Il est maintenant acquis que le système immunitaire occupe une place importante dans l’évolution les cancers (Hanahan et al., 2011). La compréhension actuelle de la réponse immunitaire adaptative en fait une cible de choix dans ce contexte. Il est apparu que les lymphocytes T CD4+, acteurs majeurs de la composante adaptative du système immunitaire, présentent des actions distinctes sur le contrôle de la croissance tumorale. Ainsi, les lymphocytes Th2 et Tfh, tous deux activateurs des lymphocytes B dans des conditions de lutte contre des infections pathogéniques, présentent des rôles ambivalents dans un contexte de cancer. En effet, de nombreuses études montrent que la présence de Th2 est corrélée à une progression de la maladie (notamment via l’action de l’IL-4 qu’ils sécrètent) (Koller et al., 2010 ; Roca et al., 2012) alors que les Tfh, seraient plutôt associés à un bon pronostic pour les patients (Gu-Trantien et al., 2013, 2017).Nos investigations actuelles nous ont permis de mettre en avant une caractéristique nouvelle de la biologie des lymphocytes Tfh. En effet, les Tfh expriment l’Hemathopoietic Prostaglandin D2 synthase (HPGDS). Cette enzyme de la voie de biosynthèse des eicosanoïdes est responsable de la production de Prostaglandine D2 (PGD2). Plusieurs travaux montrent que les cellules Th2 expriment le récepteur CRTH2, spécifique de la PGD2. Cette molécule agit sur ces cellules comme chemoattractant et permet également une augmentation de leur production cytokinique. Ainsi, nous posons l’hypothèse d’une communication potentielle entre lymphocytes Tfh et Th2 via la PGD2. Le projet présenté ici est alors axé sur la compréhension des mécanismes moléculaires et cellulaires sous-jacent à cette communication au sein des deux sous-types ainsi que sur son impact dans un contexte de cancer. Ce projet ayant également pour but de mettre en avant la PGD2 comme nouvelle cible thérapeutique dans le cancer. / It is now accepted that the immune system plays a critical role in cancers evolution (Hanahan et al., 2011). In this context, current understanding of the adaptive immune response made it a prime target. T CD4 cells, the main players of the adaptive immune system component, are known to possess distinct roles in the control of tumour growth. Thereby, Th2 and Tfh cells, both known to activate B cells in pathogenic infections, present antagonistic roles in cancer. Indeed, numerous studies demonstrate that Th2 cells are correlated with disease progression (especially via IL-4 secretion) (Koller et al., 2010 ; Roca et al., 2012), whereas Tfh cells are associated with a good prognosis for the patients (Gu-Trantien et al., 2013, 2017) despite the actual limited amount of available data.Our current researches highlighted a new property of the biology of Tfh cells. We found that Tfh cells are able to express the Hemathopoietic Prostaglandin D2 synthase (HPGDS), an eicosanoid pathway enzyme involved in Prostaglandin D2 (PGD2) production. Moreover, different studies revealed that Th2 cells expressed CRTH2, the specific PGD2 receptor. PGD2 is known as a chemoattractant molecule for Th2 cells and lead to the increase of their cytokine secretion. We hypothesized that Tfh communicate with Th2 cells via PGD2 signalling. The present project is focused on the understanding of the underlying molecular and cellular mechanisms involved in this cross-talk and their impact in cancer. The last aim of this work is to favor the development of PGD2 as a new cancer therapeutic target.

No description available.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.

As conexinas são as principais proteínas componentes das junções do tipo GAP no fígado. Existem poucos estudos sobre o papel das junções GAP nas lesões hepáticas, principalmente nas células não-parenquimatosas. O processo de isquemia e de reperfusão (IR) é um fenômeno complexo que está muito presente na prática clínica e envolve inúmeras vias metabólicas, e acredita-se que a comunicação celular parece ser uma via importante de interação entre células hepáticas, que respondem às alterações da homeostase e insultas. O objetivo da pesquisa foi comprovar a participação da conexina Cx43 na lesão de IR hepática, em um modelo experimental em roedores. A utilização de animais deficientes para Cx43 (\"Knockout\" heterozigotos Cx43+/-) permitiu verificar a evolução da lesão de IR, na deficiência desta conexina. A lesão de IR foi induzida pelo clampeamento do pedículo hepático durante uma hora. Realizou-se então a comparação dos níveis de enzimas hepáticas entre os animais \"Knockout\" e o grupo controle. Com isso se comprovou que a presença da Cx43 melhora o prognóstico em lesões hepáticas. Atualmente, com medicações capazes de bloquear ou aumentar a presença da Cx43 e, tendo em vista os altos índices de doenças hepáticas no Brasil e no mundo, esta é uma importante contribuição nesta área do conhecimento / Connexins are the main protein components of the gap junctions in the liver. There are few studies on the role of gap junctions on liver injury, and they are mainly on non-parenchymal cells. The process of ischemia and reperfusion (IR) is a complex phenomenon that involves many metabolic processes, and we believe that cellular communication is an important interaction mechanism between hepatic cells that respond to alteration of homeostasis and insults. The aim of the project was to prove that connexin Cx43 plays an important role in lesions in hepatic IR in experimental rodent models. The use of Cx43 deficient models (knockout heterozygous Cx43+/-) allowed us to verify the evolution of the liver injury from IR in the deficiency of this connexin. The IR lesion was induced by clamping the hepatic pedicle for one hour. We then carried out a comparison of the levels of hepatic enzymes between the knockout mice and the control group. This demonstrated that the presence of Cx43 altered the prognosis for liver injury. Given that there are medications capable of blocking or increasing the presence of Cx43 selectively or not, and bearing in mind the high rates of hepatic pathologies in Brazil and the rest of the world, this article contributes to understanding of the participation of connexin 43 in liver injury during ischemia and reperfusion

As conexinas são as principais proteínas componentes das junções do tipo GAP no fígado. Existem poucos estudos sobre o papel das junções GAP nas lesões hepáticas, principalmente nas células não-parenquimatosas. O processo de isquemia e de reperfusão (IR) é um fenômeno complexo que está muito presente na prática clínica e envolve inúmeras vias metabólicas, e acredita-se que a comunicação celular parece ser uma via importante de interação entre células hepáticas, que respondem às alterações da homeostase e insultas. O objetivo da pesquisa foi comprovar a participação da conexina Cx43 na lesão de IR hepática, em um modelo experimental em roedores. A utilização de animais deficientes para Cx43 (\"Knockout\" heterozigotos Cx43+/-) permitiu verificar a evolução da lesão de IR, na deficiência desta conexina. A lesão de IR foi induzida pelo clampeamento do pedículo hepático durante uma hora. Realizou-se então a comparação dos níveis de enzimas hepáticas entre os animais \"Knockout\" e o grupo controle. Com isso se comprovou que a presença da Cx43 melhora o prognóstico em lesões hepáticas. Atualmente, com medicações capazes de bloquear ou aumentar a presença da Cx43 e, tendo em vista os altos índices de doenças hepáticas no Brasil e no mundo, esta é uma importante contribuição nesta área do conhecimento / Connexins are the main protein components of the gap junctions in the liver. There are few studies on the role of gap junctions on liver injury, and they are mainly on non-parenchymal cells. The process of ischemia and reperfusion (IR) is a complex phenomenon that involves many metabolic processes, and we believe that cellular communication is an important interaction mechanism between hepatic cells that respond to alteration of homeostasis and insults. The aim of the project was to prove that connexin Cx43 plays an important role in lesions in hepatic IR in experimental rodent models. The use of Cx43 deficient models (knockout heterozygous Cx43+/-) allowed us to verify the evolution of the liver injury from IR in the deficiency of this connexin. The IR lesion was induced by clamping the hepatic pedicle for one hour. We then carried out a comparison of the levels of hepatic enzymes between the knockout mice and the control group. This demonstrated that the presence of Cx43 altered the prognosis for liver injury. Given that there are medications capable of blocking or increasing the presence of Cx43 selectively or not, and bearing in mind the high rates of hepatic pathologies in Brazil and the rest of the world, this article contributes to understanding of the participation of connexin 43 in liver injury during ischemia and reperfusion

Signal transduction is a crucial event that enables cells to sense and respond to cues from their immediate environment. Guanylyl cyclase C (GC-C) is a member of the family of receptor guanylyl cyclases. GC-C is a single transmembrane protein that responds to its ligands by the production of the second messenger cGMP. The guanylin family of peptides, (including the bacterially produced heat-stable enterotoxin ST) is the ligand for GC-C, elevates intracellular cGMP levels and activates downstream pathways. GC-C regulates the cystic fibrosis transmembrane conductance regulator (CFTR) by inducing phosphorylation by protein kinase G, resulting in chloride ion and fluid efflux. GC-C also regulates cell cycle progression through cGMP-gated Ca2+ channels. These functions are seen in the intestinal epithelium, the primary site for GC-C expression. GC-C as a molecule has been studied in detail, but its functioning in the context of other signaling pathways remains unknown. The aim of the present investigation was to understand the regulation of signal transduction by GC-C and its cross-talk with other signaling pathways operating in the cell. Molecular events that commonly connect components in a signaling pathway are protein phosphorylation and protein-protein interaction. These two aspects are explored in this thesis. The possibility of tyrosine phosphorylation of GC-C has been explored earlier in our laboratory. In vitro studies indicated that the residue Tyr820 was a site for phosphorylation by the Src family of non-receptor tyrosine kinases and those studies also suggested that phosphorylated Tyr820 could bind to the SH2 domain of Src. We generated a nonphosphorylatable mutant of GC-C, GC-CY820F, and a phosphomimetic mutant GC-CY820E to study the effect of phosphorylation of Tyr820, on the functioning of GC-C. A stable cell line of HEK293:GC-CY820F cells was generated and compared with HEK293:GC-CWT. Dose response to ST in the two cell lines showed that cGMP accumulation by GC-CY820F was greater than that of GC-CWT, although the EC50 remained unchanged. The phosphomimetic GC-CY820E mutant receptor was non-responsive to ST. Further in HEK293 cells, phosphorylation of GC-CWT by constitutively active v-Src resulted in decreased ST stimulation and this effect of v-Src was reduced with GC-CY820F. Inhibition of ST stimulation brought about by v-Src required catalytically active Src, as the kinase inactive v-SrcK295R did not inhibit ST stimulation. These results were corroborated by in vitro studies by using the recombinant catalytic domain of GC-C expressed in insect cells and by phosphorylation using a purified kinase, Hck. Observations suggested that phosphorylation of Tyr820 in the catalytic domain of GC-C compromises the guanylyl cyclase activity of GC-C. T84 and Caco-2 colon carcinoma cells endogenously express GC-C. The effect of tyrosine phosphorylation of GC-C was studied by using HgCl2, a known activator of Src kinases, and by the inhibition of protein tyrosine phosphatases using pervanadate, an irreversible inhibitor. Both these ways of achieving increased tyrosine phosphorylation resulted in decreased ST-stimulated cGMP production by GC-C, as suggested from v-Src transfection studies. This decrease was reversed by using a Src kinase specific inhibitor PP2, confirming the role of Src kinases in the inhibition of GC-C activity. Interestingly, in Caco-2 cells that differentiate in culture, the effect of pervanadate on the inhibition of ST-stimulated GC-C activation was dependent on the differentiation stage. Crypt-like cells showed higher inhibition with pervanadate. As they matured into villus-like cells, the effect of pervanadate on GC-C activation was gradually lost. This effect also correlated with a decrease in the expression of Lck, suggesting that in the context of the intestine there could be differential regulation of tyrosine phosphorylation of GC-C along the crypt-villus axis. Intestinal ligated loop assays in rats demonstrated that ST-induced fluid accumulation in the intestine was abrogated on pervanadate treatment. Reduction in this fluid accumulation by pervanadate was not observed with 8-Br-cGMP, a cell permeable analogue of cGMP. This indicated that tyrosine phosphorylation of proteins is important for ST-induced fluid accumulation, and perhaps pervanadate modulates this by phosphorylation of GC-C, thereby causing a reduction in fluid accumulation. Earlier in vitro studies on Src-SH2 binding from the laboratory had suggested the possibility of activation of Src family kinases by GC-C. The activation status of Src kinases was monitored by using phosphorylation-state specific antibody, pSFK416. ST stimulation in T84 cells increased Tyr416 phosphorylation of Src kinases in a time dependent manner, indicating that Src kinases are activated downstream of GC-C. This activation of Src kinases was also seen with the endogenous ligand of GC-C, uroguanylin. Interestingly, 8-Br-cGMP a cell permeable analogue of cGMP that is known to mimic other cellular effects of GC-C, namely Cl-secretion and cell cycle progression, did not activate Src kinases, suggesting that the mechanism of Src kinase activation by GC-C could be independent of cGMP. Binding affinities of Src, Lck, Fyn and Yes SH2 domains to Tyr820 phosphorylated GCC peptide were in the nM range, indicating a high affinity of interaction. In vitro GST-SH2 pull down experiments suggested that phosphorylation of Tyr820 in full length GC-C allows interaction of GC-C to the SH2 domain of Src. These studies suggest a dual cross-talk between Src kinases and GC-C; Src phosphorylation inhibits GC-C signaling and stimulation of GC-C by its ligands activates Src kinases. Interaction of proteins containing SH2 and SH3 domains are commonly found in signaling molecules. In accordance with the observation that there are three PXXP motifs in GCC, many SH3 domains could interact with GC-C. GC-C appears to show a preference to bind the SH3 domains of Fyn, Hck, Abl tyrosine kinases, Grb2 and Crk adaptor proteins, the α-subunit of P85 PI3 kinase, PLC-γ and cortactin to various extents. The SH3 domains of spectrin and Nck did not show any detectable interaction with GC-C. In SH3 pull-down assays, the N-terminal SH3 domain of Crk, CrkSH3 (N), bound GC-C maximally, suggesting that Crk is a good candidate for interaction with GC-C. By overlay analysis, the region of GC-C that binds CrkSH3 (N) was narrowed down to the catalytic domain of GC-C containing a ‘PGLP’ motif. Mutations were generated in GC-C at this site to generate GC-CP916Q and GC-CW918R. These mutations compromised the binding of full length receptor to CrkSH3 (N). In cells, CrkII and GC-C co-transfection inhibited the ST stimulation of GC-C. A CrkII mutant, that has compromised binding through its SH3 domain, did not inhibit the activity of GC-C. CrkII from T84 cells co-immunoprecipitated with GC-C and interestingly, the phosphorylated form of CrkII did not, indicating that GC-C - Crk interaction could be regulated by the phosphorylation of Crk. In summary, this study places GC-C, in the context of tyrosine kinase signaling pathway and interaction with the adaptor protein Crk. These studies suggest that GC-C signal transduction can be altered by cross-talk with other signaling events in the cell. Reversible phosphorylation of tyrosine residues inhibits the activity of GC-C, and this is mediated by Src family kinases. Src kinases themselves are activated on stimulation of GC-C by its ligands, possibly because of SH2 domain interaction with GC-C. Association of Crk by its SH3 domain regulates GC-C functioning primarily by inhibiting ST-stimulated cGMP production. This opens up the possibility of GC-C signaling through a multimeric complex involving other binding partners of Crk, and these cross-talks involving GC-C with the two proto-oncogenes, Src and Crk, might have far reaching consequences in the regulation of cellular functions.

Chez les angiospermes, la reproduction passe par la double fécondation. Le tube pollinique délivre deux cellules spermatiques au sein du gamétophyte femelle. Une cellule féconde la cellule œuf pour produire un zygote; l’autre féconde la cellule centrale pour produire l’endosperme. Pour assurer un succès reproductif, le développement du gamétophyte femelle au sein de l’ovule doit établir un patron cellulaire qui favorise les interactions avec le tube pollinique et les cellules spermatiques. Pour ce faire, un dialogue doit s’établir entre les différentes cellules de l’ovule lors de son développement, de même que lors de la fécondation. D’ailleurs, plusieurs types de communications intercellulaires sont supposées suite à la caractérisation de plusieurs mutants développementaux. De même, ces communications semblent persister au sein du zygote et de l’endosperme pour permettre la formation d’un embryon viable au sein de la graine. Malgré les développements récents qui ont permis de trouver des molécules de signalisation supportant les modèles d’interactions cellulaires avancés par la communauté scientifique, les voies de signalisation sont de loin très incomplètes. Dans le but de caractériser des gènes encodant des protéines de signalisation potentiellement impliqués dans la reproduction chez Solanum chacoense, l’analyse d’expression des gènes de type RALF présents dans une banque d’ESTs (Expressed Sequence Tags) spécifiques à l’ovule après fécondation a été entreprise. RALF, Rapid Alcalinization Factor, est un peptide de 5 kDa qui fait partie de la superfamille des «protéines riches en cystéines (CRPs)», dont les rôles physiologiques au sein de la plante sont multiples. Cette analyse d’expression a conduit à une analyse approfondie de ScRALF3, dont l’expression au sein de la plante se limite essentiellement à l’ovule. L’analyse de plantes transgéniques d’interférence pour le gène ScRALF3 a révélé un rôle particulier lors de la mégagamétogénèse. Les plantes transgéniques présentent des divisions mitotiques anormales qui empêchent le développement complet du sac embryonnaire. Le positionnement des noyaux, de même que la synchronisation des divisions au sein du syncytium, semblent responsables de cette perte de progression lors de la mégagamétogénèse. L’isolement du promoteur de même que l’analyse plus précise d’expression au sein de l’ovule révèle une localisation sporophytique du transcrit. La voie de signalisation de l’auxine régule également la transcription de ScRALF3. De surcroît, ScRALF3 est un peptide empruntant la voie de sécrétion médiée par le réticulum endoplasmique et l’appareil de Golgi. En somme, ScRALF3 est un important facteur facilitant la communication entre le sporophyte et le gamétophyte pour amener à maturité le sac embryonnaire. L’identification d’un orthologue potentiel chez Arabidopsis thaliana a conduit à la caractérisation de AtRALF34. L’absence de phénotype lors du développement du sac embryonnaire suggère, cependant, de la redondance génétique au sein de la grande famille des gènes de type RALF. Néanmoins, les peptides RALFs apparaissent comme d’importants régulateurs lors de la reproduction chez Solanum chacoense et Arabidopsis thaliana. / In angiosperms, reproduction occurs through double fertilization. The pollen tube delivers two sperm cells into the female gametophyte. A first sperm cell fertilizes the egg cell to produce a zygote, while the other fertilizes the central cell to produce the endosperm. To ensure reproductive success, the development of the female gametophyte within the ovule must establish a cellular pattern allowing interaction with the pollen tube and sperm cells. To this end, a dialogue must be established amongst the various cells of the ovule during its development, as well as during fertilization. Several types of communication are suggested by the analysis of developmental mutants. These communications must persist in the zygote and endosperm to allow the formation of a viable embryo within the seed. Recent developments have helped to find signaling molecules that support cell interaction models developed by the scientific community, but the signaling pathways are far from complete. In order to characterise genes encoding signaling proteins which are potentially active during reproduction in Solanum chacoense, I undertook the expression analysis of the RALF-like genes present in a bank of ESTs (Expressed Sequence Tags) specific to the ovule after fertilization. RALF, Rapid Alcalinization Factor, is a 5 kDa peptide that is part of the superfamily of Cysteine Rich Proteins (CRPs), which play a wide variety physiological roles within the plant. This expression analysis led to a detailed analysis of ScRALF3, whose expression in the plant is largely restricted to the ovule. The analysis of ScRALF3 RNAi transgenic plants revealed a function during megagametogenesis. The transgenic plants exhibit abnormal mitotic divisions that prevent the maturity of the embryo sac. The positioning of the nuclei, as well as the timing of divisions in the syncytium, appear to be responsible for the arrest of development during megagametogenesis. Isolation of the promoter as well as more accurate analysis of transcript expression reveals localisation within the ovule sporophytic tissue. The auxin signaling pathway is also involved in the regulation of ScRALF3 expression. ScRALF3 is a secreted peptide passing via the endoplasmic reticulum and the Golgi apparatus. In summary, ScRALF3 may be an important factor facilitating communication between the gametophyte and the sporophyte to allow maturation of the embryo sac. The identification of a potential orthologue in Arabidopsis thaliana led to the characterisation of AtRALF34. The lack of a phenotype during embryo sac development, however, suggests that genetic redundancy within the family of RALF-like genes is very complex. Nevertheless, the RALF peptides appear to be important regulators during reproduction in Solanum chacoense and Arabidopsis thaliana.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and “licenses” the APCs to prime CD8+ T cell responses. While other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naïve CD8+ T cells. We find an approximately 50% reduction in expansion and cytokine production of TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization and are not protected from a tumor challenge. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. We also investigated whether CD40-CD40L interactions are involved in the generation, maintenance, and function of memory CD8+ T cells. Using a virus infection system as well as a dendritic cell immunization system, we show that the presence of CD40 on DCs and other host APCs influences the survival of activated effector cells and directly affects the number of memory CD8+ T cells that are formed. In addition, memory CD8+ T cell persistence is slightly impaired in the absence of CD40. However, CD40 is not required for reactivation of memory CD8+ T cells. It seems that CD40 signals during priming also contribute to memory CD8+ T cell programming but this function can be independent of CD4+T cells, similar to what we showed for primary responses. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of primary as well as memory CD8+ T cell responses.