Myxococcus xanthus Dif proteins are chemotaxis homologues that regulate exopolysaccharide (EPS) biogenesis. Previous genetic studies suggested that Dif protein might interact with one another as do the chemotaxis proteins in enterics. The interactions among Dif proteins were since investigated with the yeast two-hybrid (Y2H) system. The results indicate that DifC interacts with both DifA and DifE. Using a modified Y2H system, DifC was shown to be able to bring DifA and DifE into a protein complex. Further Y2H experiments demonstrated that the different conserved domains of DifE likely function as their counterparts of CheA-type kinases because the putative P2 domain of DifE interacts with DifD, P5 with DifC and the dimerization domain P3 with itself. Similarly, DifA can interact with itself through its C-terminal region. In addition, DifG was found to interact with the CheY homologue DifD. These findings support the notion that Dif proteins constitute a unique chemotaxis-like signal transduction pathway in M. xanthus. In addition, KapB, a TPR (Tetratricopeptide repeats) protein, was identified as an interacting partner of DifE byY2H library screening. Further analysis demonstrated that the N-terminal half of KapB interacted with the putative P2 domain of DifE. KapB had been previously reported to interact with several Serine/Threonine (Ser/Thr) kinase pathways including the Pkn4-Pfk pathway. This pathway is implicated in glycogen metabolism in M. xanthus by a previous report. In this study, kapB as well as pfkn deletion mutants were found to overproduce EPS. It was also found that the Dif pathway is involved in glycogen metabolism because the glycogen level is altered in dif mutants. These results indicate EPS biogenesis and glycogen metabolism may be coordinately regulated. This coordination of the Dif-regulated EPS production and the Pkn4-regulated glycogen metabolism appears to involve KapB. This is the first example of a TPR protein mediating the interplays of a histidine kinase pathway and a Ser/Thr kinase pathway. / Master of Science

No description available.

The motility of neutrophils and their ability to sense and to react to chemoattractants in their environment are of central importance for the innate immunity. Neutrophils are guided towards sites of inflammation following the activation of G-protein coupled chemoattractant receptors such as CXCR2 whose signaling strongly depends on the activity of Ca²⁺ permeable TRPC6 channels. It is the aim of this study to analyze data sets obtained in vitro (murine neutrophils) and in vivo (zebrafish neutrophils) with a stochastic mathematical model to gain deeper insight into the underlying mechanisms. The model is based on the analysis of trajectories of individual neutrophils. Bayesian data analysis, including the covariances of positions for fractional Brownian motion as well as for exponentially and power-law tempered model variants, allows the estimation of parameters and model selection. Our model-based analysis reveals that wildtype neutrophils show pure superdiffusive fractional Brownian motion. This so-called anomalous dynamics is characterized by temporal long-range correlations for the movement into the direction of the chemotactic CXCL1 gradient. Pure superdiffusion is absent vertically to this gradient. This points to an asymmetric ‘memory’ of the migratory machinery, which is found both in vitro and in vivo. CXCR2 blockade and TRPC6-knockout cause tempering of temporal correlations in the chemotactic gradient. This can be interpreted as a progressive loss of memory, which leads to a marked reduction of chemotaxis and search efficiency of neutrophils. In summary, our findings indicate that spatially differential regulation of anomalous dynamics appears to play a central role in guiding efficient chemotactic behavior.

Primäre und sekundäre Lymphome des zentralen Nervensystems (ZNS), PCNSL und SCNSL, sind schwer behandelbar und mit einer ungünstigen Prognose assoziiert. Der ZNS-Tropismus dieser Lymphome hängt mit einer Dysregulation von Genen zusammen, die für Immunabwehr, Chemotaxis, Zellmigration und die Blut-Hirn-Schranke (BBB) relevant sind. Zur Untersuchung der molekularen Mechanismen wurde das Eμ-myc-Mausmodell genutzt, um die Faktoren zu analysieren, die zur ZNS-Tropie beitragen, insbesondere jene, die die BBB destabilisieren und die Immunantwort beeinflussen. Eμ-myc-Lymphome wurden in C57BL/6-Mäuse transplantiert und ZNS-positive von ZNS-negativen Lymphomen histologisch unterschieden. RNA-Sequenzierungen identifizierten Signalwege, die mit dem ZNS-Tropismus korreliert sind. Der NF-κB-Signalweg zeigte hierbei besondere Relevanz: Seine Hemmung in ZNS-positiven Lymphomen verringerte die ZNS-Tropie und stabilisierte die BBB durch den Erhalt der Tight-Junction-Proteine. Zudem ergab sich eine Hochregulation von Gfrα-1 in ZNS-positiven Lymphomen, was eine verstärkte Zellmigration zu hirn-konditioniertem Medium bewirkte. Durch die Aktivierung von Astrozyten und GDNF-Hochregulation wurde die BBB zusätzlich destabilisiert. ZNS-trope Lymphome induzierten Immunmodulationen benachbarter Zellen, wie Mikroglia und Astrozyten, und verstärkten den ZNS-Tropismus durch post-seneszente Signaturen. Das Zusammenspiel zwischen Lymphomzellen und Astrozyten, die Aktivierung der GDNF/Gfrα-1/RET-Signalachse und die BBB-Störung gelten als zentrale Mechanismen der ZNS-Tropie bei DLBCL-ähnlichen Lymphomen. Die gezielte Modulation dieser molekularen Pfade könnte die ZNS-Beteiligung bei aggressiven B-Zell-Lymphomen reduzieren. Weitere Untersuchungen könnten therapeutische Ansätze zur Minderung der ZNS-Tropie und Verbesserung der klinischen Ergebnisse bieten. / Primary and secondary CNS lymphomas (PCNSL and SCNSL) are difficult to treat, with a poor prognosis. CNS tropism in these lymphomas involves immune evasion, chemotaxis, cell migration, and blood-brain barrier (BBB) integrity. This study aimed to identify factors behind CNS tropism using the Eμ-myc mouse lymphoma model to compare CNS-tropic and non-tropic lymphomas. Methods: Eμ-myc lymphomas were transplanted into immunocompetent mice, classified as CNS (+) or CNS (-) by histology. RNA sequencing identified pathways linked to CNS tropism, focusing on NF-κB, which was suppressed using IκBα∆N in CNS (+) lymphomas in vitro and in vivo. BBB disruption was assessed through astrocyte activation and ZO-1 degradation, while BBB permeability was evaluated with the Evans Blue assay. Lymphoma migration towards GDNF-conditioned medium was tested via Boyden chambers, with GDNF/Gfrα-1/RET pathway involvement assessed by knockdown and RET inhibition. Immune modulation was analyzed by PD-L1 expression in astrocytes and microglia co-cultured with CNS lymphomas. Results: RNA sequencing showed upregulation of NF-κB targets, pro-inflammatory cytokines, and SASP in CNS (+) lymphomas. NF-κB inhibition prevented CNS tropism and preserved BBB integrity by blocking ZO-1 degradation. CNS (+) lymphomas displayed Gfrα-1 upregulation and migration toward brain-conditioned medium. Astrocyte activation and GDNF upregulation by CNS (+) lymphomas further impaired BBB stability. CNS (+) lymphomas induced immune modulation in neighboring astrocytes and microglia, with a post-senescence signature linked to CNS tropism. Interactions between lymphoma cells, astrocytes, the GDNF/Gfrα-1/RET axis, and BBB disruption are key to CNS tropism in DLBCL-like lymphomas. Targeting these pathways may help prevent CNS involvement in aggressive B-cell lymphomas, offering potential therapeutic avenues to improve patient outcomes.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

Shewanella oneidensis est une bactérie aquatique capable de chimiotactisme, c'est-à-dire d'orienter sa nage en réponse aux signaux qu'elle perçoit. Mon travail de thèse s'est focalisé sur l'étude du système chimiotactique de cette bactérie. Mon premier objectif a été d'identifier de nouveaux substrats induisant une réponse chimiotactique ainsi que les chimiorécepteurs les détectant. Une approche à haut débit utilisant une banque de substrats, couplée à une recherche de substrats plus spécifiques, a permis d'identifier de nouveaux signaux. Nous avons confirmé que S. oneidensis est attirée par les accepteurs alternatifs d'électrons ainsi que par certains métaux. Nous avons montré qu'elle est attirée par le malate et le chromate et repoussée par le nickel et le cobalt. Nous avons identifié deux MCPs potentiellement impliqués dans la détection du malate et un dans la détection du chromate.Mon second objectif a été de comprendre le rôle du système chimiotactique dans la formation d'un biofilm flottant : la pellicule. Nous avons montré que le développement de la pellicule est un processus en trois étapes déclenché par la détection de l'oxygène en condition statique, probablement par aérotactisme. Nous avons mis en évidence l'implication inattendue du chimiotactisme dans la formation de la pellicule. En effet des mutants délétés de gènes codant pour des éléments du système chimiotactique ne forment pas de pellicule normale. Le développement de la pellicule met en jeu d'autres signaux chimiotactiques, générés au sein de la pellicule, qui pourraient intervenir dans la localisation des cellules et l'homogénéisation de la pellicule au cours de sa maturation. / Shewanella oneidensis is an aquatic bacterium capable of chemotaxis, meaning that it can change its direction in response to detected signals. My thesis focused on the study of the chemotaxis system of this bacterium.The first objective of my thesis was to identify new substrates inducing a chemotactic response and the chemoreceptors involved in their detection. A high-throughput technique involving a library of solutes coupled with a search of more specific compounds allowed to identify new signals. We confirmed that S. oneidensis is attracted toward alternative electron acceptors and by several metals. We showed that S. oneidensis is attracted toward malate and also chromate. Finally, we identified two repellents, nickel and cobalt. After construction of deletion mutants, we identified two MCPs potentially involved in malate detection and one in chromate detection.The second objective of my thesis was to understand the role of the chemotaxis system in the formation of a floating biofilm: the pellicle. We first characterized the pellicle development through time, revealing that it is a three-step process. We then highlighted the unexpected role of the chemotaxis system in pellicle formation. Indeed mutants deleted of genes coding for chemotaxis elements are not able to form a pellicle or form an abnormal one. We showed that oxygen is the main signal triggering pellicle formation in static condition and that it is probably detected through aerotaxis. Pellicle development also involves other chemotactic signals produced in the pellicle which could be used in the localization of cells at the air-liquid interface but also in the homogenization of the pellicle.

Tempol (4-hidroxi-2,2,6,6-tetrametil piperidina-1-oxil) e outros nitróxidos cíclicos reduzem a injúria tecidual em modelos animais de inflamação por mecanismos que não são completamente entendidos. A mieloperoxidase (MPO) tem um papel fundamental na produção de oxidantes por neutrófilos e, portanto, é um importante alvo para anti-inflamatórios. Ao amplificar o potencial oxidativo do H2O2, a MPO produz HOCl e radicais livres através de seus intermediários oxidantes MPO-I [MPO-porfirina•+-Fe(IV)=O] e MPO-II [MPO-porfirina-Fe(IV)=O]. Esses fatos nos levaram a sintetizar e avaliar a capacidade inibitória sobre a atividade clorinante da MPO in vitro e in vivo do tempol e de três derivados hidrofóbicos substituídos na posição 4 do anel piperidina [(4-azido, 4-benzenosulfonil e 4-(4-fenil-1H-1,2,3-triazol-1-il)]. In vitro, todos os nitróxidos inibiram a clorinação da taurina mediada pela MPO a pH 7,4 com valores similares de IC50 (1,5-1,8 µM). As constantes cinéticas das reações do tempol com MPO-I (k = 3,5 x 105 M-1 s-1) e MPO-II, cuja cinética indicou um comportamento de saturação (K= 2,0 x 10-5 M; k = 3,6 x 10-2 s-1), foram determinadas. Modelagens cinéticas indicaram que, em presença de taurina, MPO não produz HOCl livre. Tomados conjuntamente, os resultados indicaram que o tempol age principalmente como um inibidor reversível da MPO por levar ao acúmulo de MPO-II e de complexo [MPO-II-tempol] que não participam do ciclo clorinante. Para examinar se nitróxidos inibem a atividade da MPO in vivo selecionamos como modelo a inflamação aguda induzida pela carragenina na pata de ratos. A atenuação da inflamação na pata mostrou correlação com a lipofilicidade do nitróxido em tempos iniciais, mas as diferenças nos efeitos foram pequenas (menor que 2 vezes) quando comparadas com as diferenças de lipofilicidade (maior que 200 vezes). Nenhuma inibição da atividade da MPO foi evidente in vivo porque os níveis de atividade nas patas dos ratos correlacionaram com os níveis de MPO. Do mesmo modo, em animais não-tratados ou tratados com nitróxidos, todos os parâmetros empregados para monitorar a inflamação (edema, níveis de proteínas oxidadas e nitradas e exsudação plasmática) correlacionaram com os níveis de MPO. Os efeitos dos nitróxidos in vivo foram também comparados com aqueles da hidrazida do ácido 4-aminobenzóico (ABAH) e da colchicina. Tomados conjuntamente, os estudos in vivo indicaram que os nitróxidos atenuam a inflamação induzida pela carragenina principalmente por inibirem a migração celular. De acordo com essa conclusão, estudos in vivo, mostraram que tempol diminuiu a migração de neutrófilos humanos e de cultura (HL-60 diferenciadas em neutrófilos) ativados com PMA ou fMLP com IC50 25 µM. Nesse caso, a polimerização da actina é inibida apenas quando as células são pré-incubadas com tempol por 30 min. Nesse intervalo de tempo, o tratamento com tempol promove a formação de O2•- via flavoenzimas de maneira dependente da concentração. Subsequente ativação dos neutrófilos de cultura com PMA resulta também em produção intracelular de O2•- e H2O2 que é inibida pelo pré-tratamento com tempol (IC50 = 38 µM). Esses estudos preliminares sugerem que o tempol interfere na migração celular de neutrófilos por atenuar a formação intracelular de ROS. A importância da atividade peroxidásica da superóxido dismutase (hSOD1) in vivo é certamente muito mais restrita do que a da MPO em processos inflamatórios no geral. Todavia, essa atividade peroxidásica da hSOD1 pode ter um papel na na patogenia da esclerose lateral amiotrófica (ELA). Por essa razão, os efeitos do tempol sobre as consequências da atividade peroxidásica da hSOD1 (oxidação, dimerização, desenovelamento e agregação da enzima) também foram examinadas. Foi demonstrado que o tempol não interfere no ciclo catalítico da hSOD1, porém, inibe a dimerização da enzima, protegendo-a de desenovelamento e agregação. O nitróxido foi consumido no processo reagindo com o radical triptofanila no peptídeo 31VWGSIK36 como comprovado por MS. No conjunto, nossos estudos contribuem para esclarecer os múltiplos mecanismos pelos quais nitróxidos podem inibir processos oxidativos e inflamatórios. / Tempol (4-hydroxy-2 ,2,6,6-tetramethyl piperidine-1-oxyl) and other cyclic nitroxides reduce tissue injury in animal models of inflammation by mechanisms that are not fully understood. Myeloperoxidase (MPO) plays a key role in the production of oxidants by neutrophils and therefore is an important target for anti-inflammatory drugs. By amplifying the oxidative potential of H2O2, MPO produces HOCl and free radicals through its oxidizing intermediates MPO-I [MPO-porphyrin•+-Fe (IV)=O] and MPO-II [MPO-porphyrin-Fe (IV)=O]. In this context, we synthesized tempol and three more hydrophobic derivatives substituted in position 4 of the piperidine ring [(4-azido-4 benzenesulfonyl and 4-(4-phenyl-1H-1,2,3-triazol-1-yl)] and evaluated their ability to inhibit the chlorinating activity of MPO in vitro and in vivo. In vitro, all the nitroxides inhibited the chlorination of taurine mediated by MPO at pH 7.4 with similar IC50 values (1.5 to 1.8 µM). The kinetic constants of the reactions of tempol with MPO-I (k = 3.5 x 105 M-1 s-1) and MPO-II, whose kinetic indicated a saturation behavior (K = 2.0 x 10-5 M; k = 3.6 x 10-2 s-1), were determined. Kinetic modeling indicated that in the presence of taurine, MPO does not produce free HOCl. Taken together, the results indicated that tempol acts primarily as a reversible inhibitor of MPO leading to accumulation of MPO-II and of the complex [MPO-II-tempol], which do not participate within chlorinating cycle. To examine whether nitroxides inhibit the activity of MPO in vivo, the acute inflammation induced by carrageenan in the rat paw was selected as model. The attenuation of inflammation in paws was correlated with the lipophilicity of the nitroxide in early times, but the differences in effects were small (less than 2-fold) when compared to the differences in lipophilicity (higher than 200 times). No inhibition of MPO activity was evident because the levels of activity in rat paws correlated with the levels of MPO. Similarly, in animals not treated or treated with nitroxides, all parameters used to monitor inflammation (swelling, levels of oxidized and nitrated proteins and plasma exudation) correlated with the levels of MPO. The effects of nitroxides in vitro were also compared with those of 4-aminobenzoic acid hydrazide (ABAH) and colchicine. Taken together, the in vivo studies indicated that nitroxides attenuate carrageenan-induced inflammation mainly by inhibiting cell migration. According to this conclusion, in vitro studies showed that tempol decreased migration of human and culture neutrophils (HL-60 differentiation to neutrophils) activated with PMA or fMLP with IC50 = 25 µM. In this case, actin polymerization is inhibited only when cells are preincubated with tempol for 30 min. During this time interval, treatment with tempol promotes the formation of O2•- via flavoenzymes in a concentration-dependent manner. Subsequent activation of culture neutrophils with PMA also results in intracellular production of O2•- and H2O2, which is inhibited by pretreatment with tempol (IC50 = 38 µM). These preliminary studies suggest that tempol interfere in neutrophil chemotaxis by attenuating the formation of intracellular ROS. The relevance of the peroxidase activity of superoxide dismutase (hSOD1) in vitro is certainly much narrower than that of MPO. Nevertheless, this peroxidase activity may have a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Therefore, the effects of tempol on the consequences of the hSOD1 peroxidase activity (oxidation, dimerization, unfolding and aggregation of the enzyme) were also examined. Tempol inhibited the dimerization of hSOD1, protecting it from unfolding and aggregation, but not interfered in its catalytic cycle. The nitroxide was consumed in the process by reacting with the tryptophanyl radical of the segment 31VWGSIK36 as evidenced by MS. Overall, our studies contribute to clarify the multiple mechanisms by which nitroxides can inhibit oxidative and inflammatory processes.

No description available.

<p>x</p> / <p>Platelet-derived growth factors (PDGF), a family of potent mitogens and chemoattractants for cells of mesenchymal origin, elicit their biological effects through the binding of two related receptor tyrosine kinases, denoted α- and β-receptors. The binding of PDGF to the receptors causes receptor dimerization and autophosphorylation on tyrosine residues. Src homology 2 (SH2) domain-containing proteins then bind the phosphorylated receptors, mediating further propagation of the signal. This thesis describes how the interaction between the PDGF receptors and some of their downstream targets can modify the cellular response to PDGF.</p><p>The tyrosine phosphatase SHP-2 has been implicated in activation of the Ras/MAPK pathway downstream of several receptor tyrosine kinases. We found that SHP-2 binds to phosphorylated Y763 in the PDGF β-receptor, in addition to the already reported binding to Y1009. Cells expressing PDGF β-receptors with Y763 and Y1009 mutated to phenylalanine exhibited decreased Ras-GTP loading and reduced activation of Erk2 in response to PDGF. Whereas these cells did not show any change in the mitogenic response to PDGF, the PDGF-induced chemotaxis was significantly reduced in cells expressing mutant compared to wild-type receptor.</p><p>The phosphorylation of Y771 of the PDGF β-receptor had been shown to be significantly lower in the αβ-heterodimeric receptor compared to in the ββ-homodimer, causing reduced binding of RasGAP to the heterodimer and increased Ras/MAPK activation. We could demonstrate that the reduced phosphorylation of Y771 is due to dephosphorylation by tyrosine phosphatases, including SHP-2.</p><p>SHP-2 had been shown to associate with the docking protein Gab1 after growth factor stimulation. We showed that the adaptor protein Grb2 was required for PDGF mediated phosphorylation of Gab1, and that phosphorylated Gab1, Grb2 and SHP-2 create a complex upon PDGF stimulation. Using a cell system with an inducible Gab1 expression, we further demonstrated that Gab1 increased SHP-2 activity in response to PDGF, without affecting the interaction between SHP-2 and the b-receptor. Induction of Gab1 correlated with an increase in both PDGF-induced Erk and p38 MAPK activation, whereas Akt activation was unaffected. The latter finding was in line with our observation that PDGF had no effect on the interaction between Gab1 and p85 of PI3’-kinase. The increase in MAPK activity after Gab1 induction and PDGF treatment did not correlate with an increase in PDGF-induced mitogenicity; instead these cells displayed more pronounced actin reorganization in response to PDGF.</p><p>In conclusion, our data indicate that SHP-2 regulates the PDGF response both through direct dephosphorylation of the receptor and through its interaction with Gab1. PDGF stimulated activation of SHP-2 seems to be correlated not only with mitogenesis, but also with reorganization of the actin cytoskeleton and cell migration.</p>

x / Platelet-derived growth factors (PDGF), a family of potent mitogens and chemoattractants for cells of mesenchymal origin, elicit their biological effects through the binding of two related receptor tyrosine kinases, denoted α- and β-receptors. The binding of PDGF to the receptors causes receptor dimerization and autophosphorylation on tyrosine residues. Src homology 2 (SH2) domain-containing proteins then bind the phosphorylated receptors, mediating further propagation of the signal. This thesis describes how the interaction between the PDGF receptors and some of their downstream targets can modify the cellular response to PDGF. The tyrosine phosphatase SHP-2 has been implicated in activation of the Ras/MAPK pathway downstream of several receptor tyrosine kinases. We found that SHP-2 binds to phosphorylated Y763 in the PDGF β-receptor, in addition to the already reported binding to Y1009. Cells expressing PDGF β-receptors with Y763 and Y1009 mutated to phenylalanine exhibited decreased Ras-GTP loading and reduced activation of Erk2 in response to PDGF. Whereas these cells did not show any change in the mitogenic response to PDGF, the PDGF-induced chemotaxis was significantly reduced in cells expressing mutant compared to wild-type receptor. The phosphorylation of Y771 of the PDGF β-receptor had been shown to be significantly lower in the αβ-heterodimeric receptor compared to in the ββ-homodimer, causing reduced binding of RasGAP to the heterodimer and increased Ras/MAPK activation. We could demonstrate that the reduced phosphorylation of Y771 is due to dephosphorylation by tyrosine phosphatases, including SHP-2. SHP-2 had been shown to associate with the docking protein Gab1 after growth factor stimulation. We showed that the adaptor protein Grb2 was required for PDGF mediated phosphorylation of Gab1, and that phosphorylated Gab1, Grb2 and SHP-2 create a complex upon PDGF stimulation. Using a cell system with an inducible Gab1 expression, we further demonstrated that Gab1 increased SHP-2 activity in response to PDGF, without affecting the interaction between SHP-2 and the b-receptor. Induction of Gab1 correlated with an increase in both PDGF-induced Erk and p38 MAPK activation, whereas Akt activation was unaffected. The latter finding was in line with our observation that PDGF had no effect on the interaction between Gab1 and p85 of PI3’-kinase. The increase in MAPK activity after Gab1 induction and PDGF treatment did not correlate with an increase in PDGF-induced mitogenicity; instead these cells displayed more pronounced actin reorganization in response to PDGF. In conclusion, our data indicate that SHP-2 regulates the PDGF response both through direct dephosphorylation of the receptor and through its interaction with Gab1. PDGF stimulated activation of SHP-2 seems to be correlated not only with mitogenesis, but also with reorganization of the actin cytoskeleton and cell migration.