Plasmon coupling is known to cause distance dependent red-shifts of the characteristic plasmon resonance and localize strong electric fields to the gap between individual nanoparticles. These effects form the basis of nanoscale plasmonic sensors designed by creating specic structures of coupled nanoparticles. The simplest of these structures, a nanoparticle dimer, can easily be assembled through molecular self-assembly, resulting in a structure called a plasmon ruler. These plasmon rulers are crucial tools for the measurement of nanoscale distances, but the impact of the molecular linker on the plasmonic response of the coupled system remains insufficiently understood. In this dissertation, plasmons rulers composed of 40 nm gold nanoparticles are utilized to systematically investigate the potential effects of one molecular linker, DNA, on the strength of the plasmon coupling at a variety of interparticle separations. The strength of the plasmon coupling is determined based on the shifting of the plasmon resonance, which, at separations below 2.7 nm, is significantly blue-shifted when compared to expected values from electromagnetic simulations and experiments without DNA linkers. This deviation indicates a reduced charge accumulation on the nanoparticles in the gap region and is ascribed to DNA-mediated charge transfer. Enhancements to the charge transfer capabilities of the DNA were also investigated, through the deposition of interstitial palladium nanocrystals on the DNA linkers. The deposition of these nanocrystals results in a variety of structural changes to the plasmon rulers, associated with blue- and red-shifts of the plasmon resonance relative to electromagnetic simulations without gap material and experimental spectra of structures without molecular or metallic linkers. The relative blue-shift of the resonance results from a variety of scenarios, including short interparticle separations bridged by DNA or palladium nanocrystals, the build-up of palladium nanocrystals within the gap, or the incorporation of discrete palladium nanoparticles in the DNA linkers. The underlying mechanisms of the observed spectral shifts are analyzed. The red-shifted resonances resulted from a significant build-up of palladium nanocrystals in the gap, effectively linking the gold nanoparticles and forming a hybrid nanorod-like structure.

Streptococcus pneumoniae é causa de doenças como pneumonia, otite, meningite e sepse. As vacinas hoje disponíveis têm cobertura limitada porque são baseadas no polissacarídeo capsular, que varia com os mais de 90 sorotipos da bactéria, além de levarem à substituição de sorotipos na população por outros não presentes nas formulações. Visando reduzir o custo e aumentar a cobertura, têm-se estudado vacinas baseadas em proteínas que ofereceriam proteção independente de sorotipo. Este trabalho teve por objetivo a obtenção, avaliação da estabilidade e da resposta imune em camundongos de híbridos de duas proteínas de S. pneumoniae: a proteína de superfície do pneumococo (PspA) e a pneumolisina geneticamente detoxificada (PdT), sem ou com espaçadores moleculares, rígido ou flexível, entre as moléculas. Os genes dos híbridos com espaçadores foram clonados através da técnica de overlap extension PCR. O gene das proteínas foi expresso em E. coli e as proteínas obtidas foram reconhecidas por anticorpos produzidos contra a célula inteira de pneumococo através de Western Blot. A purificação dos híbridos foi feita utilizando homogeneizador de alta pressão, precipitação de impurezas com detergente catiônico CTAB e diferentes etapas cromatográficas. A estabilidade foi analisada periodicamente através de SDS-PAGE e Western Blot. O híbrido sem espaçador mostrou-se instável, por isso a presença de atividade proteolítica foi investigada através de diversos ensaios para detecção dessas enzimas, mostrando que a instabilidade não decorreu de hidrólise por proteases. Os fragmentos resultantes da quebra tiveram a porção N-terminal sequenciada para identificar o sítio de clivagem, que estava localizado na junção das duas proteínas. Esse sítio foi retirado das construções seguintes e espaçadores moleculares foram incluídos entre os dois antígenos. A molécula com espaçador flexível foi obtida na forma solúvel durante o cultivo, porém durante a purificação ocorreu precipitação irreversível. O clone para produção do híbrido com espaçador rígido permitiu a obtenção da proteína, que foi purificada e teve a estabilidade avaliada periodicamente à 4° C e -20° C por Western Blot empregando anticorpos contra célula inteira de pneumococo, indicando que a introdução do espaçador rígido aumentou a estabilidade do híbrido em relação à molécula sem espaçador. Além disso, diferentes concentrações de estabilizantes foram avaliadas, mostrando que em presença de trealose 1M ou glicerol 50% o híbrido com linker rígido permaneceu estável por pelo menos 4 meses a 4° C. A molécula com espaçador rígido produziu níveis de anticorpos em camundongos comparáveis aos níveis obtidos com a molécula sem espaçador, que foram capazes de proteger 100% dos animais em ensaio de desafio letal intranasal e inibir a atividade hemolítica da pneumolisina. O aumento de estabilidade do híbrido alcançado com a inserção do linker rígido juntamente com a retirada do sítio de clivagem e com a presença de estabilizantes permitem que esta molécula seja utilizada numa vacina pneumocócica proteica. Como estudos vêm demonstrando que seria necessário mais de uma proteína para formular esta nova vacina, a produção deste híbrido traz a grande vantagem de obtenção de dois antígenos em um único processo de produção, o que pode resultar em uma diminuição do custo da dose. / Streptococcus pneumoniae is cause of diseases like pneumonia, otitis, menngitis and sepsis. The vaccines available nowadays has limited coverage because they are based on the capsular polysaccharyde, which varies among the more than 90 bacteria serotypes and they lead to serotype substitution on the population for others not present on the vaccine formulations. In order to reduce the cost and increase the coverage, vaccines based on pneumococcal proteins that would offer serotype independent protection have been studied. The objective of this thesis was the obtainment, stability evaluation and immune response evaluation in mice of hybrids composed of two proteins of S. pneumoniae: pneumococcal surface protein A (PspA) and genetically detoxified pneumolysin (PdT), with or without molecular linkers, rigid and flexible, between the molecules. The genes with molecular linkers were cloned using the overlap extension PCR technique. The genes were expressed in E. coli and the proteins were recognized by anti pneumococcal whole cell in Western Blot. The hybrids were purified using high pressure homogeneizer, precipitation of impurities using cationic detergent CTAB and different chromatography steps. The stability was periodically analyzed through SDS-PAGE and Western Blot. The hybrid without linker was unstable and the presence of proteolytic activity was investigated through several methods for protease activity detection, showing that instability was not due to protease hydrolysis. The N-terminal portion of the degraded protein fragments was sequenced in order to identify the cleavage site, which was localized exactly between the two proteins. This site was removed from the other hybrids and molecular linkers were included between the two antigens. The molecule with flexible linker was obtained on the soluble form during expression, but during purification it precipitated irreversibly. The molecule with rigid linker were expressed, purified and had its stability analyzed periodically at 4° C and -20° C by Western Blot using anti pneumococcal whole cell, indicating that the insertion of the rigid linker increased the hybrid stability when compared with the hybrid without linker. Besides that, different stabilizers in different concentrations were evaluated and it was found that the presence of trehalose 1 M or 50% glycerol stabilized the hybrid with rigid linker for at least 4 months at 4 ° C. The molecule with rigid linker produced levels of antibodies in mice comparable to the hybrid without linker. These antibodies were able to protect 100% of animals from lethal intranasal challenge and inhibit the haemolytic activity of pneumolysin. The stability increase due to the rigid linker together with the removal of the cleavage site and the stabilizers presence allow this hybrid molecule to be used on a novel pneumococcal vaccine. Since studies have shown that it would be necessary more than one protein to formulate this new vaccine, the production of this hybrid brings the great advantage of producing two antigens in one single process, which can decrease the dosage cost.

Au cours de ce travail, une série de plateformes constituées d'un espaceur central connecté à deux complexes de MnII à haut spin a été synthétisée. De nombreux ligands ont été étudiés et greffés sur un ensemble d'espaceurs de longueur variant entre 1,5 et 5,5 nm. La distance Mn-Mn a été mesurée avec succès par résonance paramagnétique électronique (RPE) impulsionnelle à haut champ en utilisant la méthode PELDOR (Pulsed Electron-Electron Double Resonance). L'emploi de complexes de MnII avec de faibles valeurs d'éclatement en champ nul (ECN) a permis d'améliorer la sensibilité de cette méthode. Pour les plateformes constituées d'un espaceur polyproline, un bon accord a été observé entre la distribution de la distance Mn-Mn obtenue par PELDOR et par dynamique moléculaire, mais des composantes plus courtes dans la distribution ont été détectées pour certains paramètres expérimentaux. Ces observations ont été rationalisées en tenant compte du terme pseudo-séculaire de l'Hamiltonien dipolaire, non négligeable pour les systèmes étudiés où les spins observés et détectés sont similaires. Lorsqu'un espaceur rigide est employé, l'interaction pseudo-séculaire est bien plus marquée, ce qui se traduit par une distribution de distances plus large que prévu par la dynamique moléculaire. L'étude de nouveaux centres paramagnétiques pour la méthode PELDOR comme les radicaux trityl persistants a également été entreprise. Le tenseur g de ces radicaux a été déterminé avec précision par RPE à haut champ en utilisant MnII comme référence. Des calculs de DFT (Density Functional Theory) ont été effectués pour comprendre la relation entre la structure et le spectre RPE de ces radicaux trityl. / In this work, the synthesis of a set of platforms that incorporate a central linker of varying length connected to two high-spin MnII complexes has been performed. Several ligands were screened and efficient synthetic methodologies were developed to graft them on various spacers covering the 1.5 – 5.5 nm range. The Mn-Mn distance has been successfully measured using high-field pulsed electron paramagnetic resonance (EPR) spectroscopy, more precisely pulsed electron-electron double resonance (PELDOR). We showed that the use of MnII complexes with low zero-field splitting (ZFS) parameters led to an improved sensitivity. For flexible polyproline-based platforms, distances and distribution profiles obtained with PELDOR were in good agreement with molecular dynamics (MD) estimations, but additional features in the distance distributions could be observed under specific conditions. These finding were rationalized by taking into account the pseudo-secular term of the dipolar Hamiltonian, which was found to be non-negligible for the studied platforms, where pumped and detected spins are very similar. When the linker was rigid, the influence of the pseudo-secular interaction was much more prominent, leading to distance profiles with a higher width than predicted by MD calculations. Other emergent spin labels for pulsed EPR-based distance measurements such as persistent substituted trityl radicals were studied and their g-tensor was accurately measured using high-field EPR with MnII as an internal reference. Density functional theory (DFT) calculations were performed to understand the relationship between the structure and the EPR properties of the studied trityl radicals.