L'anticorps recombinant anti-CD4 13B8.2 a démontré des capacités de lyse dépendante du complément (CDC) et d'inhibition de la prolifération cellulaire dans le cadre du traitement des tumeurs hématopoïétiques CD4+. Bien qu'il ait été démontré que son action antiproliférative passe par une modulation du facteur de transcription NF-(kappa)B, les mécanismes précis y conduisant restent encore inexpliqués. Au sein du laboratoire, de précédentes études ont démontré que l'anticorps anti-CD4 13B8.2 induit une réorganisation et une modulation des protéines dans les rafts, en y concentrant le CD4 et en y excluant les protéines ZAP70, SLP76, PLC(nu)1 et Vav1. Nous avons tenté d'expliquer les mécanismes qui participent à la modulation des protéines au sein des microdomaines membranaires lipidiques, et nous proposons que la voie de signalisation Carma1 soit impliquée dans les effets observés sur le facteur de transcription NF-(kappa)B. Cette étude démontre que les effets biologiques de l'anticorps recombinant 13B8.2 passent par une modulation de la voie Carma1, Bcl10, MALT1. L'anticorps 13B8.2 induit une réorganisation de ces protéines en dehors des microdomaines membranaires lipidiques avec une dissociation du complexe qui ne peut plus transmettre la signalisation conduisant à l'activation de NF-(kappa)B. Nous avons également mis en évidence que les réorganisations protéiques au niveau de la membrane, induites par l'anticorps anti-CD4 13B8.2 s'accompagnent d'une modulation des espèces lipidiques des microdomaines. Ainsi, nous observons une augmentation du niveau des acides gras de type C18 :0, et une augmentation de l'activité d'une sphingomyelinase acide qui conduit à une augmentation du taux de céramide. Parallèlement l'augmentation de céramide, induite par une sphingomyelinase bactérienne induit la réorganisation de ZAP70 hors des microdomaines, de façon comparable à l'anticorps 13B8.2. Cette meilleure connaissance des mécanismes de signalisation de l'anticorps anti-CD4 13B8.2, permettra de dégager de nouvelles approches thérapeutiques en vue de la modulation de la prolifération des cellules T et de leur sensibilisation à la chimiothérapie. Ce travail ouvre la voie à des possibilités de thérapies ciblant les microdomaines lipidiques. / The recombinant anti-CD4 antibody 13B8.2 demonstrated capabilities complement dependent lysis (CDC) and inhibition of cell proliferation in the treatment of hematopoietic tumors CD4 +. Although it has been demonstrated that its antiproliferative activity through modulation of transcription factor NF-kB, the precise mechanisms leading to them remain unexplained. Within the laboratory, previous studies have shown that anti-CD4 13B8.2 induced a reorganization and modulation of proteins in rafts by concentrating CD4 and by excluding proteins ZAP70, SLP76, Vav1 and PLC(nu)1 . We tried to explain the mechanisms involved in the modulation of proteins within lipid microdomains, and we propose that the signaling pathway is involved in Carma1 observed effects on the transcription factor NF-kB. This study demonstrates that the biological effects of recombinant antibody 13B8.2 pass through a modulation of the channel Carma1, Bcl10, MALT1. 13B8.2 antibody induces a reorganization of these proteins outside the lipid membrane microdomains with a dissociation of the complex that can no longer transmit signals leading to activation of NF-kB. We also demonstrated that the protein reorganization at the membrane induced by anti-CD4 13B8.2 accompanied by modulation of lipid microdomains species. Thus, we see an increased level of fatty acids like C18: 0, and increased activity of acid sphingomyelinase leads to an increased rate of ceramide. Parallel increase of ceramide, induced by bacterial sphingomyelinase induced reorganization of ZAP70 outside microdomains similarly to the antibody 13B8.2. A better understanding of signaling mechanisms of the anti-CD4 13B8.2, will identify new therapeutic approaches for modulating the proliferation of T cells and their sensitization to chemotherapy. This work paves the way for potential therapies targeted to lipid microdomains.

La reconnaissance d'antigènes par le TCR active des protéines tyrosine kinases qui phosphorylent d'autres substrats intracellulaires dont LAT. Ceci engendre l'activation de molécules telles que PKCθ et CARMA-1. La mutation LatY136F associe des TCR "estropiés" dans le développement de cellules T effectrices générant des désordres lymphoprolifératifs. Nous avons essayé de comprendre les gènes aggravant ou empêchant cette lymphoprolifération en utilisant la mutagénèse ENU. Nous avons identifié une mutation appelée Basilic empêchant le déroulement de la pathologie LatY136F. Basilic est une mutation du gène Rltpr qui constitue une phénocopie de Cd28-/- sur fond sauvage et sur fond LatY136F. Rltpr est un une nouvelle protéine ayant de multiples domaines, qui appartient à la famille CARMIL et qui est exprimée dans les cellules T et B. L'objectif de ce travail était d'élucider les mécanismes au cours desquels CD28 et Rltpr coopèrent avec le TCR pour différencier des cellules T naïves en cellules T effectrices. Ce travail visait aussi à caractériser Rltpr, dont la structure/fonction et l'interactome sont encore inconnus. En utilisant des techniques de microscopie confocale, nous avons montré que la localisation et le recrutement de Rltpr et de RltprBas à la synapse immunologique sont tous deux CD28-dépendants. Les deux molécules colocalisent avec CD28 tout au long du processus d'activation. En outre, Rltpr est essentiel pour la translocation à la synapse de PKCθ et CARMA-1, qui sont induits lors de la co-stimulation par CD28. Ces résultats permettent une meilleure compréhension du fin réglage du système immunitaire adaptatif qui est mis en place lors de l'activation. / TCR recognition of antigens triggers the activation of protein tyrosine kinases that phosphorylate other intracellular substrates including LAT. LAT phosphorylation leads to the activation of PKCθ and CARMA-1. The point mutation LatY136F associates TCRs with crippled signaling abilities to the development of effector T cells generating lymphoproliferative disorders (LPDs). We tried to shed light on genes exacerbating or preventing the LatY136F LPD by using an ENU mutagenesis screening. We identified one point mutation called Basilic that prevents the unfolding of the LatY136F pathology. Basilic is a point mutation of the Rltpr gene and is a phenocopy of a Cd28-/- mutation both on a wild-type and on a LatY136F background. Rltpr is a newly-discovered, multidomain protein belonging to the CARMIL family that is expressed in T and B cells. The objective of the present work was to elucidate the mechanisms during which CD28 and Rltpr cooperate withthe TCR to differentiate naïve into effector T cells. I also aimed at characterizing the Rltpr molecule, whosestructure/function and interactome are still largely unknown. Using confocal microscopy in collaborationwith Takashi Saito's group and Christoph Wülfing we showed that the localization and the recruitment ofboth Rltpr and RltprBas at the immune synapse are CD28-dependent. The two molecules colocalize with CD28all along the activation process. Moreover, Rltpr is essential for the synapse translocation of PKCθ andCARMA-1, which are induced upon CD28 costimulation. Those results allow a better understanding of theadaptive immune system fine tuning upon activation.

Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 RalfoGP_DISSERT_partes_autorizadas.pdf: 608925 bytes, checksum: dbfcce5de284f89c5cdaee179cdd9bc2 (MD5) Previous issue date: 2010-12-02 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The microorganisms play very important roles in maintaining ecosystems, which explains the enormous interest in understanding the relationship between these organisms as well as between them and the environment. It is estimated that the total number of prokaryotic cells on Earth is between 4 and 6 x 1030, constituting an enormous biological and genetic pool to be explored. Although currently only 1% of all this wealth can be cultivated by standard laboratory techniques, metagenomic tools allow access to the genomic potential of environmental samples in a independent culture manner, and in combination with third generation sequencing technologies, the samples coverage become even greater. Soils, in particular, are the major reservoirs of this diversity, and many important environments around us, as the Brazilian biomes Caatinga and Atlantic Forest, are poorly studied. Thus, the genetic material from environmental soil samples of Caatinga and Atlantic Forest biomes were extracted by direct techniques, pyrosequenced, and the sequences generated were analyzed by bioinformatics programs (MEGAN MG-RAST and WEBCarma). Taxonomic comparative profiles of the samples showed that the phyla Proteobacteria, Actinobacteria, Acidobacteria and Planctomycetes were the most representative. In addition, fungi of the phylum Ascomycota were identified predominantly in the soil sample from the Atlantic Forest. Metabolic profiles showed that despite the existence of environmental differences, sequences from both samples were similarly placed in the various functional subsystems, indicating no specific habitat functions. This work, a pioneer in taxonomic and metabolic comparative analysis of soil samples from Brazilian biomes, contributes to the knowledge of these complex environmental systems, so far little explored / Os microorganismos desempenham importantes fun??es na manuten??o dos ecossistemas, o que explica o enorme interesse em compreender as rela??es existentes entre estes organismos, bem como entre eles e o meio. Estima-se que o n?mero total de c?lulas procari?ticas na Terra seja entre 4 e 6 x 1030, constituindo um enorme pool biol?gico e gen?tico a ser explorado. Apesar de atualmente apenas 1% de toda essa riqueza poder ser cultivada por t?cnicas laboratoriais padr?o, ferramentas metagen?micas permitem o acesso ao potencial gen?mico de amostras ambientais de forma independente de cultivo, e em associa??o com tecnologias de sequenciamento da terceira gera??o, a cobertura amostral se torna ainda maior. Solos, em particular, s?o os maiores reservat?rios dessa diversidade, e muitos ambientes importantes ao nosso redor, como os biomas brasileiros Caatinga e Mata Atl?ntica, s?o pouco estudados. Sendo assim, o material gen?tico ambiental de amostras de solo dos biomas Caatinga e Mata Atl?ntica foi extra?do atrav?s de t?cnicas diretas, pirosequenciado, e as seq??ncias geradas foram analisadas atrav?s de programas de bioinform?tica (MEGAN, MG-RAST e WEBCarma). Perfis taxon?micos comparativos das amostras mostraram que os filos Proteobacteria, Actinobacteria, Acidobacteria e Planctomycetes foram os mais representativos. Em adi??o, fungos do filo Ascomycota foram identificados predominantemente na amostra de solo de Mata Atl?ntica. Perfis metab?licos mostraram que, apesar da exist?ncia de diferen?as ambientais, sequ?ncias de ambas as amostras foram inseridas similarmente nos diversos subsistemas funcionais, n?o indicando fun??es habitat espec?ficas. Este trabalho, pioneiro em an?lises taxon?micas e metab?licas comparativas de amostras de solo de biomas brasileiros, contribui para o conhecimento destes sistemas ambientais complexos, at? ent?o pouco explorados

Theories of socio-cultural change regarding the transition from the Chalcolithic to the Bronze Age in Cyprus have since the nascence of prehistoric archaeology on the island been the subject of lively debate between archaeologists who argue for internal versus external evolution processes. Yet, despite all efforts, a coherent model explaining the evident material culture differences between the two epochs remains elusive, an indication that the current polarization of theories masks inherent complexities of the archaeological record. Moving beyond the internal/external dichotomy, the present thesis argues for one such explanatory model and approaches this notable transition from three distinct and less explored aspects, namely chronology, pottery analysis and topography. Starting with chronology, the thesis assesses previous chronological schemata, examines issues of methodology, performs an in-depth data quality analysis, and, on the basis of the creative dialogue between absolute and relative dating data, proposes a novel chronology for the island. This chronology transcends linearity by adopting cultural period overlaps and differential regional adoption of technologies. Moving to spatial matters, the study disentangles space-time systematics for sites dating from the Middle Chalcolithic to the Philia Phase. In effect, it establishes a ceramic typology for the Chalcolithic that is applicable to the entire island; clarifies and records in a custom-made recording system, dubbed CARMA (Cyprus ARchaeological MAterials Relational Database System), the research history and material assemblages of each site; situates sites in the physical landscape of Cyprus and performs socio-spatial analyses, where the results of pottery analysis are interwoven with the spatial relationships between sites. The last analysis provides positive evidence for cultural uniformity in the Middle Chalcolithic, for the emergence of regional cultures in the Late Chalcolithic and the abandonment of settlements at the beginning of the Philia Phase, and for the co-existence of spatially distinct cultures during the Philia Phase. Lastly, the results of the chronology and spatial studies inform the data synthesis in the final section, where a different narrative of socio-cultural change is developed. This argues for the emergence of divergences already in the Late Chalcolithic, for the co-existence and uneven bi-directional interaction of indigenous and foreign populations during the Philia Phase, and for the development of regionalism in the Early Bronze Age as a result of variable adoption of technologies, entanglement and resistance to cultural identity assimilation.