Esta tese descreve estudos realizados na tentativa de purificar e caracterizar enzimas envolvidas na BL de fungos, além de trabalhos conduzidos a fim de investigar o mecanismo da bioluminescência de fungos. Inicialmente, tentou-se isolar as duas enzimas supostamente responsáveis pala reação bioluminescente em fungos. Parâmetros de atividade ótima (pH e temperatura) e comportamento cinético foram investigados. Todavia, com a descoberta de que a luciferina fúngica é o derivado hidroxilado da hispidina (3-hidróxihispidina), novas estratégias foram abordadas. Os esforços se concentraram na purificação da luciferase, visto que a hidroxilase não faz parte do sistema bioluminescente de fungos. Avaliação da interação da luciferase fúngica com a luciferina ou derivados dela sugeriram comportamento relativamente promíscuo da enzima. Os resultados indicaram que a reação luciferina-luciferase é favorecida em meio básico (pH ~8), a ~20 °C. Ensaios com 18O2 revelaram que a inserção de oxigênio na molécula de luciferina produz um intermediário cuja descarboxilação gera a oxiluciferina. Paralelamente, a síntese da hispidina in vitro a partir de ácido cafeico na presença de malonil-CoA e de extrato de micélios bioluminescentes resultou na emissão de luz, confirmando que a luciferina é reciclada no processo. / This work describes studies performed to purify and characterize enzymes responsible for the fungal bioluminescence. Also, it shows important data that contributes to understand the mechanism for bioluminescence reaction in fungi. First, we tried to isolate two enzymes suspected of being involved on fungal bioluminescence. Optimum activity parameters (pH and temperature) and kinetic behavior were investigated. However, the discovery that fungal luciferin is the hispidin derivative 3-hydroxyhispidin demanded adaptations in the project. First of all, concentrates efforts to luciferase purification was priority, since hydroxylase is not part of the bioluminescent system of fungi. Studies on the luciferase interaction with different substrates showed some promiscuity for the enzyme. The results indicated higher intensity of light from luciferin-luciferase reaction in alkaline solutions (pH ~ 8) at ~ 20 °C. The reaction in medium with 18O2 revealed that insertion of oxygen into the luciferin structure produces an intermediate whose decarboxylation generates oxyluciferin. In parallel, the in vitro synthesis of hispidin using caffeic acid and malonyl-CoA with the mycelium extract resulted in the emission of light, confirming that luciferin is recycled in the process.

<p>The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system.</p><p>The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.</p>

The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system. The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.

Antares est un projet de télescope à neutrinos qui sera placé au large de Toulon, à 2500 m de profondeur. L'interaction d'un neutrino avec la matière produit un muon dont la lumière Cerenkov, émise lors de sa propagation, est détectée par 900 photomultiplicateurs répartis sur 12 lignes. Les sursauts gamma (GRB) sont des phénomènes cosmiques violents, observés une fois par jour. Dans le modèle du Boulet de Canon le sursaut est produit par l'interaction d'un jet, composé de boulet de canon (CB), avec le reste d'une supernova (SNR). Des chocs se propagent vers l'avant (dans le SNR) et vers l'arrière (dans le CB), aux abords desquels, <br />les neutrinos sont produits. Une estimation de la production de neutrinos est donnée et, est étudiée sur un large espace des paramètres. Pour un GRB typique, entre 0,002 et 0,3 $\nu_{\mu}$ cm$^2$ sont produits. Selon les angles de vue, Antares pourra détecter entre 1 et 10 $\nu_{\mu}$ par an, en corrélation avec les GRB. <br />Le bruit de fond optique ambiant a été enregistré par le prototype d'une ligne d'Antares. L'analyse porte sur l'influence du bruit de fond sur le détecteur ainsi que sur l'activité des organismes qui en sont responsables. Par exemple il apparaît une périodicité entre 17,6 et 20,4 h compatible avec celle des mouvements des masses liquides imposés par la force de Coriolis à la latitude d'Antares.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus has long been considered a master circadian pacemaker that drives rhythms in physiology and behavior in mammals. The recent discovery of self-sustained and cell-autonomous circadian oscillators in peripheral tissues has challenged this position. This dissertation tested the general hypothesis that the SCN has properties that distinguish it from other oscillators, thereby positioning it atop a circadian hierarchy. The general approach was to compare the consequences of altering the molecular circadian clock on tissue-autonomous rhythmicity in mice. In the first experiments, the role of the SCN as a master clock was tested by manipulating the expression of a circadian gene in the brain. Specifically, the expression of the short period tau mutation of casein kinase-1-epsilon (CK1ε) was controlled in an anatomically- and a temporally-specific manner via a tetracycline transactivator regulatory system. This inducible expression of CK1εtau affected the period of activity rhythms when expressed in the SCN, but did not affect the tissue-autonomous rhythmic properties in the peripheral tissues. Second, real-time bioluminescence imaging of tissues from PER2::LUCIFERASE mice revealed that period and phase of different circadian oscillators were tissue specific. Various circadian gene mutations (Cry1-/-, Cry2-/-, Cry1-/-;Cry2-/-, Clock∆19/∆19) produced little difference in rhythmic properties between the SCN and peripheral oscillators, although Cry1-/- SCN had more robust and persistent rhythms compared with the periphery. Third, the loss of Bmal1, which produces behavioral arrhythmicity, eliminated rhythms in the peripheral tissues, but not in the SCN. Bmal1-/- SCN rhythms were highly variable in period and amplitude, fitting a stochastic, but not a deterministic model of rhythm generation. Unlike mutations in other circadian genes, rhythmicity was completely abolished in single SCN neurons in Bmal1-/- mice, indicating that rhythms in Bmal1-/- SCN tissue are a property of the tissue organization rather than an averaging of single-cell autonomous rhythms. The SCN, therefore, has a unique anatomical organization that contributes to long-term stability and temporal organization of the circadian hierarchy.

Scientific research using nanotechnology is a relatively recent development with a variety of potential applications in many fields of science. Within this field of research, many new products, with improved performances, have been developed. Despite increased research on its toxicity to ecosystem, the knowledge about this area is still limited. To evaluate the toxicity and genotoxicity of different sizes silica nanoparticles (SiNP)to the environment, different species, on different trophic levels (Vibrio fisheri, Raphidocelissubcapitata, Daniorerio and Allium cepa) were exposed to TM40 (22 nm), HS30 (12 nm), SM30 (7 nm) with concentrations ranging from0.19 to 163.8 g/L (TM40) and 0.29 to 122.85 g/L (HS30 and SM30), and the following parameters were monitored during exposure: production of bioluminescence (V. fischeri), growth rate (R. subcapitata), embryonic development and DNA damage (D. rerio) and germination rate, growth and DNA damage (A. cepa). Within each test SiNPpresent a size dependent chronic toxicity. The bioluminescence test present a EC50 of 29.11, 32.34 and 4.58 g/L for TM40, HS30 and SM30, respectively. For the growth rate assay the EC50 was 9.32, 9.07 and 7.93 g/L for TM40, HS30 and SM30, respectively. And for the zebra fish embryonic development test for TM40, HS30 and SM30, the EC50 was 5.85, 1.13 and 2.68 g/L respectively. All particles also induce phytotoxicity in A.cepa, growth and germination reduce significatively when expose to SiNP. Futhermoregenotoxic effects were also induced by the particles for both A.cepaand D. rerio. Therefore, SiNP can cause toxicity to the environment and size can strongly influence this toxicity / Com uma variedade de aplicações potenciais, em diversos campos da ciência, as pesquisas científicas utilizando nanotecnologia são de desenvolvimento relativamente recente. Dentro deste campo de pesquisa, vários novos produtos, com desempenhos melhorados têm sido desenvolvidos. Apesar do aumento de pesquisas sobre a toxicidade dessas tecnologias à biota, o conhecimento sobre esta área ainda é limitado. Visando avaliar a toxicidade e genotoxicidade denanopartículasde sílica (SiNP) no meio ambiente diferentes espécies pertencentes a diversos níveis tróficos (Vibriofisheri, Raphidocelissubcapitata, DaniorerioandAllium cepa) foram expostos a Ludox TM40 (22 nm), Ludox HS30 (12 nm) e Ludox SM30 (7 nm). As espécies de teste foram expostas a concentrações de nanopartículas (NP) variando de 0.29 a 163.8 g/L (TM40) e 0.19 a 122.85 g/L (HS30 e SM30) e os seguintes parâmetros monitorizados durante a exposição: a produção de bioluminescência (V. fischeri), o crescimento taxa (R. subcapitata), inibição de alimentação (D. magna), desenvolvimento embrionário e dano ao DNA (D. rerio) e taxa de germinação, crescimento e danos ao DNA (A. cepa). Nos testes feitos com as SiNPfoi observado que a toxicidade é dependente do tamanho da partícula. O ensaio de bioluminescência apresentou um EC50 de 29.11, 32.34 e 4.58 g/L para TM40, HS30 e SM30, respectivamente. Para o ensaio de taxa de crescimento o EC50 foi 9.32, 9.07 e 7.93 g/Lpara TM40, HS30 e SM30, respectivamente. E para o teste de desenvolvimento embrionário com peixe zebra, para o TM40, HS30 e SM30 o EC50 foi de 5.85, 1.13 e 2.68g/L, respectivamente. Todas as partículas também induziram fitotoxicidade em A. cepa, crescimento e germinação reduziram significativamente quando o organismo foi exposto a SiNP. Efeitos genotóxicos também foram induzir pelas partículas, tanto para A. cepa quanto paraD. rerio. Portanto, as SiNP podem causar toxicidade ao ambiente e o tamanho pode influenciar fortemente a essa toxicidade

Cette thèse traite de problèmes inverses de sources dans deux cas : les sources fixes en 2D et 3D équations elliptiques et une source non-stationnaire dans une équation de diffusion. Dans le cadre de ce travail, nous considérons des sources ponctuelles (monopôles, dipôles et sources multipolaires) et des sources ayant support compact dans un nombre fini de petits sous-domaines qui modèlent les sources dans les problèmes EEG/MEG et le problème de tomographie par bioluminescence (BLT). Le but de cette thèse est de proposer des méthodes d’identification robustes qui permettent de déterminer leur nombre, leurs intensités et leurs positions. Des méthodes algébriques directes sont utilisées pour identifier les sources fixes et une méthode quasi-algébrique mélangée avec un problème d’optimisation est utilisé pour récupérer les sources avec des intensités variables dans le temps. Des résultats numériques sont effectués afin de mettre en évidence la robustesse de nos algorithmes d’identification. / This thesis deals with inverse source problems in 2 cases : stationary sources in 2D and 3D elliptic equations and a non-stationary source in a diffusion equation. the main form of sources considered are pointwise sources (monopoles, dipoles and multipolar sources) having compact support within a finite number of small subdomains modeling EEG/MEG problems and Bioluminescence Tomography (BLT) problems. The purpose o this thesis is mainly to propose robust identification methods that enable us to reconstruct the number, the intensity and the location of the sources. Direct algebraic methods are used to identify the stationary siurces and a quasi-algebraic method mixed with an optimieation method is employed to recover sources with time-variable intensities. Numerical results are shown to prove the robustness of our identification algorithms.

Visando a construção de três biossensores bacterianos para os metais mercúrio, arsênio e chumbo, Cupriavidus metallidurans CH34 foi escolhida para obtenção dos fragmentos de DNA (por PCR) correspondentes aos operons mer, ars e pbr. Os fragmentos foram inseridos à montante do gene EGFP, no plasmídeo pBB-EGFP, obtendo três novos plasmídeos: pGHg, pGAs e pGPb. Estes foram clonados em C. metallidurans CH34 e Escherichia coli DH5<font face=\"Symbol\">&#945;. As linhagens recombinantes foram submetidas a várias situações de cultivo e diferentes intensidades de fluorescência, detectadas em microscopia e quantificadas por citometria de fluxo. As linhagens recombinantes C. metallidurans CH34 / pGHg, E. coli DH5<font face=\"Symbol\">&#945; / pGHg, C. metallidurans CH34 / pGAs e C. metallidurans CH34 / pGPb mostraram-se eficazes e, consequentemente, poderão ser utilizadas em rápidos diagnósticos de amostras contaminadas por mercúrio, arsênio e chumbo. / Aiming at the construction of three bacterial biosensors for metals mercury, arsenic and lead, Cupriavidus metallidurans CH34 was chosen to obtain the DNA fragments (by PCR) corresponding to operons mer, ars and pbr. The fragments were inserted upstream of the EGFP gene, in plasmid pBB-EGFP, getting three new plasmids: pGHg, pGAs and pGPb. They were cloned in C. metallidurans CH34 and Escherichia coli DH5<font face=\"Symbol\">&#945;. The recombinant strains were subjected to various conditions of cultivation. Different intensities of fluorescence were detected microscopy and quantified by flow cytometry. The recombinant strains C. metallidurans CH34 / pGHg, E. coli DH5<font face=\"Symbol\">&#945; / pGHg, C. metallidurans CH34 / pGAs and C. metallidurans CH34 / pGPb were effective and therefore may be used in rapid diagnosis of samples contaminated by mercury, arsenic and lead.

<p>Bilder borttagna av upphovsrättsliga skäl.</p>

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