Šioje daktaro disertacijoje buvo nagrinėjami paviršiaus plazmonų rezonanso (PPR) ir elektrocheminiai imuniniai jutikliai žmogaus augimo hormonui (ŽAH) ir antikūnams prieš žmogaus augimo hormoną (anti-ŽAH) nustatyti bei pritaikyti juos realių mėginių analizei. Šiame darbe buvo nagrinėjami nekryptingas ir kryptingas antikūnų imobilizavimo būdai PPR jutiklio lusto paviršiuje ir tirtas antikūno-antigeno komplekso formavimasis. Buvo nustatyta, kad tinkamiausias iš tirtų anti-ŽAH imobilizavimo PPR jutiklio lusto paviršiuje būdų yra kryptingas anti-ŽAH fragmentų gautų redukuojant anti-ŽAH 2-merkatoetilaminu (2-MEA) imobilizavimas. Atominės jėgos mikroskopijos pagalba buvo stebėti įvairiais būdais modifikuotų PPR jutiklio lusto paviršių skirtumai, kurie parodė, ar fragmentai gauti redukuojant anti-ŽAH buvo tinkamai prijungti prie auksinio PPR jutiklio lusto paviršiaus, o imuninis kompleksas susiformavo dėl specifinės antikūno-antigeno sąveikos. PPR ŽAH imuninis jutiklis pasižymi maža aptikimo riba (3,4 nmol/L), stabilumu, atrankamu. Aktyvus PPR jutiklio lusto paviršius visiškai regeneruojamas, o tai leidžia imuninio jutiklio daugkartinį panaudojimą. Tiesioginio ir netiesioginio tipo PPR anti-ŽAH imuniniai jutikliai buvo tiriami kombinuojant du analizinius metodus – PPR ir elektrocheminius (impulsinė amperometrija (PA) ir ciklinė voltamperometrija (CV)). Anti-ŽAH imuniniai jutikliai pasižymi maža aptikimo riba (0,056, 0,051 ir 0,027 nmol/L, atitinkamai CV, PPR ir PA), stabilumu... [toliau žr. visą tekstą] / In this doctoral thesis the development of surface plasmon resonance (SPR) and electrochemical (EL) immunosensors has been examined and used for the detection of human growth hormone (HGH) and antibodies against human growth hormone (anti-HGH). The designed immunosensors were applied for the real sample analysis. In this research work random and oriented techniques of anti-HGH immobilization on the SPR sensor chip were compared and the interaction between immobilized anti-HGH and HGH was evaluated. Fragments of anti-HGH immobilization via their native thiol-groups directly coupled to the SPR chip is the most suitable technique for the development of an SPR immunosensor for the HGH detection. Fragments of antibody were obtained by chemical reduction of 2-mercaptoethylamine (2-MEA). In our study atomic force microscopy has been successfully applied for the imaging of the the differently modified surfaces. The achieved results indicate how much adequately the fragments of anti-HGH molecules bound onto the gold substrate and how immune complexes were formed. SPR HGH immunosensor is suitable for HGH detection with a limit of detection (3.4 nmol/L), good stability and selectivity. Furthermore, active SPR-chip surface is fully regenerated that allows multiply usage of designed immunosensor. Direct and indirect anti-HGH immunosensor was studied by combining two analytical methods – SPR and EL (pulsedamperometry (PA) and cyclic voltammetry (CV)). The limit of detection values of... [to full text]

Šioje daktaro disertacijoje buvo nagrinėjami paviršiaus plazmonų rezonanso (PPR) ir elektrocheminiai imuniniai jutikliai žmogaus augimo hormonui (ŽAH) ir antikūnams prieš žmogaus augimo hormoną (anti-ŽAH) nustatyti bei pritaikyti juos realių mėginių analizei. Šiame darbe buvo nagrinėjami nekryptingas ir kryptingas antikūnų imobilizavimo būdai PPR jutiklio lusto paviršiuje ir tirtas antikūno-antigeno komplekso formavimasis. Buvo nustatyta, kad tinkamiausias iš tirtų anti-ŽAH imobilizavimo PPR jutiklio lusto paviršiuje būdų yra kryptingas anti-ŽAH fragmentų gautų redukuojant anti-ŽAH 2-merkatoetilaminu (2-MEA) imobilizavimas. Atominės jėgos mikroskopijos pagalba buvo stebėti įvairiais būdais modifikuotų PPR jutiklio lusto paviršių skirtumai, kurie parodė, ar fragmentai gauti redukuojant anti-ŽAH buvo tinkamai prijungti prie auksinio PPR jutiklio lusto paviršiaus, o imuninis kompleksas susiformavo dėl specifinės antikūno-antigeno sąveikos. PPR ŽAH imuninis jutiklis pasižymi maža aptikimo riba (3,4 nmol/L), stabilumu, atrankamu. Aktyvus PPR jutiklio lusto paviršius visiškai regeneruojamas, o tai leidžia imuninio jutiklio daugkartinį panaudojimą. Tiesioginio ir netiesioginio tipo PPR anti-ŽAH imuniniai jutikliai buvo tiriami kombinuojant du analizinius metodus – PPR ir elektrocheminius (impulsinė amperometrija (PA) ir ciklinė voltamperometrija (CV)). Anti-ŽAH imuniniai jutikliai pasižymi maža aptikimo riba (0,056, 0,051 ir 0,027 nmol/L, atitinkamai CV, PPR ir PA), stabilumu... [toliau žr. visą tekstą] / In this doctoral thesis the development of surface plasmon resonance (SPR) and electrochemical (EL) immunosensors has been examined and used for the detection of human growth hormone (HGH) and antibodies against human growth hormone (anti-HGH). The designed immunosensors were applied for the real sample analysis. In this research work random and oriented techniques of anti-HGH immobilization on the SPR sensor chip were compared and the interaction between immobilized anti-HGH and HGH was evaluated. Fragments of anti-HGH immobilization via their native thiol-groups directly coupled to the SPR chip is the most suitable technique for the development of an SPR immunosensor for the HGH detection. Fragments of antibody were obtained by chemical reduction of 2-mercaptoethylamine (2-MEA). In our study atomic force microscopy has been successfully applied for the imaging of the the differently modified surfaces. The achieved results indicate how much adequately the fragments of anti-HGH molecules bound onto the gold substrate and how immune complexes were formed. SPR HGH immunosensor is suitable for HGH detection with a limit of detection (3.4 nmol/L), good stability and selectivity. Furthermore, active SPR-chip surface is fully regenerated that allows multiply usage of designed immunosensor. Direct and indirect anti-HGH immunosensor was studied by combining two analytical methods – SPR and EL (pulsedamperometry (PA) and cyclic voltammetry (CV)). The limit of detection values of... [to full text]

Ces dernières décennies, on a assisté à l’augmentation du nombre de technologies et de concepts permettant l’analyse des interactions intermoléculaires. Dans ce contexte, les puces à fluorescence restent les plus fréquemment utilisées. Cependant, cette technologie bien que très sensible et multiplexée, ne permet pas d’avoir accès aux paramètres cinétiques, indispensables au calcul des constantes d’affinité et la recherche de systèmes alternatifs s’impose. Dans cette optique, la résonance plasmonique de surface par imagerie (SPRi) est considérée comme une véritable option. Cette technologie se caractérise par l’absence de marquage et permet de suivre en temps réel d’infimes variations de masses consécutives à des interactions intermoléculaires sur la surface du prisme. L’obtention de constantes d’affinité est ainsi possible. En revanche, la SPRi présente un certain nombre de limites, principalement au niveau de la sensibilité et du multiplexage. Les objectifs de la thèse ont ainsi consisté à combler en partie ces différentes limites. La chimie de greffage basée sur l’utilisation d’oligonucléotides modifiés par un thiol a permis d’améliorer le multiplexage et de déposer plus de 1000 spots par cm² sur la surface d’or du prisme. Dans le même temps, la modification de la surface avec des colloïdes d’or et des dendrimères a permis pour des interactions ADN/ADN, d’atteindre une limite de détection de 2 nM (d’où un gain de 200%). En parallèle de ces travaux, diverses applications biologiques ont été effectuées. Une première étude a consisté à rechercher des ligands spécifiques des structures G-quadruplex des télomères. Une seconde étude s’est portée sur le complexe de partition bactérien. Par des études de criblage les bases impliquées dans l’interaction avec une protéine indispensable à la partition du plasmide F chez E.coli ont été identifiées. L’ensemble de ces travaux ont montré le fort potentiel de la SPRi et les applications potentielles qui en découlent sont nombreuses. / During the last decades a large number of technologies have been developed to analyze intermolecular interactions. In this context, the fluorescence biochips remain the most frequently used. Although this technology is very sensitive and multiplexed, it does not allow access to the kinetic parameters, essential to the calculation of the constants of affinity. Therefore, the research for alternative systems is essential. In this way, the Surface Plasmon Resonance imaging (SPRi) is considered as an opportunity. It is an optical detection process that can occur when a polarized light hits a prism covered by a thin metal layer. Under certain conditions free electrons at the surface of the biochip absorb incident light photons and convert them into surface plasmon waves. Perturbations at the surface of the biochip, such as an interaction between probes immobilized on the chip and targets, induce a modification of resonance conditions which can be measured. It is a label free technology which allows intermolecular interactions in real time and gives access to the kinetics parameters. However, SPRi is limited in sensitivity and multiplexing. The objectives of my PhD were to circumvent these various limits. Thus, we validated the immobilization of DNA probes on gold surface using thiol-modified oligonucleotide probes. Deposition carried out on non-modified gold surface, does not require electrical stimulation and expensive specific robotic devices. The thiol modification of the probes was shown to be very stable at room temperature, contrary to pyrrole and diazonium probes that need to be prepared just prior to their spotting. We demonstrate that thiol-modified oligonucleotide probes spotted on a gold surface of the SPRi-prisms are very robust and reproducible. We also demonstrated that this simple chemistry is compatible with high density arrays fabrication bearing more than 1000 spots using a classical spotter. Furthermore, the modification of the prism surface with gold colloids and dendrimers allowed for DNA/DNA interactions, to reach a detection limit of 2 nM. In parallel of this work, various biological applications were carried out and validate our previous developments. A first study was to screen G-quadruplex specific ligands to inhibit telomerase activity. We demonstrated that SPRi technology is particularly well adapted to the screening of interaction of small molecules with DNA probes and is sensitive enough to permit distinction between interactions with different DNA structures. The second study was on the bacterial partition complex. We study the DNA binding requirement involved in SopB-sopC specific interactions and analysed at the nucleotide level the bases involved in the binding efficiency and essential for the partition All this PhD work improved the SPRi technology and demonstrated its great potential in biological applications.

Le CD36 est un récepteur de type éboueur de classe B exprimé à la surface de nombreux types cellulaires dont les macrophages, les cellules endothéliales de la microvasculature et les plaquettes. Ce récepteur multiligand est impliqué dans plusieurs processus pathologiques notamment l’athérosclérose, l’angiogénèse et la malaria via la liaison spécifique de ligands comme les lipoprotéines oxydées de basse densité, la thrombospondine-1 et la protéine PfEMP-1, respectivement. Les peptides de la relâche de l’hormone de croissance (GHRP) ont été identifiés comme les premiers ligands synthétiques du CD36. Afin de développer de nouveaux ligands synthétiques du CD36, l’établissement d’une méthode de criblage est essentiel pour découvrir des composés avec une liaison de haute affinité pour ce récepteur. Pour y parvenir, nous avons surexprimé le domaine extracellulaire du CD36 humain dans les cellules d’insectes Sf9. La protéine soluble purifiée par chromatographie d’affinité fut immobilisée à la surface d’une plaque de résonance de plasmons de surface (SPR) pour les études de liaison. La méthodologie développée a permis de caractériser les ligands du CD36 en déterminant leurs constantes de dissociation (KD), et d’établir une relation structure-activité des ligands de la famille des azapeptides, des composés dérivés du GHRP-6. Afin de valider la méthode par spectroscopie SPR, une corrélation a été établie entre les valeurs de KD obtenues en SPR et les valeurs d’CI50 de courbes d’inhibition de la phosphorylation des MAP kinases JNK1/2 induite par un phospholipide oxydé, le POVPC, en présence de concentrations croissantes de ligands du CD36 dans les macrophages RAW 264.7. / CD36 is a class B scavenger receptor expressed at the cell surface of macrophages, endothelial cells and platelets, among others. This multiligand receptor is implicated in various diseases such as atherosclerosis, angiogenesis and malaria through the specific binding of ligands, such as oxidized low-density lipoproteins, thrombospondin-1 and the PfEMP-1 protein, respectively. Growth hormone-releasing peptides (GHRP) were identified as the first CD36 synthetic ligands. In order to identify new CD36 synthetic ligands, the development of a high-throughput method is essential to unveil compounds of high binding affinity. We have expressed a recombinant CD36 ectodomain protein in Sf9 insect cells. The soluble and affinity purified protein was immobilized on a surface plasmon resonance (SPR) sensor for binding studies. Synthetic ligands were analyzed by SPR spectroscopy for determination of their respective dissociation constant (KD). A structure-activity relationship of CD36 ligands was established. To validate the SPR binding signal, a good correlation was observed between KD and the IC50 values obtained from the inhibition curves of the MAPK kinase JNK1/2 phosphorylation induced by an oxidized phospholipid, the POVPC, in the presence of increasing concentrations of CD36 ligands in RAW 264.7 macrophage cells.

Les réseaux de nanotrous sont des structures plasmoniques ayant un énorme potentiel en tant que transducteurs pour la conception de biocapteurs. De telles structures sont prometteuses pour l’élaboration de biocapteurs capable d’effectuer du criblage à haut débit. L’intérêt de travailler avec des réseaux de nanotrous est dû à la simplicité d’excitation des polaritons de plasmons de surface en transmission directe, à la sensibilité et à la facilité de fabrication de ces senseurs. L’architecture de tels réseaux métalliques permet la conception de nanostructures ayant de multiples propriétés plasmoniques. L’intensité, la signature spectrale et la sensibilité du signal plasmonique sont grandement affectées par l’aspect physique du réseau de nanotrous. L’optimisation du signal plasmonique nécessite ainsi un ajustement du diamètre des trous, de la périodicité et de la composition métallique du réseau. L'agencement de l'ensemble de ces paramètres permet d'identifier une structure optimale possédant une périodicité de 1000 nm, un diamètre des nanotrous de 600-650 nm et un film métallique ayant une épaisseur de 125 nm d'or. Ce type de transducteur a une sensibilité en solution de 500-600 nm/RIU pour des bandes plasmoniques situées entre 600-700 nm. L'intérêt de travailler avec cette structure est la possibilité d'exciter les plasmons de polaritons de surface (SPPs) selon deux modes d'excitation : en transmission exaltée (EOT) ou en réflexion totale interne par résonance des plasmons de surface (SPR). Une comparaison entre les propriétés plasmoniques des senseurs selon les modes d'excitation permet de déterminer expérimentalement que le couplage de la lumière avec les ondes de SPP de Bloch (BW-SPPs) en transmission directe résulte en un champ électromagnétique davantage propagatif que localisé. D'un point de vue analytique, la biodétection de l'IgG en SPR est 6 fois plus sensible par rapport au mode EOT pour une même structure. Une étude du signal plasmonique associé au BW-SPP pour un certain mode de diffraction démontre que la distance de pénétration de ces structures en EOT est d'environ 140 nm. La limite de détection de l'IgG humain pour un réseau de nanotrous de 1000 nm de périodicité est d'environ 50 nM en EOT. Ce mémoire démontre la viabilité des réseaux de nanotrous pour effectuer de la biodétection par criblage à haut débit lors de prochaines recherches. L'investigation de l'effet de l'angle d'excitation en transmission exaltée par rapport au signal plasmonique associé au mode (1,0) d'un réseau de nanotrous de 820 nm d'or démontre que la sensibilité en solution n'est pas proportionnelle à la sensibilité en surface du senseur. En fait, une optimisation de l'angle d'incidence pour le mode (1,0) de diffraction des BW-SPP permet d'amplifier la sensibilité en surface du senseur jusqu'à 3-fois pour un angle de 13,3°. Ce mémoire démontre ainsi la nécessité d'optimiser l'angle d'excitation et les propriétés physiques du senseur afin de développer un transducteur de grande sensibilité basé sur l'excitation en transmission de réseaux de nanotrous. / This research aims at developing a multiplexed biosensor for protein detection based on the nanohole array technology. Gold nanohole arrays exhibit distinct plasmonics properties depending on the excitation mode of the surface plasmon polaritons (SPPs). The interest of working with nanohole arrays is related to their high sensitivity, ease of fabrication and simple setup of excitation in transmission. The architecture of nanohole arrays leads to a nanostructure having multiple plasmonics properties. The intensity, the spectral signature and the sensitivity of the plasmonic signal were highly affected by the shape of the nanohole arrays. Varying the diameter of the holes, the periodicity and the metallic composition of the array were used to optimize the plasmonic signal. The optimal structure was found to have a periodicity of 1000 nm, a diameter of 600-650 nm and a metallic film with a thickness of 125 nm of gold. Such a transducer exhibits a bulk refractive index sensitivity of 500-600 nm/RIU for plasmonic bands absorbing around 600-700 nm. Surface plasmon resonance (SPR) in the Kretschmann configuration and enhanced optical transmission (EOT) mode were compared using large gold nanohole arrays (1000 nm periodicity, 600 nm diameter and 125 nm depth) in order to assess their relative analytical performance. Biodetection of IgG was found to be 6 times more sensitive with SPR in the Kretschmann configuration than in EOT mode for the same structure. The decay length of the electromagnetic field in EOT mode was determined experimentally to be around 140 nm with a layer-by-layer polyelectrolyte deposition. This results suggests that the plasmonic properties of EOT for nanohole arrays is much more associated to a Bloch wave SPPs mode rather than a localized SPR. Variation of the incident angle of excitation of the BW-SPPs in transmission leads to a higher surface sensitivity for the (1,0) diffraction mode for gold nanohole arrays of 820 nm periodicity. Optimization of the physical properties and the excitation angle of the nanohole arrays is essential in order to develop a transducer having a potential towards multiplexed biosensors.

L'objectif principal de ces travaux de thèse est de développer des modèles membranaires biomimétiques pour la reconstitution et l'étude d'interactions protéine/membrane. Dans ce but, deux approches sont adoptées : l'une mettant en œuvre une plateforme basée sur des nanoparticules de silice/Au recouvertes de lipides et l'autre comprenant la formation de bicouches lipidiques découplées d'un support solide d'or. Dans la première approche, nous avons synthétisé des particules de silice de taille nanométrique contenant des grains d'or inclus dans la matrice silicique. Ces nanoparticules sont ensuite recouvertes par différents phospholipides. Les propriétés plasmoniques acquises grâce aux grains d'or sont caractérisées puis utilisées pour suivre l'interaction avec les lipides et/ou les protéines. Le suivi de ces interactions est également visualisé par analyse de la mobilité électrophorétique des particules. La deuxième stratégie développée, consiste à assembler un système membranaire sur une surface solide d'or. Dans un premier temps, une couche de calmoduline est liée à la surface de manière stable. Dans un deuxième temps, une bicouche est formée au-dessus de la couche de calmoduline par deux méthodes. La première méthode consiste à ancrer la bicouche directement sur la couche de protéine par un mécanisme faisant intervenir des lipides chélateurs. Alors que dans la deuxième méthode les lipides sont liés à la surface et découplés grâce à l'utilisation d'une surface d'or modifiée par de la cystéamine et à des lipides fonctionnalisés. L'ancrage est assuré par des groupements succinimidyl et le découplage par des polymères de polyéthylène glycol porté sur un même lipide. Dans les deux stratégies, un réservoir sub-membranaire est créé entre la bicouche étanche et le support. Le suivi des constructions moléculaires est réalisé par résonance plasmonique de surface et analyse du retour de fluorescence. De plus le système est implémenté par des électrodes afin d'étudier l'effet d'application de potentiel sur la bicouche. Après caractérisation, le modèle membranaire est validé par la reconstitution de la translocation de la toxine CyaA de Bordetella pertussis. Cette protéine dispose en effet d'un mécanisme d'internalisation singulier qui permet d'explorer tout le potentiel de notre modèle membranaire. / The main objective of this work is to develop biomimetic membrane models for the reconstitution and study of protein/membrane interaction. Two devices were designed: one operate a nanometric platform composed of phospholipids coated lipid silica/Au nanoparticles, while the other including tethered lipid bilayer reconstitution on a gold surface. The first approach needs the synthesis of nanometer sized gold/silica particles and that are subsequently coated with different phospholipids. The plasmonic properties provided by gold seeds are characterized and they are of utility to follow the interaction between lipids and/or proteins at the surface. Following of these interactions was also realized with electrophoretic mobility analysis. The second biomimetic device involves a membrane assembly on a gold surface. In a first time, a calmodulin layer is bound on the surface. In a second time, a lipid bilayer is assembled above the calmodulin layer by two approaches. In the first approach the lipid bilayer is anchored on the protein layer with chelators lipid and His-Tag bearing by the proteins. While, in the second approach, lipids are bound on the surface and tethered with the use of a cysteamin modified gold surface and functionalized lipids. The anchorage is realized by succinimidyl group and the tethering by polyethylene glycol group wearing by one kind of lipid. A sub-membrane reservoir is created under the lipid bilayer. The biomimetic model formation was followed by plasmonic resonance and fluorescence recovery after photobleaching. After their characterization the tethered model is validated by reconstitution of a particular mechanism: the CyaA toxin from Bordetella pertussis translocation.

A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clones foram expressas em E.coli para estabelecer o método para a purificação. Com a obtenção dos fragmentos Fab purificados, foi caracterizada a sua afinidade ao antígeno e especificidade, em ensaios de inibição por digoxina, digitoxina, digoxigenina e ouabaina. Os parâmetros cinéticos da ligação dos fragmentos Fab dos 4 clones e do DigiFab® foram avaliados por SPR. Nas condições experimentais, não foram verificadas diferenças significativas entre os produtos dos 4 clones e o comercial, demonstrando o potencial dos fragmentos Fab monoclonais obtidos como antídoto à digoxina. / Digoxin is a medication indicated for heart failure treatment. Its therapeutic window is narrow, being responsible for intoxication cases. The only antidote available for the detoxification is a polyclonal antibody - DigiFab® in Fab format. Its use is effective, but costly. Bacterial clones producing anti-digoxin monoclonal Fab fragments were previously obtained by our group using phage display technology. In this work the Fab variants of the 4 clones were expressed in E.coli to establish the purification method. The purified fragments were characterized regarding the affinity to the antigen and the specificity through inhibition assays with digoxin, digitoxin, digoxigenin and ouabain. The binding kinetic parameters of Fab fragments of the 4 clones and the commercial product to Dig-BSA conjugate were assessed by SPR. Under the experimental conditions no significant differences were observed among the 4 clones and the commercial product, demonstrating the potential of monoclonal Fab fragments as an antidote to digoxin.

FGF2 (Fibroblast Growth Factor 2), o fundador da família FGF, tem funções regulatórias na mitogênese, diferenciação, morfogênese e reparo tecidual. Diversas espécies moleculares de FGF2 compartilham uma seqüência C-terminal comum de 155 aminoácidos, pois se originam de diferentes sítios de iniciação de leitura de um único mRNA. O menor, o FGF2-18kDa, é liberado extracelularmente para se ligar a receptores específicos (FGFRs) para disparar as funções parácrinas e autócrinas pelas quais este fator é conhecido. Por outro lado, as espécies maiores (FGF2-21, 22, 22,5 e 34kDa) são intracelulares se ligam a parceiros moleculares desconhecidos para exercer funções intrácrinas ainda indefinidas. O objetivo desta tese foi produzir espécies recombinantes do FGF2-18 e FGF2-22,5, na forma de proteínas de fusão, para analisar funções biológicas e mecanismos de sinalização. Nas células malignas Y1 de camundongo, os recombinantes de FGF2-18kDa (FGF2-18, His-FGF2-18 e His-FGF2-18-ProA) dispararam uma resposta antagônica estimulando as vias de sinalização mitogênica, mas bloqueando o ciclo celular. Nos fibroblastos não tumorigênicos Balb3T3, estes mesmos recombinantes de FGF2-18kDa dispararam apenas a resposta mitogênica clássica. Todos os efeitos biológicos destes recombinantes de FGF2-18kDa foram bloqueados pelo inibidor específico da proteína quinase de tirosina dos FGFRs, PD173074, demonstrando que são respostas intermediadas pelos FGFRs. Portanto, os domínios estruturais adicionados aos recombinantes de FGF2-18kDa não impediram que estas proteínas se ligassem e ativassem os FGFRs. Por outro lado, o recombinante His-FGF2-22,5 dispara apenas as vias de sinalização mitogênica em ambas as células Y1 e 3T3, mas este efeito biológico não é inibido por PD173074. Estes resultados sugerem que a seqüência N-terminal de 55 resíduos, rica em aminoácidos básicos, impede que o FGF2-22,5kDa se ligue e/ou ative os FGFRs. Entretanto, o recombinante His-FGF2-22,5ProA dispara a resposta antagônica característica do FGF2-18kDa. As implicações destes últimos resultados é que o domínio de ProA adicionado ao C-terminal torna o FGF2-22,5kDa um bom ligante dos FGFRs. A interação física entre ligante e receptor das formas recombinantes His-FGF2-18kDa (ou His-FGF2-18ProA) e FGF2-22,5kDa com os putativos FGFRs foi analisada através da técnica de SPR e os resultados mostram KDs aproximados (Kd18=21, 488.10-9 e Kd22,5=20,70393.10-9), enquanto que o número de sítios ligantes em vesículas microssomais das células é significantemente inferior para o FGF2-22,5kDa. Estes resultados são compatíveis com a existência de receptores diferentes para FGF2-18kDa e FGF2-22,5kDa, uma hipótese ainda a ser definitivamente corroborada. Em conclusão, o FGF2-18kDa, mesmo em formas recombinantes como proteína de fusão, dispara todos os efeitos biológicos descritos para FGF2, através dos FGFRs. Diferentemente, o FGF2-22,5kDa, como fator parácrino, só desencadeou a resposta mitogênica clássica de FGF2, provavelmente através de receptores diferentes dos FGFRs. Os resultados e conclusões desta tese têm um potencial indiscutivelmente relevante para a biologia molecular do câncer, com implicações possíveis em terapia oncológica / FGF2 (Fibroblast Growth Factor 2), the founder of the FGF family, has regulatory functions in mitogenesis, differentiation, morphogenesis and tissue repair. Multiple FGF2 molecular species, sharing a C-terminal sequence of 155 amino acids, are translated from different iniciation sites of the same mRNA. The smaller, the FGF2-18kD, is extracellularly released to bind to specific membrane receptors (FGFRs), performing paracrine and autocrine functions. On the other hand, the larger FGF2s (21, 22, 22.5 and 34kDa) are intracellular species that bind to unknown partners to play still undefined intracrine roles. The aim of this thesis was to produce recombinant species of FGF2-18kDa and FGF2-22,5kDa, in the form of fusion proteins, to analyze functions and signaling mechanisms. In mouse Y1 malignant cells, FGF2-18kD recombinants (FGF2-18kDa and His-FGF2-18kDaProA) triggered an antagonistic response activating mitogenic signaling pathways, but blocking the cell cycle. However, in non tumorigenic Balb3T3 fibroblasts, these same FGF2-18kD recombinants only elicited the classical mitogenic response. All biological effects of these FGF2-18kD recombinants were blocked by the specific inhibitor of FGFR-protein-tyrosine-kinases, PD173074, demonstrating that these responses are mediated by FGFRs. Therefore, the new peptide domains added to FGF2-18kD did not prevent these recombinant fusion proteins to bind and activate FGFRs. Conversely, the recombinant His-FGF2-22,5kDa triggered only mitogenic signaling pathways in both Y1 and Balb3T3 cells, a biological effect not inhibited by PD173074. These results suggested that the additional basic-rich N-terminal sequence of 55 amino acid residues, found in FGF2-22,5kDa, prevents this FGF2 species from binding and / or activate FGFRs. However, surprisingly, the recombinant His-FGF2-22kDaProA triggered the antagonistic response characteristic of FGF2-18kDa. These results imply that the ProA-domain added to the C-terminal end rendered the FGF2-22,5kDaProA a good ligand of FGFRs. The physical interaction between recombinants of both His-FGF2-18kD and His-FGF2-22kDa with putative FGFRs, analyzed by SPR, yielded close KD values (KD18=21, 5.10-9 e K D22,5=20,7.10-9), while the number of binding sites in cell microsomal vesicles were significantly lower for the His-FGF2-22,5kDa. These results are consistent with the existence of different receptors for FGF2 and FGF2-18kD-22,5kDa, a hypothesis that has yet to be definitively confirmed. In conclusion, FGF2-18kD, even as recombinant fusion proteins, triggered all biological effects of FGF2, through FGFRs. Conversely, the FGF2-22, 5kDa only triggered the classical mitogenic response, probably via receptors other than FGFRs. The results and conclusions of this thesis are potentially of great interest in cancer molecular biology, with implications in oncologic therapy.

Os dispositivos biossensores foram primeiramente pesquisados por Clark e Lyons, em 1962, visando medir o nível de glicemia. Atualmente, há uma popularização dos medidores de glicemia e diversas tecnologias foram desenvolvidas, principalmente, para aplicações em diagnósticos médicos e monitoramento ambiental. No entanto, há uma ampla quantidade de aplicações para biossensores ainda não disponível no mercado. A imobilização de espécies bioativas que interagem diretamente com o analito, chamada de superfície bioativa, é frequentemente considerada a etapa mais difícil no desenvolvimento de biossensores para novas aplicações. O objetivo deste trabalho é demonstrar a caracterização da camada bioativa desenvolvida pela técnica de construção de camadas auto-organizadas usando o princípio da adsorção eletrostática. Para isto, foram utilizadas três diferentes concentrações de soluções compostas por ligações covalentes entre nanotubos de carbono de paredes múltiplas e três diferentes biomateriais, Polietilenoimina, Ácido Desoxirribonucléico e enzimas do tipo Hidrolase de Organofosforados. As diferentes soluções polieletrolíticas foram caracterizadas pela técnica de ressonância de plasmons de superfície e simultaneamente formaram camadas auto-organizadas na superfície de um eletrodo de carbono vítreo. A atividade das enzimas foi analisada por voltametria cíclica para as diferentes concentrações das soluções utilizadas. Os resultados mostraram que a caracterização por ressonância de plasmons de superfície promove o controle da adsorção eletrostática. A variação do ângulo de ressonância de plasmons de superfície corresponde à intensidade e à saturação das interações eletrostáticas entre as camadas. Biossensores desenvolvidos com soluções de maior concentração, consequentemente, ampliaram os limites de detecção e elevaram a resolução do sinal elétrico. Em aplicações para medir baixas concentrações do analito, o adequado controle das concentrações das soluções poli eletrolíticas reduz os custos no desenvolvimento de biossensores especialmente, com relação ao custo de enzimas isoladas. / The biosensor devices were first researched by Clark and Lyons in 1962, aiming to measure the blood glucose level. Currently, there is a popularization of blood glucose meters and several technologies have been developed mainly for applications in medical diagnostics and environmental monitoring. However, there is a wide range of applications for biosensors not yet available. The immobilization of bioactive species that interact directly with the analyte, called bioactive surface, is often considered the most difficult step in the biosensors development for new applications. The objective of this work is to demonstrate the characterization of the bioactive surface developed by self-assembled monolayers technique using the electrostatic adsorption principle. For this, we used three different concentrations of solutions composed of covalent bonds between multi-walled carbon nanotubes and three different biomaterials, Polyethyleneimine, Deoxyribonucleic Acid and Organophosphorous Hydrolase enzymes. These enzymes catalyze the hydrolysis of phosphorus compounds such as organophosphorous neurotoxins. The different polyelectrolyte solutions were characterized by the technique of surface plasmon resonance, and simultaneously, self-assembled monolayers were formed on the surface of a glassy carbon electrode. The enzymes activity was analyzed by cyclic voltammetry for the different concentrations of the solutions used. The results showed that the surface plasmon resonance characterization promote the control of electrostatic adsorption. The variation of the surface plasmon resonance angle corresponds the intensity and saturation of electrostatic interactions between the layers. Biosensors developed with solutions of higher concentration, consequently, widened the detection limits, and increased the resolution of the electrical signal. In applications to measure low concentrations of analyte, adequate control of the concentrations of polyelectrolytes solutions reduces costs in the development of biosensors especially, the cost of isolated enzymes.

Os dispositivos biossensores foram primeiramente pesquisados por Clark e Lyons, em 1962, visando medir o nível de glicemia. Atualmente, há uma popularização dos medidores de glicemia e diversas tecnologias foram desenvolvidas, principalmente, para aplicações em diagnósticos médicos e monitoramento ambiental. No entanto, há uma ampla quantidade de aplicações para biossensores ainda não disponível no mercado. A imobilização de espécies bioativas que interagem diretamente com o analito, chamada de superfície bioativa, é frequentemente considerada a etapa mais difícil no desenvolvimento de biossensores para novas aplicações. O objetivo deste trabalho é demonstrar a caracterização da camada bioativa desenvolvida pela técnica de construção de camadas auto-organizadas usando o princípio da adsorção eletrostática. Para isto, foram utilizadas três diferentes concentrações de soluções compostas por ligações covalentes entre nanotubos de carbono de paredes múltiplas e três diferentes biomateriais, Polietilenoimina, Ácido Desoxirribonucléico e enzimas do tipo Hidrolase de Organofosforados. As diferentes soluções polieletrolíticas foram caracterizadas pela técnica de ressonância de plasmons de superfície e simultaneamente formaram camadas auto-organizadas na superfície de um eletrodo de carbono vítreo. A atividade das enzimas foi analisada por voltametria cíclica para as diferentes concentrações das soluções utilizadas. Os resultados mostraram que a caracterização por ressonância de plasmons de superfície promove o controle da adsorção eletrostática. A variação do ângulo de ressonância de plasmons de superfície corresponde à intensidade e à saturação das interações eletrostáticas entre as camadas. Biossensores desenvolvidos com soluções de maior concentração, consequentemente, ampliaram os limites de detecção e elevaram a resolução do sinal elétrico. Em aplicações para medir baixas concentrações do analito, o adequado controle das concentrações das soluções poli eletrolíticas reduz os custos no desenvolvimento de biossensores especialmente, com relação ao custo de enzimas isoladas. / The biosensor devices were first researched by Clark and Lyons in 1962, aiming to measure the blood glucose level. Currently, there is a popularization of blood glucose meters and several technologies have been developed mainly for applications in medical diagnostics and environmental monitoring. However, there is a wide range of applications for biosensors not yet available. The immobilization of bioactive species that interact directly with the analyte, called bioactive surface, is often considered the most difficult step in the biosensors development for new applications. The objective of this work is to demonstrate the characterization of the bioactive surface developed by self-assembled monolayers technique using the electrostatic adsorption principle. For this, we used three different concentrations of solutions composed of covalent bonds between multi-walled carbon nanotubes and three different biomaterials, Polyethyleneimine, Deoxyribonucleic Acid and Organophosphorous Hydrolase enzymes. These enzymes catalyze the hydrolysis of phosphorus compounds such as organophosphorous neurotoxins. The different polyelectrolyte solutions were characterized by the technique of surface plasmon resonance, and simultaneously, self-assembled monolayers were formed on the surface of a glassy carbon electrode. The enzymes activity was analyzed by cyclic voltammetry for the different concentrations of the solutions used. The results showed that the surface plasmon resonance characterization promote the control of electrostatic adsorption. The variation of the surface plasmon resonance angle corresponds the intensity and saturation of electrostatic interactions between the layers. Biosensors developed with solutions of higher concentration, consequently, widened the detection limits, and increased the resolution of the electrical signal. In applications to measure low concentrations of analyte, adequate control of the concentrations of polyelectrolytes solutions reduces costs in the development of biosensors especially, the cost of isolated enzymes.