Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-19T11:45:26Z No. of bitstreams: 1 aline-stelling-zanatta.pdf: 2385669 bytes, checksum: 6f759289878ca2d698465044b392ae3f (MD5) / Made available in DSpace on 2012-11-19T11:45:26Z (GMT). No. of bitstreams: 1 aline-stelling-zanatta.pdf: 2385669 bytes, checksum: 6f759289878ca2d698465044b392ae3f (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Desde o trabalho de Köhler e Milstein (1975), hibridomas tem sido cultivados para obtenção de anticorpos monoclonais com finalidade de uso em pesquisa, diagnóstico e terapia. O método tradicional de obtenção de anticorpos monoclonais em altas concentrações é através de indução de ascite em camundongos. Estatécnica vem sendo substituída por cultivos de hibridomas em altas concentrações celulares. Neste trabalho, foram cultivados hibridomas secretores de anticorpos monoclonais anti-dengue tipo 2 em frascos T, garrafas rotatórias (roller) e frascos do tipo spinner, utilizando-se o meio DMEM, suplementado com soro fetal bovino a 10%, e o meio comercial livre de soro animal Ex-Cell® TiterHigh TM (Sigma). Ao longo dos diferentes cultivos, foram avaliadas a concentração celular, viabilidade celular e as concentrações de nutrientes (glicose eglutamina), metabólitos (lactato e amônio) e produto (IgG). A partir dos resultados obtidos, foram calculadas as grandezas representativas do metabolismo celular: concentração máxima de células (X máx), taxa específica de crescimento celular (µexp), tempo de duplicação (td) e coeficientes de rendimento de glicose em células (YX/glc), glutamina em células (Y X/gln), células em produto (YP/X), glutamina em amônio (YNH4/gln), glicose em lactato (Ylac/glc), glicose em produto (YP/glc) e glutamina em produto (YP/gln). O meio livre de soro mostrou ser capaz de fornecer melhores condições para o crescimento celular (alcançando 4 x 106 céls/mL), mantendo a viabilidade por um período maior de tempo, nos três sistemas decultivo testados. Quanto à formação de produto, no meio livre de soro, os hibridomas também secretaram altas concentrações de IgG, alcançando níveis de 3 µg/mL. Os melhores resultados de crescimento e viabilidade celular foram observados em garrafas rollera 40 rpm (após adaptação a rotações inferiores) e a produção de IgG foi maior em garrafas rollera 16 rpm (também após adaptação a rotações inferiores) e em frascos do tipo spinner a 50 rpm (após adaptação a rotações inferiores em garrafas rolleraté 40 rpm). Quando foram comparadas as concentrações de IgG entre os sobrenadantes de cultivo e três amostras de fluido ascítico do mesmo hibridoma, foi observado que o fluido ascítico continha concentrações 10 a 20 vezes maiores que as obtidas nos sobrenadantes de cultivo. Entretanto, como os volumes de sobrenadantes de cultivo são significativamente maiores do que os de fluido ascítico de camundongos, infere-se que é viável a substituição da produção in vivopela obtenção do anticorpo monoclonal estudado neste trabalho em sistemas agitados, utilizando-se meio livre de soro animal. Contudo, sugere-se a condução de experimentos adicionais para confirmação da total viabilidade da obtenção de anticorpos monoclonais anti-dengue tipo2 in vitroutilizando o processo proposto no presente trabalho. / Since Köhler and Milstein’s work (1975), hybridoma cells have been cultured to obtain monoclonal antibodies for research, diagnostic and therapeutic purposes. The traditional method to obtain high concentrations (5 to 10 mg/mL) of the monoclonal antibodies is the induction of ascite in mice. This technique is being replaced by high cell density cultivations. In this work, hybridoma secreting anti-dengue type 2 monoclonal antibodies were cultivated in T flasks, roller bottles and spinner flasks, using DMEM medium supplemented with fetal bovine serum at 10%, and the commercial serum-free medium Ex-Cell® TiterHigh TM (Sigma). Cell concentration, cell viability, as well as concentration of nutrients (glucose and glutamine), metabolites (lactate and amonium) and product (IgG) were evaluated along culture time in the different media and culture systems. Based on these data, variables that reflect the cell metabolism were calculated: maximum cell concentration (Xmáx), specific cell growth rate (µexp), duplication time (td), as well as the yield coefficients of glucose to cells (YX/glc), glutamine to cells (YX/gln), cells to product (YP/X), glutamine to ammonium (Y NH4/gln), glucose to lactate (Ylac/glc), glucose to product (YP/glc) and glutamine to product (YP/gln). Among the culture media, the serum-free medium showed to provide better conditions for cell growth (reaching 4 x 106 cells/mL), keeping high cell viabilities for a longer period, in all three tested culture systems. Concerning product formation, hybridoma also released high IgG concentrations (3 µg/mL) in the serum-free medium. Among the culture systems, the best results for cell growth and viability were found inroller bottles at 40 rpm (after adaptation under lower rotation rates) and IgG production was higher in roller bottles at 16 rpm (after adaptation under lower rotation rates) and in spinner flasks at 50 rpm (after adaptation under lower rotation rates in roller bottles, up to 40 rpm). The IgG concentrations ascitic fluid presented concentrations 10 to 20 times higher thanthose obtained in culture supernatants. However, since the volumes of culture supernatant obtained in relatively simple, small-scale culture systems are significantly higher than thoseof mice ascitic fluids, the replacement of in vivoproduction for in vitroIgG production in stirred systems, using serum-free media, seems to be feasible. Nevertheless, additional experiments should be carried out to confirm the feasibility of switching the production of anti-dengue type 2 monoclonal antibodies for in vitrosystems, using the process proposed in this work.

L’imagerie médicale a longtemps été limitée à cause des performances médiocres des fluorophores organiques. Récemment la recherche sur les nanocristaux semi-conducteurs a grandement contribué à l’élargissement de la gamme d’applications de la luminescence dans les domaines de l’imagerie et du diagnostic. Les points quantiques (QDs) sont des nanocristaux de taille similaire aux protéines (2-10 nm) dont la longueur d’onde d’émission dépend de leur taille et de leur composition. Le fait que leur surface peut être fonctionnalisée facilement avec des biomolécules rend leur application particulièrement attrayante dans le milieu biologique. Des QDs de structure « coeur-coquille » ont été synthétisés selon nos besoins en longueur d’onde d’émission. Dans un premier article nous avons modifié la surface des QDs avec des petites molécules bi-fonctionnelles portant des groupes amines, carboxyles ou zwitterions. L’effet de la charge a été analysé sur le mode d’entrée des QDs dans deux types cellulaires. À l’aide d’inhibiteurs pharmacologiques spécifiques à certains modes d’internalisation, nous avons déterminé le mode d’internalisation prédominant. L’endocytose par les radeaux lipidiques représente le mode d’entrée le plus employé pour ces QDs de tailles similaires. D’autres modes participent également, mais à des degrés moindres. Des disparités dans les modes d’entrée ont été observées selon le ligand de surface. Nous avons ensuite analysé l’effet de l’agglomération de différents QDs sur leur internalisation dans des cellules microgliales. La caractérisation des agglomérats dans le milieu de culture cellulaire a été faite par la technique de fractionnement par couplage flux-force (AF4) associé à un détecteur de diffusion de la lumière. En fonction du ligand de surface et de la présence ou non de protéines du sérum, chacun des types de QDs se sont agglomérés de façon différente. À l'aide d’inhibiteur des modes d’internalisation, nous avons corrélé les données de tailles d’agglomérats avec leur mode d’entrée cellulaire. Les cellules microgliales sont les cellules immunitaires du système nerveux central (CNS). Elles répondent aux blessures ou à la présence d’inflammagènes en relâchant des cytokines pro-inflammatoires. Une inflammation non contrôlée du CNS peut conduire à la neurodégénérescence neuronale et est souvent observée dans les cas de maladies chroniques. Nous nous sommes intéressés au développement d’un nanosenseur pour mesurer des biomarqueurs du début de l’inflammation. Les méthodes classiques pour étudier l’inflammation consistent à mesurer le niveau de protéines ou molécules relâchées par les cellules stressées (par exemple monoxyde d’azote, IL-1β). Bien que précises, ces méthodes ne mesurent qu’indirectement l’activité de la caspase-1, responsable de la libération du l’IL-1β. De plus ces méthode ne peuvent pas être utilisées avec des cellules vivantes. Nous avons construit un nanosenseur basé sur le FRET entre un QD et un fluorophore organique reliés entre eux par un peptide qui est spécifiquement clivé par la caspase-1. Pour induire l’inflammation, nous avons utilisé des molécules de lipopolysaccharides (LPS). La molécule de LPS est amphiphile. Dans l’eau le LPS forme des nanoparticules, avec des régions hydrophobes à l’intérieure. Nous avons incorporé des QDs dans ces régions ce qui nous a permis de suivre le cheminement du LPS dans les cellules microgliales. Les LPS-QDs sont internalisés spécifiquement par les récepteurs TLR-4 à la surface des microglies. Le nanosenseur s’est montré fonctionnel dans la détermination de l’activité de la caspase-1 dans cellules microgliales activées par le LPS. Éventuellement, le senseur permettrait d’observer en temps réel l’effet de thérapies ciblant l’inflammation, sur l’activité de la caspase-1. / Medical imaging based on fluorescence has suffered from the poor photostability and mediocre performance of organic fluorophores. The discovery and subsequent improvements in nanocrystal synthesis and functionalization has greatly benefited the applications in medical imaging and the development of nanocrystal-based sensors for diagnostics. QDs are semi-conductor nanocrystals which have similar sizes as proteins (2-10 nm). They are highly luminescent, and can be made to emit at any desired wavelength by varying their size and composition. The surface of QDs can be easily functionalized with biomolecules. Hence, it is interesting to study how QDs interact in the biological world. Highly luminescent core-shell QDs emitting at different wavelengths were prepared according to our needs. In a first study, the surface of the QDs was modified with various small bi-functional thiolated ligands (carboxylated, aminated and zwitterionic). The modified-QDs of nearly identical sizes were administered in vitro to study the impact of surface charge and cell type on the mode and extent of cell uptake and elimination. Using specific inhibitors of cell uptake we determined which modes contributed to the internalization of the QDs. Endocytosis mediated by lipid rafts represented the predominant pathway for the internalization of QDs. However, other modes contributed to a lesser degree, depending on the surface ligand. We then analyzed the effect of QD agglomeration in cell culture media on its cellular uptake by microglia. Thorough characterization of QD agglomerate size distribution was conducted by asymmetrical flow field-flow fractionation (AF4) with a dynamic light scattering detector. Depending on the type of surface ligand and if serum proteins were present, the agglomeration pattern of the QDs was significantly different. With inhibitors of specific modes of cell uptake, we showed that the size distribution data, obtained by AF4, correlated with the modes of cell uptake. Microglia cells are immune cells of the central nervous system (CNS). They respond to injury or the presence of inflammagens by producing pro-inflammatory cytokine. Inflammation in the CNS may lead to loss of neurons, and can found in many chronic diseases. We were interested in building nanosensors to measure the onset of inflammation. Current methods to study inflammation consist in measuring levels of certain proteins or chemicals released by stressed cell (e.g. Western blot or ELISA assay for IL-1β). Although precise, these methods measure indirectly the activity of the enzyme responsible for releasing IL-1β, i.e. caspase-1. Moreover, these methods cannot be applied to live cells. We designed a sensor based on FRET between a QD and a dye linked by a peptide specifically cleaved by the caspase-1. To induce inflammation, we applied lipopolysaccharides (LPS), which are endotoxins present in Gram negative bacteria responsible for sceptic shock. The LPS form nanoparticles due to their amphiphilicity. The interior hydrophobic regions were used to load hydrophobic QDs, making the LPS luminescent. The microglia internalized LPS-QD predominantly through TLR-4 membrane receptors. We describe how the LPS induce inflammation and demonstrated the functionality of the QD-based sensor. Eventually, the sensor could be used to monitor in real time the action of therapeutics against inflammation.