs potentiels.La contribution de cette thèse au projet se situe sur plusieurs plans. Tout d’abord, il était demandé de définir les paramètres caractérisant la vision résiduelle de chaque utilisateur. En effet, chaque patient a ses propres conditions de confort lumineux, qui dépendent en particulier de l’état d’avancement de sa pathologie : à chaque étape de l’évolution de leur maladie, les patients ont des limites spécifiques de luminosité minimale en dessous de laquelle ils ne perçoivent plus les détails dans une scène, et aussi de la luminosité maximale au-dessus de laquelle ils ressentent gêne et douleur. La définition de ces limitations en luminosité va permettre de paramétrer le dispositif et l’adapter à chaque utilisateur. Mais il n’existe pas de méthode pour mesurer ces niveaux de luminance limite. Nous avons donc participé à la conception et au développement de tests dédiés, et à la réalisation d’essais sur des sujets déficients visuels, dans le cadre d’un essai clinique piloté par le CHU de Nîmes et l’ARAMAV (institut spécialisé dans la rééducation fonctionnelle basse vision), pour l’aspect médical. Nous avons également proposé un nouveau test pour mesurer la sensibilité au contraste chromatique, toujours dans le but de mieux adapter les images affichées à la vision des utilisateurs.Nous avons ensuite travaillé sur la mise au point d’un prototype du dispositif (caméras et visiocasque de réalité virtuelle). Pour cela, nous avons dû choisir les équipements de capture et d’affichage d’images. Un travail de calibration colorimétrique sur ces équipements nous permet de relier grandeurs numériques (code RGB) et grandeurs physiques (luminance et chrominance). Cette étape est nécessaire pour réaliser les tests précités dans des conditions physiquement connues. Elle nous permet également de définir les caractéristiques physiques que devront posséder les équipements qui seront choisis pour réaliser le produit final, s’ils sont différents de ceux utilisés pendant nos travaux.Enfin, nous avons abordé la question des traitements à appliquer au signal capturé par la caméra. Nous avons proposé des traitements en temps réel sur la luminosité dans le but d’augmenter la luminosité dans les zones sombres de l’image et de baisser la luminosité dans les zones qui éblouissent le patient. Nous avons montré les limitations de l’imagerie classique et la nécessité de travailler sur des images HDR (high dynamic range) Nous avons comparé plusieurs méthodes pour permettre l’affichage de ces images HDR sur les écrans de plus faible dynamique, en recherchant les caractéristiques de l’image que ces méthodes doivent préserver au mieux, et en prenant en compte les performances visuelles des utilisateurs potentiels. Nous avons aussi proposé des traitements sur la couleur en augmentant le contraste et la saturation pour rendre les images mieux perceptibles par les patients qui souffrent de troubles de vision des couleurs. / This thesis is part of a project conducted by the LGI2P research center (IMT Mines Ales). The project's final aim is to help people with vision disorders suffering from retinitis pigmentosa and glaucoma get back to work and improve their daily lives. The final device is designed to help people in their mobility activities: detecting obstacles, searching for visual signals, by addressing problems related to dazzling and haemeralopia affecting these potential users.The research of this thesis has several contributions to the project. First of all, parameters characterizing the residual vision of each user had to be defined. Indeed, each patient has his own light comfort conditions, which depend in particular on his pathology's progress: at each stage of the evolution of their disease, patients have specific minimum luminosity limits below which they no longer perceive the details in a scene, and also on the maximum luminosity above which they feel discomfort and pain. The definition of these limitations in luminosity will make it possible to parameterize the device and adapt it to each user. But there is no method to measure these limiting luminance levels. We have therefore participated in the design and development of specialized tests, and in the conduct of trials on visually impaired subjects, as part of a clinical trial led by the Nîmes University Hospital and the ARAMAV (institute specializing in low vision functional rehabilitation), for medical research. We have also proposed a new test to measure sensitivity to chromatic contrast, always with the aim of better adjusting the images displayed to users' vision.Then, we developed a prototype of the device (cameras and virtual reality video headset). In order to achieve these results, we had to choose the image capture and display equipment. A colorimetric calibration work on these equipments allowed us to link digital quantities (RGB code) and physical quantities (luminance and chrominance). This stage is required to perform the above tests under physically known conditions. It also allowed us to define the physical characteristics of the equipment that would be selected to produce the final product, whether they are different from those required during our work.Finally, we discussed the processing to be applied to the signal captured by the camera. We have proposed real-time brightness treatments to increase brightness in dark areas of the image and decrease brightness in areas that dazzle the patient. We have presented the limitations of conventional imaging and the necessity to work on HDR (high dynamic range) images. We have compared several methods to allow the display of these HDR images on screens with lower dynamic range, looking for the image characteristics that these methods should better preserve, and taking into consideration the visual performance of potential users. We have also suggested color treatments by increasing contrast and saturation to make images more perceptible to patients with color vision disorders.

People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing. To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries. The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.

Nanopartikel basierte colorimetrische Assays bieten vielfältige Anwendungsmöglichkeiten vom Nachweis ionischer oder molekularer Spezies bis hin zu komplexen Strukturen, wie zum Beispiel Zellen oder Bakterien. Trotz der großen Anzahl von Anwendungsbeispielen in der Literatur gibt es nur sehr wenige Beispiele für den Fall, wenn Analyt und Sensorpartikel etwa gleich groß sind. Jedoch nimmt das Interesse an dem Nachweis solcher Analyten, wie zum Beispiel von Viren, in der Bioverfahrenstechnik, der Umwelttechnik und Medizin stetig zu. In der Dissertation wird anhand von drei unterschiedlichen Anwendungsbeispielen die lückenlose Anwendbarkeit des Nanopartikel basierten colorimetrischen Sensorprinzips für verschiedene Analyten aus dem gesamten interessierenden Größenspektrum nachgewiesen. Für die Detektion von Ionen bzw. Ionenkomplexen ist mit der Funktionalisierung der Goldnanopartikel mit bakteriellen Zellhüllenproteinen ein spezifisches Nachweisverfahren für spezielle Ionenkomplexe entwickelt worden. Erste Ergebnisse zum Nachweis von toxischen Arsen(V)-Komplexen in wässriger Lösung belegen das enorme Potential. Als Nachweisgrenze konnten Werte im Bereich herkömmlicher sensitiver Messverfahren ermittelt werden. Durch Untersuchung der Wechselwirkung von Au- und AuxAg1-x–Nanopartikeln mit dendritischen Polymerstrukturen mit Abmessungen im Größenbereich zwischen 2 und 5 nm konnten tiefere Einblicke in das Aggregationsverhalten der Partikel bei der Ausbildung größerer komplexer Strukturen erzielt werden, die eine sensorische Umsetzung ermöglichen. Derartige Betrachtungen existieren bisher nur für Ionen oder kleinere molekulare Spezies. Der Nachweis von Analyten mit zu den Nanopartikeln vergleichbarer Größe wird am Beispiel zweier unterschiedlicher viraler Proben, dem Rinder-Coronavirus (BCV) und einem humanen Influenzavirus (H3N2) in grundlegenden Experimenten demonstriert. Die Durchführung dieser Arbeiten mit prozessnahem Virusprobenmaterial ist mit besonderen Herausforderungen an die Signalgewinnung und Interpretation verbunden. Aufbauend auf diesen experimentellen Ergebnissen konnte in einer skalenübergreifenden Betrachtung für Analytgrößen vom Subnanometerbereich bis hin zu einigen hundert Nanometern die lückenlose, universelle Anwendbarkeit des colorimetrischen Sensorprinzips demonstriert werden. Diese Betrachtungen ermöglichen generelle Aussagen zur Lage des Farbumschlagbereiches sowohl in Abhängigkeit von der Analytgröße als auch der Funktionalisierung, der Konzentration und der Größe der Nanopartikel.

Submitted by Fred Willians Calonego null (fwcalonego@ig.com.br) on 2017-12-20T15:47:02Z No. of bitstreams: 1 Calonego FW_Doutorado 2017.pdf: 5105344 bytes, checksum: b332f3457105ae9be3d54a958181484a (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2017-12-20T16:13:09Z (GMT) No. of bitstreams: 1 calonego_fw_dr_bot.pdf: 5105344 bytes, checksum: b332f3457105ae9be3d54a958181484a (MD5) / Made available in DSpace on 2017-12-20T16:13:09Z (GMT). No. of bitstreams: 1 calonego_fw_dr_bot.pdf: 5105344 bytes, checksum: b332f3457105ae9be3d54a958181484a (MD5) Previous issue date: 2017-11-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No Brasil, a espécie pioneira Schizolobium parahyba (Vell.) Blake, conhecida como Guapuruvu, tem potencial para os projetos de restauração ecológica da Floresta Atlântica, e quando atingem cerca de 15 anos podem ser retiradas mediante plano de manejo. Entretanto, uma característica indesejável da sua madeira é a presença acentuada de lenho juvenil e uma das suas limitações é a baixa durabilidade natural. Uma forma de minimizar este problema é a modificação térmica logo após a sua secagem. Porém, esses tratamentos provocam mudanças na cor da madeira. Assim, o objetivo desse estudo foi avaliar o efeito do tratamento de modificação térmica nas propriedades físicas, químicas e colorimétricas e na resistência dos lenhos juvenil e adulto de S. parahyba aos organismos xilófagos e ao intemperismo. Para tanto, foram usadas tábuas provenientes de toras de Guapuruvu, com cerca de 15 anos de idade, retiradas de uma área de recuperação florestal da Faculdade de Ciências Agronômicas, da UNESP, de Botucatu-SP. Cada tábua foi seccionada de modo a fornecer peças controle e outras destinadas para os tratamentos de modificação térmica, com temperaturas finais de 180ºC, 200ºC e 220ºC. Corpos de prova foram retirados para a caracterização tecnológica dos lenhos juvenil e adulto através dos ensaios de propriedades físicas, químicas, de caracterização colorimétrica da madeira e de resistências aos fungos de podridão parda Gloeophyllum trabeum e de podridão branca Pycnoporus sanguineus, ao cupim de madeira seca Cryptotermes brevis e ao intemperismo. Os resultados mostraram que: (1) o aumento da temperatura de modificação térmica promove decréscimos significativos de até 9,1% na massa específica aparente a 21ºC e 65% de umidade relativa da madeira de S. parahyba, e reduz em até 51,1% e 54,1% os seus respectivos teores de umidade de equilíbrio e inchamento volumétrico; (2) a massa específica básica e os coeficientes de retratibilidades não variam com os tratamentos térmicos e não são indicados para avaliar a qualidade da madeira modificada termicamente; (3) a modificação térmica provoca reduções significativas de até 25,0% nos teores de holocelulose e um aumento proporcional de até 41,7% e 286,5% nos respectivos teores de lignina e de extrativos totais da madeira; (4) a cor da madeira sem tratamento é classificada como branco amarelada, pois apresenta alta claridade (L* de 80,41 a 80,85) e presença marcante do pigmento amarelo (b* de 19,06 a 20,07), e o tratamento térmico provoca o seu escurecimento e avermelhamento; (5) a madeira sem tratamento é classificada como não resistente ao cupim de madeira seca e ao fungo de podridão parda, e, de resistência moderada ao fungo de podridão branca, sendo que os tratamentos térmicos provocam reduções significativas de até 90,6% nas perdas de massa provocadas pelos fungos apodrecedores e não mudam a classe de resistência da madeira aos cupins; e (6) a madeira sem tratamento ficou mais escurecida, avermelhada e amarelada após o intemperismo, e o tratamento térmico aumenta a sua resistência aos agentes abióticos. Pode-se concluir que a modificação térmica possui um grande potencial para melhorar a estabilidade dimensional e a resistência natural da madeira de S. parahyba aos fungos apodrecedores e ao intemperismo, embora, modifique a sua cor e não altera a sua classe de resistência aos cupins. / In Brazil, the pioneer plant species Schizolobium parahyba (Vell.) Blake, known as Guapuruvu, has potential for ecological restoration projects in the Atlantic Forest, and when they reach about 15 years can be taken by the management plan. However, an undesirable feature of this wood is the pronunced presence of juvenile wood and a limitation is low biological resistance. An attractive way to mitigate this problem is this thermal modification process after its drying. However, these thermal treatments cause changes in color of wood. The aim of this study was to evaluate the effect of thermal modification on physicochemical and colorimetric properties and biological resistance of juvenile and mature woods from S. parahyba to xylophagous organisms and weathering. Boards were taken from a 15-year-old S. parahyba forest recovery area of Agronomy Sciences College from UNESP, located in Botucatu, SP, Brazil. Each board was sawed into smaller pieces measuring 0.60 m in length. One small piece was kept in its original condition (untreated wood), whereas the other pieces were reserved for the thermal treatments at final temperature of 180ºC, 200ºC, and 220ºC. Subsequently, samples were cut from all the boards (untreated and thermally modified woods) to technological characterization for both juvenile and mature wood across to tests of physical and chemical properties, colorimetric characterization of wood, decay resistance at brown-rot fungus Gloeophyllum trabeum and white-rot fungus Pycnoporus sanguineus, biological resistance to térmite dry wood Cryptotermes brevis, and weathering resistance. The results showed that: (1) the increase in the thermal modification temperature causes significant decreases of up to 9,1% in the density at 21ºC and 65% relative humidity of the S. parahyba wood and reduces the equilibrium moisture content and volumetric swellings in up to 51.1% and 54.1%, respectively; (2) the density basic and dimensional change coefficient of wood were not the most suitable for evaluating the quality of thermally modified wood; (3) the thermal modification causes significant decreases of up to 25.0% in the holocelulloses contents, an proportional increase of up to 41.7% e 286.5% in the lignin and extractives contents, respectively; (4) the color of untreated wood can be classified by yellowish white, because presents high brightness (L* of 80.41 to 80.85) and strong presence of yellow pigment (b* of 19.06 to 20.07), and the thermal treatment causes the darkening and reddening of timber; (5) the untreated wood was classified as non-durable to the dry wood termites and brown-rot fungus, and also was classified as moderate resistance to the white-rot fungus, whereas the thermal modification promotes significant reductions of up to 90.6% in the weight losses caused by rot fungi and do not change the damage caused by termites in wood; and (6) the untreated wood showed darkening, a reddish behavior and a yellowish behavior after weathering tests, and the thermal modification causes significant decreases in the weathering of wood. We concluded that the thermal modification has a great potential to improve the dimensional stability and the natural resistance of S. parahyba wood to decay fungi and weathering, although it modifies the color of wood and does not change its resistance class to the termite.

Cavity enhanced spectroscopies (CES) are currently amongst the most sensitive spectroscopic techniques available for probing gas-phase samples, however their application to the liquid-phase has been more limited. Sensitive analysis of submicrolitre liquid samples is highly desirable, as miniaturisation allows for the reaction and analysis of scarce or expensive reagents, produces less waste, and can increase the speed of separations and reactions, whilst having a small footprint and high throughput. Absorption spectroscopy is a particularly desirable technique due to its universal, label-free nature, however its application to small volume liquid samples is hampered by the associated short absorption pathlengths, which limit sensitivity. CES improve sensitivity by trapping light within a confined region, increasing the effective pathlength through the sample. Three distinct types of optical cavity were constructed and evaluated for the purposes of making optical absorption measurements on liquid samples. The first incorporated a high optical quality flow cell into a "macrocavity" formed from two dielectric mirrors separated by 51.3 cm. Cavity losses were minimised by positioning the flow cell at Brewster's angle to the optical axis, and the setup was used to perform a single-wavelength cavity ringdown spectroscopy experiment to detect and quantify nitrite within aqueous samples. The detection limit was determined to be 8.83 nM nitrite in an illuminated volume of only 74.6 nL. Scattering and reflective losses from the flow cell surfaces were found to be the largest barrier to increased sensitivity, leading us to focus on the integration of cavity mirrors within a microfluidic flow system in the work that followed. In the second set of experiments, cavity enhanced absorption spectroscopy (CEAS) measurements were performed on Thymol Blue using custom-made microfluidic chips with integrated cavity mirrors. Unfortunately, due to the plane-parallel configuration of the mirrors and the corresponding difficulty in sustaining stable cavity modes, the results were underwhelming, with a maximum cavity enhancement factor (CEF) of only 2.68. At this point, attention was focussed toward a more well-defined cavity geometry: open-access plano-concave microcavities. The microcavities consist of an array of micron-scale concave mirrors opposed by a planar mirror, with a pathlength that is tunable to sub-nanometer precision using piezoelectric actuators. In contrast to the other experimental setups described, themicrocavities allow for optical measurements to be performed in which we monitor the change of wavelength and/or amplitude of a single well-defined cavity mode in response to a liquid sample introduced between the mirrors. In the first microcavity experiment, we used 10 μm diameter mirrors with cavity lengths from 2.238 μm to 10.318 μm to demonstrate refractive index sensing in glucose solutions with a limit of detection of 3.5 x 10^-4 RIU. The total volume of detection in our setup was 54 fL. Thus, at the limit of detection, the setup can detect the change of refractive index that results from the introduction of 900 zeptomoles (500,000 molecules) of glucose into the device. The microcavity sensor was then adapted to enable broadband absorption measurements of methylene blue via CEAS. By recording data simultaneously from multiple cavities of differing lengths, absorption data is obtained at a number of wavelengths. Using 10 μm diameter mirrors with cavity pathlengths from 476 nm to 728 nm, a limit of detection, expressed as minimum detectable absorption per unit pathlength, of 1.71 cm^-1 was achieved within a volume of 580 attolitres, corresponding to less than 2000 molecules within the mode volume of the cavity. Finally, a new prototype was developed with improved cavity finesse, a much more intense and stable light source, and improved flow design. Using a single plano-concave microcavity within the array with a cavity pathlength of 839.7 nm, and 4 μm radius of curvature mirror, absorption measurements were performed on Methylene Blue. Analysis of this data indicated a CEF of around 9270, and a limit of detection based on the measured signal-to-noise ratio of 0.0146 cm^-1. This corresponds to a minimum detectable concentration of 104 nM Methylene Blue, which given the mode volume of 219 aL, suggests a theoretical minimum detectable number of molecules of 14.

Le travail présenté dans ce mémoire a pour cadre le dispositif de réalité mixte ray-on, conçu par la société on-situ. Ce dispositif, dédié à la mise en valeur du patrimoine architectural et en particulier d'édifices historiques, est installé sur le lieu de l'édifice et propose à l'utilisateur une vision uchronique de celui-ci. Le parti pris étant celui du photo-réalisme, deux pistes ont été suivies : l'amélioration du mélange réel virtuel par la reproduction de l'éclairage réel sur les objets virtuels, et la mise en place d'une méthode de segmentation d'image résiliente aux changements lumineux.Pour la reproduction de l'éclairage, une méthode de rendu basé-image est utilisée et associée à une capture haute dynamique de l'environnement lumineux. Une attention particulière est portée pour que ces deux phases soient justes photométriquement et colorimétriquement. Pour évaluer la qualité de la chaîne de reproduction de l'éclairage, une scène test constituée d'une mire de couleur calibrée est mise en place, et capturée sous de multiples éclairages par un couple de caméra, l'une capturant une image de la mire, l'autre une image de l'environnement lumineux. L'image réelle est alors comparée au rendu virtuel de la même scène, éclairée par cette seconde image.La segmentation résiliente aux changements lumineux a été développée à partir d'une classe d'algorithmes de segmentation globale de l'image, considérant celle-ci comme un graphe où trouver la coupe minimale séparant l'arrière plan et l'avant plan. L'intervention manuelle nécessaire à ces algorithmes a été remplacée par une pré-segmentation de moindre qualité à partir d'une carte de profondeur, cette pré-segmentation étant alors utilisée comme une graîne pour la segmentation finale. / The work described in this report has as a target the mixed reality device ray-on, developed by the on-situ company. This device, dedicated to cultural heritage and specifically architectural heritage, is meant to be installed on-site and shows the user an uchronic view of its surroundings. As the chosen stance is to display photo-realistic images, two trails were followed: the improvement of the real-virtual merging by reproducing accurately the real lighting on the virtual objects, and the development of a real-time segmentation method which is resilient to lighting changes.Regarding lighting reproduction, an image-based rendering method is used in addition to a high dynamic range capture of the lighting environment. The emphasis is put on the photometric and colorimetric correctness of these two steps. To measure the quality of the lighting reproduction chain, a test scene is set up with a calibrated color checker, captured by a camera while another camera is grabbing the lighting environment. The image of the real scene is then compared to the simulation of the same scene, enlightened by the light probe.Segmentation resilient to lighting changes is developed from a set of global image segmentation methods, which consider an image as a graph where a cut of minimal energy has to be found between the foreground and the background. These methods being semi-automatic, the manual part is replaced by a low resolution pre-segmentation based on the depthmap of the scene which is used as a seed for the final segmentation.

Cette thèse traite un sujet pluridisciplinaire, la conception d’un prototype de micro-capteur biologique pour la détection des anticorps de patients susceptibles d'être allergiques.Elle a pour objectif la miniaturisation de la méthode ELISA pour « Enzyme Linked Immune Sorbent Assay » en concevant en « full-custom », avec une technologie CMOS APS, un capteur colorimétrique. Ce capteur est posé en dessous un système micro fluidique contenant l’échantillon biologique à tester, le tout est illuminé via une fibre optique. Le détecteur capte la lumière qui a traversé le micro-tube contenant l’échantillon. Les courants photoniques induits sont liés à la concentration et la coloration de la solution. Le virage colorimétrique de la réaction enzymatique, due à la présence des anticorps dans le sérum, et l’évaluation quantitative de la concentration seront déterminée par la mesure de ces photo-courants. Le capteur pourrait contenir une matrice de 20x20 pixels de détecteurs de couleur ainsi que leur électronique de lecture et de commande. Pour des raisons de coûts, nous avons validé le procédé à l’aide d’une matrice de 4x4 pixels de détecteur de couleur. La réalisation du circuit a été suivie par une caractérisation électrique et colorimétrique. La caractérisation électrique a permis de valider le fonctionnement du bloc de commande du circuit ainsi que celui du pixel (l’électronique de lecture, BDJ). Les résultats de mesures concordent avec ceux de simulations. La caractérisation colorimétrique consiste à mesurer le virage colorimétrique de deux solutions différentes. Les mesures ont pu montrer que notre capteur est plus sensible que le spectromètre utilisé pour mesurer la concentration des deux solutions. Ainsi ce travail de recherche a contribué à la miniaturisation d’une bio-puce colorimétrique dédiée aux tests immunologiques basée sur la méthode ELISA. / This PhD treats a multidisciplinary subject based on the design of a biological micro-sensor prototype for the detection of antibodies of patients susceptible to be allergic.The goal has been the miniaturization of the ELISA "Enzyme Linked Immune Sorbent Assay" method, designing in integrated full-custom colorimetric sensor with a CMOS APS technology. This sensor is installed below a microfluidic system containing the biological test sample, the whole is illuminated via an optical fiber. The sensor detects the light passed through the micro reservoir containing the sample, the induced photo-currents is related to the concentration of the solution. The color change of the enzyme reaction due to the presence of antibodies in the serum, and the quantitative evaluation of the concentration will be determined by the measurement of the induced photo-currents. The sensor may contain a matrix of 20x20 color detector pixels and their reading and control electronics. For cost reasons, we validated the method using a matrix of 4 x 4 pixels of color detectors.The design of electrical device was followed by a colorimetric and electrical characterizations. The latter was used to validate the operation of the control block of the circuit as well as that of the pixel (readout electronics, BDJ). The results brought by measurements are in good agreement with those obtained through simulations. The colorimetric characterization consists in measuring the intensity of the color of two different solutions of different colors. These measurements have shown that our sensor is more sensitive than a spectrometer. Therefore, this research work has contributed to the miniaturization of a colorimetric sensor and its electronic part for the Immunoassay based on the ELISA method.

L’objectif de cette thèse, dans le cadre de l'ANR TRICETOX (CESA, 2013-2017), était de développer des méthodes alternatives aux méthodes d’analyse chimiques classiques pour la détection spécifique des herbicides de la famille des β-tricétones. Ces nouveaux outils analytiques doiventt être faciles à utiliser, rapides, peu coûteux et devant répondre aux exigences imposées pour la détection de ces herbicides (valeur critique de 0,1 µg.L-1). Les β-tricétones ont pour cible spécifique l'enzyme p-HydroxyPhénylPyruvate Dioxygénase (HPPD), impliquée dans la biosynthèse des caroténoïdes, son inhibition entraîne un blanchiment des feuilles des plantes traitées. Deux stratégies ont été envisagées, basées sur l’inhibition de l’HPPD : un bio-essai colorimétrique à cellules entières utilisant un clone recombinant d'Escherichia coli exprimant l’HPPD d’Arabidopsis thaliana, et un biocapteur enzymatique à transduction électrochimique. Le principe du bio-essai repose sur la capacité du clone recombinant à produire un pigment brun à partir de la voie de catabolisme de la tyrosine. L’addition de β-tricétones entraîne une diminution de la pigmentation dans le milieu. Après optimisation des conditions opératoires, les LODs obtenues pour la mésotrione, la tembotrione, la sulcotrione et la leptospermone sont de 0,069, 0,051, 0,038 et 20 μM, respectivement. En format biocapteur, l'enzyme HPPD purifiée a été immobilisé sur la surface d’une électrode de graphite sérigraphiée. Le biocapteur à enzyme libre a permis l’obtention de limites de détection de 1,36-10 M, 1,25.10-11 M et 1,08.10-11 M pour la sulcotrione, la tembotrione et la mésotrione, respectivement. A ce jour, des échantillons réels ont été analysés sur le bio-essai et les résultats ont été validés par HPLC. / The goal of this thesis, in the frame of the ANR TRICETOX (CESA, 2013-2017), was to develop some alternative methods to conventional chromatographic methods for the monitoring of the β-triketone herbicides. These new analytical tools have to be easy to handle, fast, low-cost, and to respond to the requirements for the detection of these herbicides (critical value of 0.1 μg.L-1). The β-triketones specifically target the 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme, involved in the carotenoids biosynthesis in plants ; its inhibition results in foliage bleaching of the treated plant. Two strategies have been considered, both based on the HPPD inhibition : a whole-cell colorimetric bioassay involving a recombinant E. coli expressing the HPPD from A. thaliana, and an electrochemical enzymatic biosensor. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble brown pigment from tyrosine catabolism. The addition of β-triketone induces the decrease of the pigment production in the medium. After optimization of the operational conditions, the LODs obtained for mesotrione, tembotrione, sulcotrione, and leptospermone were 0.069, 0.051, 0.038, and 20 μM, respectively. In the biosensor format, the purified HPPD enzyme was immobilized on the surface of a screen-printed graphite electrode. Using the free enzyme biosensor, LODs as low as 1.36-10 M, 1.25.10-11 M and 1.08.10-11 M were obtained for sulcotrione, mesotrione and tembotrione, respectively. Up to date, real samples have been analyzed using the bioassay and the results were validated by HPLC.

The one-humped or the dromedarian camel is a pseudo-ruminant mammal, well adapted to the hot and dry climates of the desert. Its ability to withstand torrid heat and extreme desiccation is of paramount importance to its survival. The studies presented in this thesis were designed to investigate and document the effect of dehydration in the presence or absence of angiotensin II (Ang II) AT1 receptor blocker (losartan) on blood constituents, electrolytes, hormones, neurotransmitters as well as liver and kidney enzymes in a subset of dehydrated camels and to compare them with hydrated camels. Additionally, we studied the response of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) and revealed for the first time the cardiac storage form of BNP in the camel heart. Dehydration induced significant increments in packed cell volume (PCV), white blood cells (WBC), gamma glutamyl-transferase (GGT), serum sodium, creatinine and urea levels, and a doubling in plasma cortisol and arginine vasopressin (AVP) levels. At the same time dehydration caused significant decrease in body weights, plasma insulin like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), and a 50% decrement in ANP and BNP levels. Moreover, dehydration with and without losartan resulted in significant changes in stress hormones and anti-oxidants in plasma, liver and kidney homogenates. Losartan on one hand enhanced the effect of dehydration resulting in significant increases in sodium, creatinine and urea levels. In addition losartan raised the  binding affinity of Ang II AT2 receptors in the small intestine with 8-fold and with 16-fold for liver AT1 receptors, indicating that Ang II AT1 and AT2 receptor binding sites were present in camel's small intestine while only AT1 receptor binding sites were found in the camel liver. One the other hand losartan resulted in significant decrease in body weights impaired the rise in anti-diuretic hormone and reduced aldosterone level. Finally, we showed that the proBNP is the storage form of BNP in the camel heart.

Toxoplasma gondii é um protozoário pertencente ao filo Apicomplexa, de distribuição mundial, e que infecta diversas espécies hospedeiros, como mamíferos e aves, possuindo como hospedeiros definitivos o gato e outros felídeos, enquanto o homem e outros animais são os seus hospedeiros intermediários. O tratamento é feito, na maioria das vezes, com uma combinação de sulfadiazina e pirimetamina, agindo na via metabólica do ácido fólico, o qual é necessário para a biossíntese de purinas, pirimidinas e certos aminoácidos. Propusemos estabelecer um protocolo de avaliação sobre esse protozoário, assim como padronizarmos uma metodologia por espectroscopia para uma rápida avaliação ou triagem de substâncias potencialmente ativas contra o parasito. Verificamos a atividade das substâncias ácido ursólico e ácido oleanóico, as quais já demonstraram atividade biológica sobre outras espécies de protozoários, como Plasmodium, Trypanosoma cruzi e Leishmania sp. em sistemas in vitro e in vivo. A metodologia colorimétrica pelo MTT, pelo Alamar Blue®, do kit CyQUANT® NF e a contagem manual em meio líquido das formas taquizoítas do parasito em ensaios biológicos in vitro mostram-se inviáveis, pois há grande dificuldade em manter a integridade do parasita em meio de cultura líquido, o qual se mostra sensível à adição de qualquer outro componente que não aqueles necessários para manter sua viabilidade em ambiente extracelular. O ácido ursólico mostrou-se potencialmente ativo in vitro sobre formas intracelulares de T. gondii. Entretanto, o contato de células infectadas com as substâncias avaliadas por um período de 48 horas não resultou em diminuição mais acentuada na porcentagem de células infectadas do que a ocorrida no tratamento de 24 horas. A comparação dos resultados do tratamento pós infecção celular por 24 horas e do pré-tratamento das formas taquizoítas houve diferenças significativas, indicando principalmente maior ação do pré-tratamento sobre T. gondii. Quando administrado na dose de 7 mg/kg/ dia a camundongos infectados, o ácido ursólico não apresentou atividade sobre o parasita. / Toxoplasma gondii is an Apicomplexa protozoan, of worldwide distribution, that infects several species, from mammalians to birds; its definitive hosts are the cats and other felines, while man and other animals are considered as intermediate hosts. Treatment is, generally, a combination of sulfadiazine and pyrimethamine, triggering the metabolic pathway of folic acid, which is necessary for certain purines, pirimidines and aminoacids biosynthesis. We have proposed an evaluation protocol on this protozoan, and also to establish a methodology by spectroscopy for rapid evaluation of pottentialy bioactive substances against the parasite. The ursolic acid and oleanoic acid bioactivity were tested. These substances have already demonstrated to be effective on another protozoan species, like Plasmodium, Trypanosoma cruzi e Leishmania sp., either in vitro or in vivo. The colorimetric methodology by MTT, Alamar Blue®, CyQUANT® NF kit and manual counting of tachyzoite forms in liquid culture medium showed to be unviable, because there is a great difficult to maintain the parasite viability in liquid culture medium, wich one is sensible to addition of any other component different of that necessary for its survival in extracelular ambient. Ursolic acid was pottentialy active on T. gondii intracellular forms. However, the infected cells in contact with the tested substances for a period of 48 hours did not show a statistical greater reduction as compared to infected cells which underwent treatment for 24 hours. Significant results were observed when comparing pre-treatment of tachyzoite forms and treatment for 24 hours post-cellular infection. Our data pointed in the direction that pre-treatment exerted a higher effectiveness. Any parasiticidal activity was observed when ursolic acid on a concentration of 7 mg/kg/day was administered to infected mice.