Abstract Epithelial cell-cell adhesions have a critical role in morphogenesis, establishment and maintenance of tissue architecture, cell-cell communication, normal cell growth and differentiation. These adhesions are disrupted during malignant transformation and tumour cell invasion. Several kinases, phosphatases and small GTPases regulate cell-cell contacts. In the present work we investigated the dynamics of cell-cell adhesion structures after microinjection of fluorophore tagged vinculin, during transformation caused by an active Src tyrosine kinase and during Helicobacter pylori infection. The regulatory role of Rac GTPase as well as the behaviour of actin and cadherin were analysed in all these conditions. Microinjection of vinculin into bovine kidney epithelial MDBK cells induced release of actin, cadherin and plakoglobin to cytoplasm of the cells, caused disruption of protein complexes at adherens and tight junctions that finally led to formation of polykaryons. Activated Rac GTPase, in turn, enhanced accumulation of cadherin to membranes and thereby diminished the formation of polykaryons, whereas inactive Rac removed cadherin from membranes. Incorporation of vinculin to lateral membranes took place also in acidifying and depolarising conditions where cell fusions were prevented. Thus, the membrane potential seemed to control fusion ability. In src-MDCK cells, activation of Src kinase led to disintegration of adherens junctions. Clusters of junctional components and bundles of actin were seen at the basal surface already within 30 min after Src activation. p120ctn was the only component of adherens junction whose relocation correlated to its phosphorylation. Inhibition of Src by a specific inhibitor PP2 restored the cubic morphology of the cells and accumulated cadherin back to lateral walls. Still p120ctn remained in cytoplasm and thereby was not responsible for the epithelial phenotype. Activation of Rac GTPase by Tiam1 also increased the amount of cadherin at lateral membranes and maintained the morphology of src-MDCK cells practically normal after activation of Src kinase. In the same way, actin cytoskeleton was reorganised in gastric carcinoma cells in response to infection with H. pylori via activation of Rac signalling pathway. Hence, Rac and cadherin seem to be the major players in the maintenance of epithelial cell morphology.

No description available.

Les protéines DOCK180 et ELMO coopèrent ensemble biochimiquement et génétiquement afin d’activer la GTPase Rac1 lors de plusieurs évènements biologiques. Toutefois, le rôle que jouent ces protéines dans la signalisation par Rac est encore mal compris. Nous émettons l’hypothèse que Dock180 agit comme activateur de Rac, alors que ELMO est requis pour l’intégration de la signalisation de Rac plutôt que son activation per se. Nous postulons que ELMO agit comme signal de localisation intracellulaire afin de restreindre de façon spatio-temporelle la signalisation de Rac en aval de Dock180, et/ou que ELMO agit comme protéine d’échafaudage entre Rac et ses effecteurs pour amplifier la migration cellulaire. Dans l’objectif nº 1, nous démontrons que le domaine PH atypique de ELMO1 est le site d’interaction principal entre cette protéine et DOCK180. De plus, nous démontrons que la liaison entre ELMO et DOCK180 n’est pas nécessaire pour l’activation de Rac, mais est plutôt essentielle pour faciliter la réorganisation du cytosquelette induite par l’activation de Rac en aval de Dock180. Ces résultats impliquent que ELMO pourrait jouer des rôles additionnels dans la signalisation par Rac. Dans l’objectif nº 2, nous avons découvert l’existence d’une homologie structurelle entre ELMO et un module d’autorégulation de la formine Dia1, et avons identifié trois nouveaux domaines dans la protéine ELMO : les domaines RBD, EID et EAD. De façon analogue à Dia1, nous avons découvert que ELMO à l’état basal est autoinhibé grâce à des intéractions intramoléculaires. Nous proposons que l’état d’activation des protéines ELMO est régulé de façon similaire aux formines de la famille Dia, c’est-à-dire grâce à des interactions avec d’autres protéines. Dans l’objectif nº 3, nous identifions un domaine RBD polyvalent chez ELMO. Ce domaine possède une double spécificité pour les GTPases de la famille Rho et Arf. Nous avons découvert que Arl4A agit comme signal de recrutement membranaire pour le module ELMO/DOCK180/Rac. Nos résultats nous permettent de supposer que d’autres GTPases pourraient être impliquées dans l’activation et la localisation de cette voie de signalisation. Nous concluons qu’à l’état basal, ELMO et DOCK180 forment un complexe dans lequel ELMO est dans sa conformation autoinhibée. Bien que le mécanisme d’activation de ELMO ne soit pas encore bien compris, nous avons découvert que, lorsqu’il y a stimulation cellulaire, certaines GTPases liées au GTP peuvent intéragir avec le domaine RBD de ELMO pour relâcher les contacts intramoléculaires et/ou localiser le complexe à la membrane. Ainsi, les GTPases peuvent servir d’ancrage au complexe ELMO/DOCK180 pour assurer une regulation spatiotemporelle adequate de l’activation et de la signalisation de Rac. / DOCK180 and ELMO cooperate biochemically and genetically to activate Rac in several biological events. However, the role of these proteins in Rac signaling is still poorly understood. We hypothesize that DOCK180 functions as a RacGEF, with ELMO binding to DOCK180 being required for integration of proper Rac signaling rather than Rac activation per se. We postulate that ELMO acts as a subcellular targeting signal for spatio-temporal restriction of DOCK180-mediated Rac signaling and/or as a scaffold for Rac effectors to enforce cell migration. In Aim #1, we elucidate that the atypical ELMO1 PH is the major DOCK180 binding site. We demonstrate that the binding of ELMO1 to DOCK180 is not necessary for Rac GTP-loading, but is instead required to facilitate Rac-GTP induced cytoskeletal changes following DOCK180 activation. These results imply additional roles for ELMO in mediating Rac signaling. In Aim #2, we reveal structural homology between ELMO and an autoregulatory module in the formin, Dia1, and identify three novel domains in ELMOs: the RBD, EID and EAD. Analogous to Dia1, we uncovered that ELMO is autoinhibited via intramolecular interactions at basal state. We propose that the activation state of ELMO proteins is regulated, much like in Dia-family formins, via interaction with other proteins. Aim #3 identifies a polyvalent RBD in ELMO with dual specificity for Rho and Arf family GTPases. We found Arl4A as a novel membrane recruitment signal for the ELMO/DOCK180/Rac module. Our results may have broad implications in the activation and localization of this pathway by additional GTPases. We conclude that, at basal levels, ELMO/DOCK180 is complexed, with ELMO in an autoinhibited state in the cytosol. Through cell stimulation, certain GTPases will be activated that now bind the ELMO RBD and alleviate the intramolecular contacts. In this way, the GTPase anchors the activated ELMO/DOCK180 module in place for proper spatio-temporal regulation of Rac activation and signaling.

Recently reported data demonstrate that Vav3, a Rho Guanine Nucleotide Exchange Factor (Rho GEF) is overexpressed in breast tumors, coexpressed with ER, necessary for proliferation in breast cancer cells, and predictive of response to neoadjuvant endocrine therapies in patients with ER+ tumors. Such data beg the question as to what roles Vav3 plays in modulation of steroid receptor activity in breast cancer and in resistance to current hormonal therapies. Using reporter assays, I provide novel evidence that Vav3 potentiates Estrogen Receptor activity and represses Androgen Receptor activity in breast cancer cells. Vav3 potentiates ligand-dependent estrogen receptor activity in the MCF-7. A truncated, constitutively active form of Vav3, caVav3 potentiates ligand dependent ER activity in both MCF-7 and T47D. Vav3 activates Rho GTPases through its GEF domain. ER potentiation by caVav3 is dependent upon GEF activity. A caVav3 mutant with defective GEF function represses basal and ligand-mediated ER activity in T47D. Although other studies have shown that Vav3 could activate various Rho GTPases, only constitutively active Rac1 mutants potentiated ER activity in both cell lines. Contrastingly, reporter assays were used to show that caVav3 inhibits ligand-mediated AR activity in the AR+ T47D cell line by both R1881 and DHT stimulation. caVav3-mediated repression of AR activity is GEF-dependent, as caVav3 GEF mutants potentiate AR activity. Constitutively active forms of Rho GTPases were found to repress AR activity to different extents, but R1881-mediated AR activity was only significantly repressed by caCdc42. My studies of the effect of androgens on AR protein by western blot show that androgens downregulate AR protein in the highly Vav3 positive T47D cell line. Previous studies have demonstrated that androgens stabilize AR protein in MCF-7, and I now provide evidence that overexpression of Vav3 or caVav3 reverses hormone-mediated AR protein stabilization in MCF-7. These data are especially relevant given recently published data that decreased AR protein levels contributed to failure of response to MPA in patients with metastatic breast cancer. Further breast cancer studies may prove Vav3 to be a potential drug target in hormone dependent, hormone independent, and metastatic disease.

We are interested in understanding the molecular mechanisms that govern synaptic growth and plasticity. Recent evidence from several laboratories suggests that small GTPases play an important role in the promotion of neurite outgrowth; however, their role in the control of synaptic growth and functional plasticity is not well understood. The goal of this thesis is to investigate the role of small GTPases (including Rac, Rho and Cdc42) in the regulation of synaptic growth in vivo, using the Drosophila larval neuromuscular junction (NMJ) synapses as a model system. Our results show that presynaptic overexpression of Rac, but not of Rho or Cdc42, positively regulates both synaptic structure and function. Genetic loss of Rac leads to embryonic lethality, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we use the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single motor neuron clones devoid of all genetic copies of Rac. Our data suggest that Rac activity is crucial for normal synaptic development. In support of this conclusion, we demonstrate that genetic removal of trio, a guanine nucleotide exchange factor (GEF) that is known to activate Rac, leads to a drastic reduction in the number of synaptic boutons. In addition, genetic removal of one copy of trio is sufficient to suppress the gain-of-function phenotype of Rac. Moreover, we demonstrate that partial removal of the fragile X mental retardation gene (dfmr1), a known suppressor of Rac, enhances the gain-of-function phenotype of Rac. Taken together, our findings support a model in which Rac signaling positively regulates synaptic growth and function at the Drosophila larval NMJ.

Les protéines DOCK180 et ELMO coopèrent ensemble biochimiquement et génétiquement afin d’activer la GTPase Rac1 lors de plusieurs évènements biologiques. Toutefois, le rôle que jouent ces protéines dans la signalisation par Rac est encore mal compris. Nous émettons l’hypothèse que Dock180 agit comme activateur de Rac, alors que ELMO est requis pour l’intégration de la signalisation de Rac plutôt que son activation per se. Nous postulons que ELMO agit comme signal de localisation intracellulaire afin de restreindre de façon spatio-temporelle la signalisation de Rac en aval de Dock180, et/ou que ELMO agit comme protéine d’échafaudage entre Rac et ses effecteurs pour amplifier la migration cellulaire. Dans l’objectif nº 1, nous démontrons que le domaine PH atypique de ELMO1 est le site d’interaction principal entre cette protéine et DOCK180. De plus, nous démontrons que la liaison entre ELMO et DOCK180 n’est pas nécessaire pour l’activation de Rac, mais est plutôt essentielle pour faciliter la réorganisation du cytosquelette induite par l’activation de Rac en aval de Dock180. Ces résultats impliquent que ELMO pourrait jouer des rôles additionnels dans la signalisation par Rac. Dans l’objectif nº 2, nous avons découvert l’existence d’une homologie structurelle entre ELMO et un module d’autorégulation de la formine Dia1, et avons identifié trois nouveaux domaines dans la protéine ELMO : les domaines RBD, EID et EAD. De façon analogue à Dia1, nous avons découvert que ELMO à l’état basal est autoinhibé grâce à des intéractions intramoléculaires. Nous proposons que l’état d’activation des protéines ELMO est régulé de façon similaire aux formines de la famille Dia, c’est-à-dire grâce à des interactions avec d’autres protéines. Dans l’objectif nº 3, nous identifions un domaine RBD polyvalent chez ELMO. Ce domaine possède une double spécificité pour les GTPases de la famille Rho et Arf. Nous avons découvert que Arl4A agit comme signal de recrutement membranaire pour le module ELMO/DOCK180/Rac. Nos résultats nous permettent de supposer que d’autres GTPases pourraient être impliquées dans l’activation et la localisation de cette voie de signalisation. Nous concluons qu’à l’état basal, ELMO et DOCK180 forment un complexe dans lequel ELMO est dans sa conformation autoinhibée. Bien que le mécanisme d’activation de ELMO ne soit pas encore bien compris, nous avons découvert que, lorsqu’il y a stimulation cellulaire, certaines GTPases liées au GTP peuvent intéragir avec le domaine RBD de ELMO pour relâcher les contacts intramoléculaires et/ou localiser le complexe à la membrane. Ainsi, les GTPases peuvent servir d’ancrage au complexe ELMO/DOCK180 pour assurer une regulation spatiotemporelle adequate de l’activation et de la signalisation de Rac. / DOCK180 and ELMO cooperate biochemically and genetically to activate Rac in several biological events. However, the role of these proteins in Rac signaling is still poorly understood. We hypothesize that DOCK180 functions as a RacGEF, with ELMO binding to DOCK180 being required for integration of proper Rac signaling rather than Rac activation per se. We postulate that ELMO acts as a subcellular targeting signal for spatio-temporal restriction of DOCK180-mediated Rac signaling and/or as a scaffold for Rac effectors to enforce cell migration. In Aim #1, we elucidate that the atypical ELMO1 PH is the major DOCK180 binding site. We demonstrate that the binding of ELMO1 to DOCK180 is not necessary for Rac GTP-loading, but is instead required to facilitate Rac-GTP induced cytoskeletal changes following DOCK180 activation. These results imply additional roles for ELMO in mediating Rac signaling. In Aim #2, we reveal structural homology between ELMO and an autoregulatory module in the formin, Dia1, and identify three novel domains in ELMOs: the RBD, EID and EAD. Analogous to Dia1, we uncovered that ELMO is autoinhibited via intramolecular interactions at basal state. We propose that the activation state of ELMO proteins is regulated, much like in Dia-family formins, via interaction with other proteins. Aim #3 identifies a polyvalent RBD in ELMO with dual specificity for Rho and Arf family GTPases. We found Arl4A as a novel membrane recruitment signal for the ELMO/DOCK180/Rac module. Our results may have broad implications in the activation and localization of this pathway by additional GTPases. We conclude that, at basal levels, ELMO/DOCK180 is complexed, with ELMO in an autoinhibited state in the cytosol. Through cell stimulation, certain GTPases will be activated that now bind the ELMO RBD and alleviate the intramolecular contacts. In this way, the GTPase anchors the activated ELMO/DOCK180 module in place for proper spatio-temporal regulation of Rac activation and signaling.

No description available.

Des ligands diketimines porteurs de substituants N-benzyl, N-9-anthrylmethyl et N-mesitylmethyl (nacnacBnH, nacnacAnH, and nacnacMesH) ont été synthétisés par condensation d’une amine et d’acétyl acétone ou son monoacétal d’éthylène glycol. La chlorination de la position 3 a été effectuée à l’aide de N-chlorosuccinimide conduisant à la formation des ligands ClnacnacBnH et ClnacnacAnH. Cette même position 3 a également été substituée par un groupement succinimide par lithiation du nacnacBnH, suivi de la réaction avec le N-chlorosuccinimide (3-succinimido-nacnacBnH). Les ligands N-aryl nacnacippH et nacnacNaphH (ipp = 2-isopropylphenyl, Naph = 1-naphthyl) ont été préparés selon les procédures reportées dans la littérature. La réaction de ces ligands avec Zn(TMSA)2 (TMSA = N(SiMe3)2) conduit à la formation des complexes nacnacAnZn(TMSA) et ClnacnacBnZn(TMSA). La protonation avec l’isopropanol permet l’obtention des complexes nacnacAnZnOiPr et ClnacnacBnZnOiPr. La réaction avec Mg(TMSA)2 permet quant à elle la formation des complexes nacnacAnMg(TMSA), nacnacMesMg(TMSA), ClnacnacBnMg(TMSA) et ClnacnacAnMg(TMSA). La protonation subséquente à l’aide du tert-butanol permet l’obtention du nacnacMesMgOtBu et du ClnacnacBnMgOtBu, alors que l’on observe uniquement une décomposition avec les ligands possédant des substituants N-anthrylmethyl. La réaction de ces diketimines avec Cu(OiPr)2 conduit aux dimères hétéroleptiques [nacnacBnCu(μ-OiPr)]2 et [3-Cl-nacnacBnCu(μ-OiPr)]2 lors de l’usage des ligands stériquement peu encombrés. Lors de l’utilisation de ligands plus encombrés, la stabilisation du complexe hétéroleptique par dimérisation n’est plus possible, conduisant, par un échange de ligand, à la formation des complexes homoleptiques Cu(nacnacipp)2 et Cu(nacnacNaph)2. Les complexes homoleptiques Cu(nacnacBn)2 et Cu(3-succinimido-nacnacBn)2 ont été obtenus à partir des ligands N-benzyl. Les ligands encore plus encombrés tels que nacnacAnH, nacnacMesH ou ceux comportant des substituants N-methylbenzyl ne présentent alors plus de réactivité avec le Cu(OiPr)2. La plupart des complexes ont été caractérisés par Diffraction des Rayons X. Les complexes homoleptiques ainsi que ceux de TMSA sont monomériques, alors que ceux formés à partir d’alkoxides se présentent sous forme de dimères à l’état solide. Tous les complexes d’alkoxides ainsi que les nacnacAnMg(TMSA)/BnOH et ClnacnacAnMg(TMSA)/BnOH présentent une réactivité modérée à haute en matière de polymérisation du rac-lactide (90% de conversion en 30 secondes à 3 heures). Le nacnacAnZnOiPr permet la synthèse d’un polymère hautement hétérotactique (Pr = 0.90) quand le ClnacnacBnMgOtBu/BnOH génère un polymère isotactique à -30°C (Pr = 0.43). Tous les autres catalyseurs produisent des polymères atactiques avec une légère tendance hétérotactique (Pr = 0.48 – 0.55). Les complexes hétéroleptiques [nacnacBnCu(μ-OiPr)]2 et [3-Cl-nacnacBnCu(μ-OiPr)]2 se révèlent être de très bons catalyseurs pour la polymérisation du rac-lactide présentant une conversion complète du monomère à température ambiante, en solution, en 0,5 à 5 minutes. Le [nacnacBnCu(μ-OiPr)]2 est actif en présence ou absence d’isopropanol, agissant comme agent de transfert de chaine à haute activité (k2 = 32 M–1•s–1) dans le dichlorométhane. Dans l’acétonitrile, le THF, le dichloromethane et le toluène, [nacnacBnCu(μ-OiPr)]2 conduit à une étroite polydispersité, possédant respectivement des kobs = 2.4(1), 5.3(5), 3.6-4.4 and 10(1) min–1. Aucune réaction parasite, telle qu’une trans-esterification, une épimerisation ou une décomposition du catalyseur, n’a été observée. Les complexes homoleptiques en présence d’alcool libre semblent présenter un équilibre avec une petite quantité de leurs équivalents hétéroleptiques, permettant une polymérisation complète, en moins de 60 min, à température ambiante. Tous les catalyseurs de cuivre présentent un haut contrôle de la polymérisation avec une polydispersité égale ou inférieure à 1.1. Les polymères obtenus sont essentiellement atactiques, avec une légère tendance à l’hétérotacticité à température ambiante et -17°C. Le [nacnacBnCu(μ-OiPr)]2 polymérise également la -butyrolactone (BL), l’-caprolactone (CL) et la -valerolactone (VL) avec des constantes respectivement égales à kobs = 3.0(1)•10–2, 1.2–2.7•10–2, et 0.11(1) min–1. Les homopolymères présentent une étroite polydispersité d’approximativement 1.1. Les polymérisations par addition séquentielle ont mis en évidence une trans-estérification (non observée dans les homopolymérisations) si BL ou CL sont introduits après un bloc lactide. / Diketimine ligands bearing N-benzyl, N-9-anthrylmethyl and N-mesitylmethyl substituents (nacnacBnH, nacnacAnH, and nacnacMesH) were prepared from condensation of amine with either acetyl acetone or its ethylene glycol monoketal. Chlorination of the 3-position was achieved using N-chlorosuccinimide, yielding ClnacnacBnH and ClnacnacAnH. The 3-position was also substituted by succinimido by lithiation of nacnacBnH followed by reaction with N-chlorosuccinimide (3-succinimido-nacnacBnH). N-aryl ligands nacnacippH and nacnacNaphH (ipp = 2-isopropylphenyl, Naph = 1-naphthyl) were prepared from literature. The ligands were reacted with Zn(TMSA)2 (TMSA = N(SiMe3)2) to yield nacnacAnZn(TMSA) and ClnacnacBnZn(TMSA). Protonation with isopropanol gave nacnacAnZnOiPr and ClnacnacBnZnOiPr. Reaction of the diketimines with Mg(TMSA)2 afforded nacnacAnMg(TMSA), nacnacMesMg(TMSA), ClnacnacBnMg(TMSA) and ClnacnacAnMg(TMSA). Subsequent protonation with tert-butanol produced nacnacMesMgOtBu and ClnacnacBnMgOtBu, but only decomposition was observed with N-anthrylmethyl substituents. Reaction of the diketimines with Cu(OiPr)2 yielded the heteroleptic [nacnacBnCu(μ-OiPr)]2 and [3-Cl-nacnacBnCu(μ-OiPr)]2 when using sterically undemanding ligands. When sterically more demanding diketimines were used, stabilization of the heteroleptic complex by dimerization was not possible, resulting in the formation of the homoleptic complexes Cu(nacnacipp)2 and Cu(nacnacNaph)2 by ligand exchange. Homoleptic complexes were also prepared with N-benzyl ligands, i. e. Cu(nacnacBn)2 and Cu(3-succinimido-nacnacBn)2. Even bulkier ligands such as nacnacAnH, nacnacMesH or N-methylbenzyl substituents failed to react with Cu(OiPr)2. Most complexes were characterized by single crystal X-ray diffraction. TMSA complexes and homoleptic complexes were monomeric, alkoxide complexes were dimeric in the solid state. All alkoxide complexes, as well as nacnacAnMg(TMSA)/BnOH and ClnacnacAnMg(TMSA)/BnOH were moderately to highly active in rac-lactide polymerization (90% conversion in 30 sec to 3 h). nacnacAnZnOiPr produced highly heterotactic polymer (Pr = 0.90), ClnacnacBnMgOtBu/BnOH produced slightly isotactic polymer at –30 °C (Pr = 0.43), all other catalysts produced atactic polymers with a slight heterotactic bias (Pr = 0.48 – 0.55). Heteroleptic complexes [nacnacBnCu(μ-OiPr)]2 and [3-Cl-nacnacBnCu(μ-OiPr)]2 are very highly active rac-lactide polymerization catalysts, with complete monomer conversion at ambient temperature in solution in 0.5 – 5 min. [nacnacBnCu(μ-OiPr)]2 specifically polymerized in the presence or absence of isopropanol as a chain-transfer reagent with very high activity (k2 = 32 M–1•s–1), in methylene chloride. While in acetonitrile, THF, dichloromethane and toluene has a kobs = 2.4(1), 5.3(5), 3.6-4.4 and 10(1) min–1, respectively. [nacnacBnCu(μ-OiPr)]2 yields narrow polydispersities and no evidence of side reactions such as transesterification, epimerization or catalyst decomposition. The homoleptic complexes in the presence of free alcohol, seem to be in equilibrium with small amounts of the respective heteroleptic complex, which are sufficient to complete polymerization in less than 60 min at room temperature. All Cu catalysts show high control of polymerization with polydispersities of 1.1 and below. The obtained polymers were essentially atactic, with a slight heterotactic bias at ambient temperature and at –17 °C. [nacnacBnCu(μ-OiPr)]2 polymerizes -butyrolactone (BL), -caprolactone (CL) and -valerolactone (VL) with rate constants of kobs = 3.0(1)•10–2, 1.2–2.7•10–2, and 0.11(1) min–1, respectively. Homopolymers showed narrow polydispersities of appr. 1.1. Sequential addition polymerizations showed evidence for transesterification (not seen in homopolymerizations) if BL or CL are introduced after a lactide block.

Um dos principais resíduos gerados nos aterros é o lixiviado, que possui elevada concentração de matéria orgânica biodegradável e refratária e de matéria inorgânica, como nitrogênio amoniacal e metais pesados. O tratamento combinado de lixiviado com esgoto sanitário tem sido utilizado em algumas estações de tratamento de esgoto (ETE) brasileiras. No entanto, o processo ainda sofre vários questionamentos e incertezas, especialmente sobre os efeitos da adição do lixiviado sobre o sistema de tratamento. Nesse contexto, o presente trabalho visou avaliar a eficiência do tratamento combinado de lixiviado/esgoto em sistema de lodos ativados, em diferentes condições. Na primeira etapa desta pesquisa, foram realizados experimentos de tratabilidade em escala de bancada (regime de batelada) utilizando as proporções volumétricas de 0% (controle), 0,2%, 2% e 5% de lixiviado em diferentes condições experimentais. Dentre eles, o Experimento 2 (lixiviado pré-tratado por alcalinização e air stripping) se mostrou mais viável tecnicamente, alcançando eficiências de remoção da DBO, da DQO e do COD acima de 97, 82, 60%, respectivamente, até a proporção de 2% de lixiviado pré-tratado. Na segunda etapa da pesquisa, foi avaliada a tratabilidade utilizando reatores em escala piloto (regime contínuo) tratando esgoto sanitário com 2% (P1) e 0% (P2-controle) de lixiviado pré-tratado, com relação microrganismo-alimento de 0,22 kgDBO/kgSSV.dia, tempo de detenção hidráulica de 24 horas e idade do lodo de 20 dias. Os resultados indicaram viabilidade do tratamento nas condições estudadas com 2% de lixiviado pré-tratado, cujas eficiências médias de remoção da DBO, da DQO, do COD foram de 93, 84 e 60%, respectivamente. Esta pesquisa foi desenvolvida com apoio financeiro (Processo N° 2010/51955-2) da Fundação de Amparo à Pesquisa de São Paulo (FAPESP). / The leachate is one of the main wastes generated in landfills, it has a high concentration of biodegradable and refractory organic matter and inorganic matter, such as ammonia and heavy metals. The combined treatment of leachate with sewage has been used in various sewage treatment plants in Brazil. However, there are still many questions and uncertainties about the process, especially the effects of adding leachate on the treatment system. In this context, this study aimed at evaluating the efficiency of combined treatment of leachate/sewage in activated sludge under different conditions. In the first stage of this research, treatability experiments were carried out in a bench scale (SBR) using the volumetric proportions of 0% (control), 0.2%, 2% and 5% leachate under different experimental conditions. The Experiment 2 (leachate pretreated by alkalinization and air stripping) was more technically feasible, achieving efficiencies of removal of BOD, COD and DOC above 97, 82, 60%, respectively, until the proportion of 2% pre-treated leachate. In the second stage of the research was evaluated the treatability using pilot-scale reactors (continuous flow) for sewage treatment with 2% (P1) and 0% (P2-control) leachate pretreated. The operational parameters adopted were food-microorganism rate of 0.22 kgDBO/ kgSSV.d, hydraulic retention time of 24 hours and the sludge retention time of 20 days. The results indicated the viability of the combined treatment with 2% leached pretreated, whose average efficiency of removing BOD, COD, DOC were 93, 84 and 60% respectively. This research was developed with financial support (case No. 2010/2-51955) of Fundação de Amparo à Pesquisa de São Paulo (FAPESP).

Rho GTPases and their regulators such as guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) represent an important class of molecules controlling dendritic spine plasticity. Although they are typically described as cytoskeletal modulators, roles for the GTPases in endocytosis and cell polarity establishment have also been defined. The neuronal proteins a1- and a2-chimaerins belong to a group of Rac and Cdc42 GAPs that inactivate these GTPases; in addition to a GAP domain, the a-chimaerins share a phosphokinase C (PKC)-like C1 domain but have distinct N-terminal domains (NTDs). My project has explored the importance of specific domains of a1-chimaerin both in induction of a morphological cellular protrusion collapse phenotype ('circularisation') and in interactions with partner proteins that may help to explain the phenotype. The results described in my thesis show that a1-chimaerin possesses a previously undescribed C-terminal domain (CTD) that is indispensable for the ability of the protein to induce collapse of protrusions, and consequent circularisation, in various cell types; moreover, an intact CTD is also important for association of a1-chimaerin with its known effector EphA4, and potentially with other undefined membrane proteins, in a C1-domain- dependent manner. In addition, my results show that a1-chimaerin associates via its NTD with the Src kinase Fyn, and via its C1 domain with the NR2A subunit of the NMDA receptor. Further experiments explored a1-chimaerin effects on EphA4 and NMDA receptor cell surface expression, as well as binding to other putative partners - including the adaptor protein p35 and the polarity protein PAR6. Finally, I have shown that inhibition of a pathway involving the Rho-associated coiled-coil containing protein kinase (ROCK) reverts circularisation induced by a1- chimaerin, and that a blocking peptide based on the CTD may be employed to partially counteract the phenotype. These results uncover a novel domain in a1-chimaerin that may have a crucial importance for the induction of cellular process collapse by a1-chimaerin with a potential relevance to the EphA4-induced dendritic spine retraction, EphA4 receptor endocytosis, and cell surface expression of NR2A-containing NMDA receptors. This suggests a model of a multi-protein signalling complex involving a1-chimaerin that coordinates cellular process remodelling, and that is likely to be important both for adult neuronal circuit plasticity and for neurodegenerative diseases.