The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.

Structural and Resting State Functional Connectivity of the Subthalamic Nucleus: Identification of Motor STN Parts and the Hyperdirect Pathway.

Deep brain stimulation (DBS) for Parkinson's disease often alleviates the motor symptoms, but causes cognitive and emotional side effects in a substantial number of cases. Identification of the motor part of the subthalamic nucleus (STN) as part of the presurgical workup could minimize these adverse effects. In this study, we assessed the STN's connectivity to motor, associative, and limbic brain areas, based on structural and functional connectivity analysis of volunteer data. For the structural connectivity, we used streamline counts derived from HARDI fiber tracking. The resulting tracks supported the existence of the so-called "hyperdirect" pathway in humans. Furthermore, we determined the connectivity of each STN voxel with the motor cortical areas. Functional connectivity was calculated based on functional MRI, as the correlation of the signal within a given brain voxel with the signal in the STN. Also, the signal per STN voxel was explained in terms of the correlation with motor or limbic brain seed ROI areas. Both right and left STN ROIs appeared to be structurally and functionally connected to brain areas that are part of the motor, associative, and limbic circuit. Furthermore, this study enabled us to assess the level of segregation of the STN motor part, which is relevant for the planning of STN DBS procedures.

Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR).

Peroxisome proliferators regulate the transcription of genes by activating ligand-dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPAR alpha and beta but not xPPAR gamma are expressed in oocytes and embryos. In the adult, expression of xPPAR alpha and beta appears to be ubiquitous, and xPPAR gamma is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic-nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in the peroxisomal beta-oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the promoter of the ACO gene. Besides the known xenobiotic activators of PPARs, such as hypolipidemic drugs, natural activators have been identified. Polyunsaturated fatty acids at physiological concentrations are efficient activators of PPARs, and 5,8,11,14-eicosatetraynoic acid (ETYA), which is the alkyne homolog of arachidonic acid, is the most potent activator of xPPAR alpha described to date. Taken together, our data suggest that PPARs have an important role in lipid metabolism.

Long-term cerebral plasticity-related gene expression : a study of the respective role of CBP and CRTC1 in CREB transcriptional activation

1.1 AbstractThe treatment of memory disorders and cognitive deficits in various forms of mental retardation may greatly benefit from a better understanding of the molecular and cellular mechanisms of memory formation. Different forms of memory have distinct molecular requirements.Short-term memory (STM) is thought to be mediated by covalent modifications of existing synaptic molecules, such as phosphorylation or dephosphorylation of enzymes, receptors or ion channels. In contrast, long-term memoiy (LTM) is thought to be mediated by growth of new synapses and restructuring of existing synapses. There is extensive evidence that changes in gene expression and de novo protein synthesis are key processes for LTM formation. In this context, the transcription factor CREB (cAMP-response element-binding protein) was shown to be crucial. Activation of CREB requires phosphorylation of a serine residue (Ser-133), and the subsequent recruitment of a coactivator called CREB-binding protein (CBP). Moreover, we have recently shown that another coactivator called CREB Regulated Transcription Coactivator 1 (CRTC1) functions as a calcium- and cAMP-sensitive coincidence detector in neurons, and is involved in hippocampal long-term synaptic plasticity. Given the importance of cAMP and calcium signaling for plasticity-related gene expression in neurons and in astrocytes, we sought to determine the respective involvement of the CREB coactivators CBP and CRTC1 in CREB-mediated transcription.We developed various strategies to selectively interfere with these CREB coactivators in mouse primary neurons and in astrocytes in vitro. However, despite several pieces of evidence implicating CBP and/or CRTC1 in the regulation of neuronal plasticity genes, we could not clearly determine the respective requirement of these coactivators for the activation of these genes. Nevertheless, we showed that calcineurin activity, which is important for CRTC1 nuclear translocation, is necessary for the expression of some CREB-regulated plasticity genes. We associated this phenomena to physiopathological conditions observed in Down's syndrome. In addition, we demonstrated that in astrocytes, noradrenaline stimulates CREB-target gene expression through β-adrenergic receptor activation, intracellular cAMP pathway activation, and CRTC-induced CREB transactivation.Defining the respective role of CREB and its coactivators CBP and CRTC1 in neuronal and astrocytic cultures in vitro sets the stage for future in vivo studies and for the possible development of new therapeutic strategies to improve the treatment of memoiy and cognitive disorders.1.2 RésuméUne meilleure connaissance des mécanismes moléculaires et cellulaires responsables de la formation de la mémoire pourrait grandement améliorer le traitement des troubles de la mémoire ainsi que des déficits cognitifs observés dans différentes formes de pathologies psychiatriques telles que le retard mental. Les différentes formes de mémoire dépendent de processus moléculaires différents.La mémoire à court terme (STM) semble prendre forme suite à des modifications covalentes de molécules synaptiques préexistantes, telles que la phosphorylation ou la déphosphorylation d'enzymes, de récepteurs ou de canaux ioniques. En revanche, la mémoire à long terme (LTM) semble être due à la génération de nouvelles synapses et à la restructuration des synapses existantes. De nombreuses études ont permis de démontrer que les changements dans l'expression des gènes et la synthèse de protéine de novo sont des processus clés pour la formation de la LTM. Dans ce contexte, le facteur de transcription CREB (cAMP-response element-binding protein) s'est avéré être un élément crucial. L'activation de CREB nécessite la phosphorylation d'un résidu sérine (Ser-133), et le recrutement d'un coactivateur nommé CBP (CREB binding protein). En outre, nous avons récemment démontré qu'un autre coactivateur de CREB nommé CRTC1 (CREB Regulated Transcription Coactivator 1) agit comme un détecteur de coïncidence de l'AMP cyclique (AMPc) et du calcium dans les neurones et qu'il est impliqué dans la formation de la plasticité synaptique à long terme dans l'hippocampe. Etant donné l'importance des voies de l'AMPc et du calcium dans l'expression des gènes impliqués dans la plasticité cérébrale, nous voulions déterminer le rôle respectif des coactivateurs de CREB, CBP et CRTC1.Nous avons développé diverses stratégies pour interférer de façon sélective avec les coactivateurs de CREB dans les neurones et dans les astrocytes chez la souris in vitro. Nos résultats indiquent que CBP et CRTC1 sont tous deux impliqués dans la transcription dépendante de CREB induite par l'AMPc et le calcium dans les neurones. Cependant, malgré plusieurs évidences impliquant CBP et/ou CRTC1 dans l'expression de gènes de plasticité neuronale, nous n'avons pas pu déterminer clairement leur nécessité respective pour l'activation de ces gènes. Toutefois, nous avons montré que l'activité de la calcineurine, dont dépend la translocation nucléaire de CRTC1, est nécessaire à l'expression de certains de ces gènes. Nous avons pu associer ce phénomène à une condition physiopathologique observée dans le syndrome de Down. Nous avons également montré que dans les astrocytes, la noradrénaline stimule l'expression de gènes cibles de CREB par une activation des récepteurs β- adrénergiques, l'activation de la voie de l'AMPc et la transactivation de CREB par les CRTCs.Définir le rôle respectif de CREB et de ses coactivateurs CBP et CRTC1 dans les neurones et dans les astrocytes in vitro permettra d'acquérir les connaissances nécessaires à de futures études in vivo et, à plus long terme d'éventuellement développer des stratégies thérapeutiques pour améliorer les traitements des troubles cognitifs.

ERK1/2 pathway is activated in degenerated Rpe65-deficient mice.

The MAPK family is composed of three majors kinases, JNK, p38 and ERK1/2, and is implicated in many degenerative processes, including retinal cell death. The purpose of our study was to evaluate the activation of ERK1/2 kinase, and its potential role in Müller cell gliosis, during photoreceptor cell death in Rpe65(-/-) mice. We assayed ERK1/2 mRNA and protein levels, and evaluated ERK1/2 phosphorylation involved in kinase activation, in 2, 4 and 6 month-old Rpe65(-/-) mice and in age-matched wild-type controls. No differences in ERK1/2 expression were detected between Rpe65(-/-) and wild-type mice, however, ERK1/2 phosphorylation was dramatically increased in the knock out mice at 4 and 6 months-of-age. Phosphorylated ERK1/2 co-localized with GFAP in the ganglion cell layer, and correlated with an increase in GFAP protein expression and retinal cell death. Accumulation of cFOS protein in the ganglion cell layer occurred concomitant with pERK1/2 activation. Müller cell proliferation was not observed. ERK1/2 activation did not occur in 2 month-old Rpe65(-/-) or in the Rpe65(-/-)/Gnat1(-/-) mice, in which no degeneration was evident. The observed activation ERK1/2 and GFAP, both markers of Müller cell gliosis, in the absence of Müller cell proliferation, is consistent with the activation of atypical gliosis occurring during the slow process of degeneration in Rpe65(-/-) mice. As Müller cell gliosis is activated in many neuronal and retinal degenerative diseases, further studies will be needed to determine whether atypical gliosis in Rpe65(-/-) mice contributes to, or protects against, the pathogenesis occurring in this model of Leber congenital amaurosis.

Efficacy of PI3K pathway inhibitors as single agents in metastatic breast cancer

L’objectiu del cribatge molecular és seleccionar pacients que es beneficiïn especialment de teràpies dirigides. S’analitza l’activitat en monoteràpia de fàrmacs inhibidors de la via de PI3K/AKT/mTOR (PI3Ki) en pacients amb càncer de mama metastàtic (CMM) i s’exploren potencials predictors de benefici clínic. La mitjana de temps a la progressió és de 2.6 mesos en 38 pacients incloses. No existeix correlació entre alteracions de la via i l’eficàcia, excepte en pacients amb mutació de PIK3CA que van millor al tractar-se amb un PI3Ki alfa-especific. Aquests resultats emfatitzen la necessitat d’un adequat cribatge molecular previ al tractament amb teràpies dirigides en CMM

GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens.

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the -10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

Neuroprotection by inhibiting the c-Jun N-terminal kinase pathway after cerebral ischemia occurs independently of interleukin-6 and keratinocyte-derived chemokine (KC/CXCL1) secretion.

BACKGROUND: Cerebral ischemia is associated with the activation of glial cells, infiltration of leukocytes and an increase in inflammatory mediators in the ischemic brain and systemic circulation. How this inflammatory response influences lesion size and neurological outcome remains unclear. D-JNKI1, an inhibitor of the c-Jun N-terminal kinase pathway, is strongly neuroprotective in animal models of stroke. Intriguingly, the protection mediated by D-JNKI1 is high even with intravenous administration at very low doses with undetectable drug levels in the brain, pointing to a systemic mode of action, perhaps on inflammation. FINDINGS: We evaluated whether D-JNKI1, administered intravenously 3 h after the onset of middle cerebral artery occlusion (MCAO), modulates secretion of the inflammatory mediators interleukin-6 and keratinocyte-derived chemokine in the plasma and from the spleen and brain at several time points after MCAO. We found an early release of both mediators in the systemic circulation followed by an increase in the brain and went on to show a later systemic increase in vehicle-treated mice. Release of interleukin-6 and keratinocyte-derived chemokine from the spleen of mice with MCAO was not significantly different from sham mice. Interestingly, the secretion of these inflammatory mediators was not altered in the systemic circulation or brain after successful neuroprotection with D-JNKI1. CONCLUSIONS: We demonstrate that neuroprotection with D-JNKI1 after experimental cerebral ischemia is independent of systemic and brain release of interleukin-6 and keratinocyte-derived chemokine. Furthermore, our findings suggest that the early systemic release of interleukin-6 and keratinocyte-derived chemokine may not necessarily predict an unfavorable outcome in this model.

The aldosterone-mineralocorticoid receptor pathway exerts anti-inflammatory effects in endotoxin-induced uveitis.

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

Absence of intestinal PPARγ aggravates acute infectious colitis in mice through a lipocalin-2-dependent pathway.

To be able to colonize its host, invading Salmonella enterica serovar Typhimurium must disrupt and severely affect host-microbiome homeostasis. Here we report that S. Typhimurium induces acute infectious colitis by inhibiting peroxisome proliferator-activated receptor gamma (PPARγ) expression in intestinal epithelial cells. Interestingly, this PPARγ down-regulation by S. Typhimurium is independent of TLR-4 signaling but triggers a marked elevation of host innate immune response genes, including that encoding the antimicrobial peptide lipocalin-2 (Lcn2). Accumulation of Lcn2 stabilizes the metalloproteinase MMP-9 via extracellular binding, which further aggravates the colitis. Remarkably, when exposed to S. Typhimurium, Lcn2-null mice exhibited a drastic reduction of the colitis and remained protected even at later stages of infection. Our data suggest a mechanism in which S. Typhimurium hijacks the control of host immune response genes such as those encoding PPARγ and Lcn2 to acquire residence in a host, which by evolution has established a symbiotic relation with its microbiome community to prevent pathogen invasion.

The PPARalpha-leukotriene B4 pathway to inflammation control.

Inflammation is a local immune response to 'foreign' molecules, infection and injury. Leukotriene B4, a potent chemotactic agent that initiates, coordinates, sustains and amplifies the inflammatory response, is shown to be an activating ligand for the transcription factor PPARalpha. Because PPARalpha regulates the oxidative degradation of fatty acids and their derivatives, like this lipid mediator, a feedback mechanism is proposed that controls the duration of an inflammatory response and the clearance of leukotriene B4 in the liver. Thus PPARalpha offers a new route to the development of anti- or pro-inflammatory reagents.

Phylogenetic analyses of C4 photosynthesis evolution in grasses: genetics of convergence and convergence of genes

Summary : During the evolutionary diversification of organisms, similar ecological constraints led to the recurrent appearances of the same traits (phenotypes) in distant lineages, a phenomenon called convergence. In most cases, the genetic origins of the convergent traits remain unknown, but recent studies traced the convergent phenotypes to recurrent alterations of the same gene or, in a few cases, to identical genetic changes. However, these cases remain anecdotal and there is a need for a study system that evolved several times independently and whose genetic determinism is well resolved and straightforward, such as C4 photosynthesis. This adaptation to warm environments, possibly driven by past atmospheric CO2 decreases, consists in a CO2-concentrating pump, created by numerous morphological and biochemical novelties. All genes encoding C4 enzymes already existed in C3 ancestors, and are supposed to have been recruited through gene duplication followed by neo-functionalization, to acquire the cell specific expression pattern and altered kinetic properties that characterize Ca-specific enzymes. These predictions have so far been tested only in species-poor and ecologically marginal C4 dicots. The monocots, and especially the grass family (Poaceae), the most important C4 family in terms of species number, ecological dominance and economical importance, have been largely under-considered as suitable study systems. This thesis aimed at understanding the evolution of the C4 trait in grasses at a molecular level and to use the genetics of C4 photosynthesis to infer the evolutionary history of the C4 phenotype and its driving selective pressures. A molecular phylogeny of grasses and affiliated monocots identified 17 to 18 independent acquisitions of the C4 pathway in the grass family. A relaxed molecular clock was used to date these events and the first C4 evolution was estimated in the Chloridoideae subfamily, between 32-25 million years ago, at a period when atmospheric CO2 abruptly declined. Likelihood models showed that after the COZ decline the probability of evolving the C4 pathway strongly increased, confirming low CO2 as a likely driver of C4 photosynthesis evolution. In order to depict the genetic changes linked to the numerous C4 origins, genes encoding phopshoenolpyruvate carboxylase (PEPC), the key-enzyme responsible for the initial fixation of atmospheric CO2 in the C4 pathway, were isolated from a large sample of C3 and C4 grasses. Phylogenetic analyses were used to reconstruct the evolutionary history of the PEPC multigene family and showed that the evolution of C4-specific PEPC had been driven by positive selection on 21 codons simultaneously in up to eight C4 lineages. These selective pressures led to numerous convergent genetic changes in many different C4 clades, highlighting the repeatability of some evolutionary processes, even at the molecular level. PEPC C4-adaptive changes were traced and used to show multiple appearances of the C, pathway in clades where species tree inferences were unable to differentiate multiple C4 appearances and a single appearance followed by C4 to C3 reversion. Further investigations of genes involved in some of the C4 subtypes only (genes encoding decarboxylating enzymes NADP-malic enzyme and phosphoenolpyruvate carboxykinase) showed that these C4-enzymes also evolved through strong positive selection and underwent parallel genetic changes during the different Ca origins. The adaptive changes on these subtype-specific C4 genes were used to retrace the history of the C4-subtypes phenotypes, which revealed that the evolution of C4-PEPC and C4-decarboxylating enzymes was in several cases disconnected, emphasizing the multiplicity of the C4 trait and the gradual acquisition of the features that create the CO2-pump. Finally, phylogenetic analyses of a gene encoding the Rubisco (the enzyme responsible for the fixation of CO2 into organic compounds in all photosynthetic organisms) showed that C4 evolution switched the selective pressures on this gene. Five codons were recurrently mutated to adapt the enzyme kinetics to the high CO2 concentrations of C4 photosynthetic cells. This knowledge could be used to introgress C4-like Rubisco in C3 crops, which could lead to an increased yield under predicted future high CO2 atmosphere. Globally, the phylogenetic framework adopted during this thesis demonstrated the widespread occurrence of genetic convergence on C4-related enzymes. The genetic traces of C4 photosynthesis evolution allowed reconstructing events that happened during the last 30 million years and proved the usefulness of studying genes directly responsible for phenotype variations when inferring evolutionary history of a given trait. Résumé Durant la diversification évolutive des organismes, des pressions écologiques similaires ont amené à l'apparition récurrente de certains traits (phénotypes) dans des lignées distantes, un phénomène appelé évolution convergente. Dans la plupart des cas, l'origine génétique des traits convergents reste inconnue mais des études récentes ont montré qu'ils étaient dus dans certains cas à des changements répétés du même gène ou, dans de rares cas, à des changements génétiques identiques. Malgré tout, ces cas restent anecdotiques et il y a un réel besoin d'un système d'étude qui ait évolué indépendamment de nombreuses fois et dont le déterminisme génétique soit clairement identifié. La photosynthèse dite en Ça répond à ces critères. Cette adaptation aux environnements chauds, dont l'évolution a pu être encouragé par des baisses passées de la concentration atmosphérique en CO2, est constituée de nombreuses nouveautés morphologiques et biochimiques qui créent une pompe à CO2. La totalité des gènes codant les enzymes Ç4 étaient déjà présents dans les ancêtres C3. Leur recrutement pour la photosynthèse Ç4 est supposé s'être fait par le biais de duplications géniques suivies par une néo-fonctionnalisation pour leur conférer l'expression cellule-spécifique et les propriétés cinétiques qui caractérisent les enzymes C4. Ces prédictions n'ont jusqu'à présent été testées que dans des familles C4 contenant peu d'espèces et ayant un rôle écologique marginal. Les graminées (Poaceae), qui sont la famille C4 la plus importante, tant en termes de nombre d'espèces que de dominance écologique et d'importance économique, ont toujours été considérés comme un système d'étude peu adapté et ont fait le sujet de peu d'investigations évolutives. Le but de cette thèse était de comprendre l'évolution de la photosynthèse en C4 chez les graminées au niveau génétique et d'utiliser les gènes pour inférer l'évolution du phénotype C4 ainsi que les pressions de sélection responsables de son évolution. Une phylogénie moléculaire de la famille des graminées et des monocotylédones apparentés a identifié 17 à 18 acquisitions indépendantes de la photosynthèse chez les graminées. Grâce à une méthode d'horloge moléculaire relâchée, ces évènements ont été datés et la première apparition C4 a été estimée dans la sous-famille des Chloridoideae, il y a 32 à 25 millions d'années, à une période où les concentrations atmosphériques de CO2 ont décliné abruptement. Des modèles de maximum de vraisemblance ont montré qu'à la suite du déclin de CO2, la probabilité d'évoluer la photosynthèse C4 a fortement augmenté, confirmant ainsi qu'une faible concentration de CO2 est une cause potentielle de l'évolution de la photosynthèse C4. Afin d'identifier les mécanismes génétiques responsables des évolutions répétées de la photosynthèse C4, un segment des gènes codant pour la phosphoénolpyruvate carboxylase (PEPC), l'enzyme responsable de la fixation initiale du CO2 atmosphérique chez les plantes C4, ont été séquencés dans une centaine de graminées C3 et C4. Des analyses phylogénétiques ont permis de reconstituer l'histoire évolutive de la famille multigénique des PEPC et ont montré que l'évolution de PEPC spécifiques à la photosynthèse Ça a été causée par de la sélection positive agissant sur 21 codons, et ce simultanément dans huit lignées C4 différentes. Cette sélection positive a conduit à un grand nombre de changements génétiques convergents dans de nombreux clades différents, ce qui illustre la répétabilité de certains phénomènes évolutifs, et ce même au niveau génétique. Les changements sur la PEPC liés au C4 ont été utilisés pour confirmer des évolutions indépendantes du phénotype C4 dans des clades où l'arbre des espèces était incapable de différencier des apparitions indépendantes d'une seule apparition suivie par une réversion de C4 en C3. En considérant des gènes codant des protéines impliquées uniquement dans certains sous-types C4 (deux décarboxylases, l'enzyme malique à NADP et la phosphoénolpyruvate carboxykinase), des études ultérieures ont montré que ces enzymes C4 avaient elles-aussi évolué sous forte sélection positive et subi des changements génétiques parallèles lors des différentes origines de la photosynthèse C4. Les changements adaptatifs sur ces gènes liés seulement à certains sous-types C4 ont été utilisés pour retracer l'histoire des phénotypes de sous-types C4, ce qui a révélé que les caractères formant le trait C4 ont, dans certains cas, évolué de manière déconnectée. Ceci souligne la multiplicité du trait C4 et l'acquisition graduelle de composants participant à la pompe à CO2 qu'est la photosynthèse C4. Finalement, des analyses phylogénétiques des gènes codant pour la Rubisco (l'enzyme responsable de la fixation du CO2 en carbones organiques dans tous les organismes photosynthétiques) ont montré que l'évolution de la photosynthèse Ça a changé les pressions de sélection sur ce gène. Cinq codons ont été mutés de façon répétée afin d'adapter les propriétés cinétiques de la Rubisco aux fortes concentrations de CO2 présentes dans les cellules photosynthétiques des plantes C4. Globalement, l'approche phylogénétique adoptée durant cette thèse de doctorat a permis de démontré des phénomène fréquents de convergence génétique sur les enzymes liées à la photosynthèse C4. Les traces génétiques de l'évolution de la photosynthèse C4 ont permis de reconstituer des évènements qui se sont produits durant les derniers 30 millions d'années et ont prouvé l'utilité d'étudier des gènes directement responsables des variations phénotypiques pour inférer l'histoire évolutive d'un trait donné.

Arbuscular mycorrhiza-specific signaling in rice transcends the common symbiosis signaling pathway.

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

Critical Role of the GM-CSF Signaling Pathway in Macrophage Pro-Repair Activities.

OBJECTIVE: Macrophages play a critical role in intestinal wound repair. However, the molecular pathways that regulate macrophage wound repair activities remain poorly understood. The aim of this study was to evaluate the role of GM-CSF receptor signaling in the wound repair activities of macrophages. METHODS: Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of Crohn's disease (CD) patients. In vitro models were used to study the repair activities of macrophages. RESULTS: We provide evidence that GM-CSF receptor signaling is required for murine macrophages to promote epithelial repair. In addition, we demonstrate that the deficient repair properties of macrophages from CD patients with active disease can be recovered via GM-CSF therapy. CONCLUSION: Our data support a critical role of the GM-CSF signaling pathway in the pro-repair activities of mouse and human macrophages. © 2014 S. Karger AG, Basel.

The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming.

We present a high-quality (>100× depth) Illumina genome sequence of the leaf-cutting ant Acromyrmex echinatior, a model species for symbiosis and reproductive conflict studies. We compare this genome with three previously sequenced genomes of ants from different subfamilies and focus our analyses on aspects of the genome likely to be associated with known evolutionary changes. The first is the specialized fungal diet of A. echinatior, where we find gene loss in the ant's arginine synthesis pathway, loss of detoxification genes, and expansion of a group of peptidase proteins. One of these is a unique ant-derived contribution to the fecal fluid, which otherwise consists of "garden manuring" fungal enzymes that are unaffected by ant digestion. The second is multiple mating of queens and ejaculate competition, which may be associated with a greatly expanded nardilysin-like peptidase gene family. The third is sex determination, where we could identify only a single homolog of the feminizer gene. As other ants and the honeybee have duplications of this gene, we hypothesize that this may partly explain the frequent production of diploid male larvae in A. echinatior. The fourth is the evolution of eusociality, where we find a highly conserved ant-specific profile of neuropeptide genes that may be related to caste determination. These first analyses of the A. echinatior genome indicate that considerable genetic changes are likely to have accompanied the transition from hunter-gathering to agricultural food production 50 million years ago, and the transition from single to multiple queen mating 10 million years ago.