Selective expression of a dominant-negative form of peroxisome proliferator-activated receptor in keratinocytes leads to impaired epidermal healing.

Many nuclear hormone receptors are involved in the regulation of skin homeostasis. However, their role in the epithelial compartment of the skin in stress situations, such as skin healing, has not been addressed yet. The healing of a skin wound after an injury involves three major cell types: immune cells, which are recruited to the wound bed; dermal fibroblasts; and epidermal and hair follicle keratinocytes. Our previous studies have revealed important but nonredundant roles of PPARalpha and beta/delta in the reparation of the skin after a mechanical injury in the adult mouse. However, the mesenchymal or epithelial cellular compartment in which PPARalpha and beta/delta play a role could not be determined in the null mice used, which have a germ line PPAR gene invalidation. In the present work, the role of PPARalpha was studied in keratinocytes, using transgenic mice that express a PPARalpha mutant with dominant-negative (dn) activity specifically in keratinocytes. This dn PPARalpha lacks the last 13 C terminus amino acids, binds to a PPARalpha agonist, but is unable to release the nuclear receptor corepressor and to recruit the coactivator p300. When selectively expressed in keratinocytes of transgenic mice, dn PPARalphaDelta13 causes a delay in the healing of skin wounds, accompanied by an exacerbated inflammation. This phenotype, which is similar to that observed in PPARalpha null mice, strongly suggests that during skin healing, PPARalpha is required in keratinocytes rather than in other cell types.

ON THE ROLE OF PERIOD-2 IN THE CIRCADIAN AND HOMEOSTATIC REGULATION OF SLEEP

Humans spend one third of their life sleeping, then we could raise the basic question: Why do we sleep? Despite the fact that we still don't fully understand its function, we made much progress in understanding at different levels how sleep is regulated. One model suggests that sleep is regulated by two processes: a homeostatic process that tracks the need for sleep and by a circadian rhythm that determines the preferred time-of-day sleep occurs. At the molecular level circadian rhythms are a property of interlocking transcriptional regula-tors referred to as clock genes. The heterodimeric transcription factors BMAL1::CLOCK/NPAS2 drive the transcription of many target genes including the clock genes Cryptochome1 (Cry1), Cry2, Period1 (Per1), and Per2. The encoded CRY/PER proteins are transcriptional inhibitors of BMAL1::CLOCK/NPAS2 thereby providing negative feedback to their own transcription. These genes seem, however, also involved in sleep homeostasis because the brain expression of clock genes, es-pecially that of Per2, increase as a function of time-spent-awake and because mice lacking clock genes display altered sleep homeostasis. The aim of first part of my doctoral work has been to advance our understanding the link that exists between sleep homeostasis and circadian rhythms investigating a possible mechanism by which sleep deprivation could alter clock gene expression by quantifying DNA-binding of the core-clock genes BMAL1, CLOCK and NPAS2 to their target chromatin loci including the E-box enhancers of the Per2 promoter. We made use of chromatin immunoprecipitation (ChIP) and quantitative poly-merase chain reaction (qPCR) to show that DNA-binding of CLOCK and BMAL1 to their target genes changes as a function of time-of-day in both liver and cerebral cortex. We then performed a 6h sleep deprivation (SD) and observed a significant decrease in DNA-binding of CLOCK and BMAL1 to Dbp. This is consistent with a decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was similarly decreased following SD. However, SD has been previously shown to in-crease Per2 expression in the cortex which seems paradoxical. Our results demonstrate that sleep-wake history can affect the molecular clock machinery directly at the level of the chromatin thereby altering the cortical expression of Dbp and Per2, and likely other targets. However, the precise dy-namic relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive. The second aim of my doctoral work has been to perform an in depth characterization of cir-cadian rhythmicity, sleep architecture, analyze the response to SD in full null-Per2 knock-out (Per2-/-) mice, and Per1-/- mice, as well as their double knock-out offspring (Per1,2-/-) and littermate wildtype (Wt) mice. The techniques used include locomotor activity recording by passive infrared (PIR) sen-sors, EEG/EMG surgery, recording, and analysis, and cerebral cortex extraction and quantification of mRNA levels by qPCR. Under standard LD12:12 conditions, we found that wakefulness onset, as well as the time courses of clock gene expression in the brain and corticosterone plasma levels were ad-vanced by about 2h in Per2-/- mice compared to Wt mice. When released under constant dark condi-tions almost all Per2-/- mice (97%) became arrhythmic immediately. From these observations, we conclude that while Per2-/- mice seem to be able to anticipate dark onset, this does not result from a self-sustained circadian clock. Our results suggest instead that the earlier onset of activity results from a labile, not-self sustained 22h rhythm linked to light onset suggesting the existence of a light-driven rhythm. Analyses of sleep under LD12:12 conditions revealed that in both Per2-/- and Per1,2-/- mice the same sleep phenotypes are observed compared to Wt mice: increased NREM sleep frag-mentation and inability to adequately compensate the loss of NREM sleep. That suggests a possible role of PER2 in sleep consolidation and recovery.

Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis.

A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

The FoxO3a gene is a key negative target of canonical Notch signalling in the keratinocyte UVB response.

Notch signalling has an important role in skin homeostasis, promoting keratinocyte differentiation and suppressing tumorigenesis. Here we show that this pathway also has an essential anti-apoptotic function in the keratinocyte UVB response. Notch1 expression and activity are significantly induced, in a p53-dependent manner, by UVB exposure of primary keratinocytes as well as intact epidermis of both mouse and human origin. The apoptotic response to UVB is increased by deletion of the Notch1 gene or down-modulation of Notch signalling by pharmacological inhibition or genetic suppression of 'canonical' Notch/CSL/MAML1-dependent transcription. Conversely, Notch activation protects keratinocytes against apoptosis through a mechanism that is not linked to Notch-induced cell cycle withdrawal or NF-kappaB activation. Rather, transcription of FoxO3a, a key pro-apoptotic gene, is under direct negative control of Notch/HERP transcription in keratinocytes, and upregulation of this gene accounts for the increased susceptibility to UVB of cells with suppressed Notch signalling. Thus, the canonical Notch/HERP pathway functions as a protective anti-apoptotic mechanism in keratinocytes through negative control of FoxO3a expression.

Regulation of mucoidy in the biological control agent Pseudomonas fluorescens CHA0 and construction of an exopolysaccharide-overproducing variant

Pseudomonas fluorescens CHA0, an effective biological control agent of soilborne plant diseases, is naturally non-mucoid. We have isolated a highly mucoid Tn5 insertion mutant of strain CHA0. The mucoid phenotype was found to be due to the overproduction of exopolysaccharide (EPS), as a result of a mutation in the mucA gene. The wild-type mucA gene was cloned by a two-step, Tn5-dependent cloning procedure previously described and the deduced amino acid sequence showed 71% identity with MucA of P. aeruginosa, a negative regulator of the alternative sigma factor AlgU (=s22, sE). As in P. aeruginosa, mucA is preceded by the algU gene encoding s22 (91% identity at the amino acid sequence level). A mucA in-frame deletion mutant of CHA0 overproduced EPS and formed mucoid colonies, whereas an algU in-frame deletion mutant showed a non-mucoid phenotype. Pyoluteorin, an antibiotic produced by P. fluorescens, was found to be entrapped in EPS of a mucoid mutant. In natural soil, mucoidy negatively affected survival of the bacteria, suggesting that under these conditions the potential to produce abundant EPS does not confer a selective advantage on the bacteria.

Regulation of the AKAP-LBC signaling complex : molecular characterization and functional implications

RÉSUMÉ Les protéines d'ancrage de la protéine kinase A (AKAPs) constituent une grande famille de protéines qui ciblent la protéine kinase A (PKA) à proximité de ses substrats physiologiques pour assurer leur régulation. Une nouvelle protéine de cette famille, appelée AKAP-Lbc, a été récemment caractérisée et fonctionne comme un facteur d'échange de nucléotides guanine (GEF) pour la petite GTPase Rho. AKAP-Lbc est régulée par différents signaux qui activent et désactivent son activité Rho-GEF. Son activation est assurée par la sous-unité alpha de la protéine G hétérotrimérique G12, tandis que son inhibition dépend de son interaction avec la PKA et 14-3-3. AKAP-Lbc est principalement exprimée dans le coeur et pourrait réguler des processus importants tels que l'hypertrophie et la différenciation des cardiomyocytes. Ainsi, il est crucial d'élucider les mécanismes moléculaires impliqués dans la régulation de son activité Rho-GEF. Le but général de ce travail de thèse est la caractérisation de deux nouveaux mécanismes impliqués dans la régulation de l'activité de AKAP-Lbc. Le premier mécanisme consiste en la régulation de l'activité de AKAP-Lbc par son homo-oligomérisation. Mes travaux montrent que l'homo-oligomérisation maintient AKAP-Lbc inactive, dans une conformation permettant à la PKA ancrée et à 14-3-3 d'exercer leur effet inhibiteur sur l'activité de AKAP-Lbc. Le second mécanisme concerne la régulation de l'activité de AKAP-Lbc via une nouvelle interaction entre AKAP-Lbc et la protéine LC3. LC3 joue un rôle crucial dans l'autophagie, un processus cellulaire qui adresse les protéines cytoplasmiques au lysosome pour leur dégradation. Ce mécanisme est particulièrement important pour le survie des cardiomyocytes durant les périodes d'absence de nutriments. Mes travaux mettent en évidence que LC3 inhibe l'activité Rho-GEF de AKAP-Lbc, ce qui suggère que, au-delà son rôle bien établi dans l'autophagie, LC3 participerait à la régulation de la signalisation de Rho. Prises ensembles, ces études contribuent à comprendre comment le complexe de signalisation formé par AKAP-Lbc régule la signalisation de Rho dans les cellules. Au-delà de leur intérêt au niveau biochimique, ces travaux pourraient aussi contribuer à élucider les réseaux de signalisation qui régulent des phénomènes physiologiques dans le coeur. ABSTRACT A-kinase anchoring proteins (AKAPs) are a group of functionally related proteins, which target the cAMP dependent protein kinase A (PKA) in close proximity to its physiological substrates for ensuring their regulation. A novel PKA anchoring protein, termed AKAP-Lbc, has been recently characterized, which also functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rho. AKAP-Lbc is regulated in a bi-directional manner by signals which activate or deactivate its Rho-GEF activity. Activation is mediated by the alpha subunit of the heterotrimeric G protein G12, whereas inhibition occurs following its interaction with PKA and 14-3-3. AKAP-Lbc is predominantly expressed in the heart and might regulate important processes such as hypertrophy and differentiation of cardiomyocytes. Therefore ít is crucial to elucidate the molecular mechanisms involved in the regulation of the Rho-GEF activity of AKAP-Lbc. The general aim of the present thesis work is the characterization of two novel molecular mechanisms involved in the regulation of the Rho-GEF activity of AKAP-Lbc. The first mechanism consists of the. regulation of AKAP-Lbc activity through its homooligomerization. I report here that homo-oligomerization maintains AKAP-Lbc inactive, under a conformation suitable for ensuring the inhibitory effect of anchored PKA and 14-33 on AKAP-Lbc activity. The second mechanism concerns the regulation of AKAP-Lbc activity through a novel interaction between AKAP-Lbc and ubiquitin-like protein LC3. LC3 is a key mediator of autophagy, which is a cellular process that targets cytosolic proteins to the lysosome for degradation. This process is particularly important for cardiomyocyte survival during conditions of nutrient starvation. Here, I show that LC3 is a negative regulator of the Rho-GEF activity of AKAP-Lbc, which suggests that, beyond its well established role in autophagy, LC3 can participate in the regulation of Rho signaling in cells. Overall, these findings contribute to understand how the AKAP-Lbc signaling complex can regulate the Rho signaling in cells. Beyond its interest at the biochemical level, this work might also contribute to elucidate the signaling network that regulate physiological events in the heart.

Regulation of mass and function of pancreatic β-cells: identification of anti-apoptotic peptides and role of GLP-1

Résumé La masse de cellules β sécrétrices d'insuline est un tissu dynamique qui s'adapte aux variations de la demande métabolique pour assurer une normoglycémie. Cette adaptation se fait par un changement de sécrétion d'insuline et de la masse totale des cellules β. Une perte complète ou partielle des cellules β conduit respectivement à un diabète de type 1 et de type 2. Les mécanismes qui régulent la masse de cellules β et maintiennent leur phénotype differencié sont encore peu connus. Leur identification est nécessaire pour comprendre le développement du diabète et développer des stratégies de traitement. La greffe d'îlots est une approche thérapeutique prometteuse pour le diabète de type 1, mais est limitée par une perte précoce des cellules β due à une apoptose induite par des cytokines. Afin d'améliorer la survie des cellules β lors de la greffe d'îlots, le premier but était de trouver des peptides pouvant bloquer l'apoptose induite par FasL et TNF-α. Pour ce faire, deux librairies de phages ont été criblées pour sélectionner des peptides se liant au Fas DD ou au TNFRl DD. Nous avons identifié six peptides différents. Cependant, aucun d'entre eux n'était capable de protéger les cellules de l'apoptose induite par FasL ou TNF-α. Deuxièmement, le GLP-1 est une hormone qui stimule la sécrétion d'insuline, et est impliquée dans la prolifération des cellules β, la différentiation, et inhibe l'apoptose. Nous avons fait l'hypothèse que le GLP-1 joue un rôle crucial dans le contrôle de la masse et de la fonction des cellules β. Afin de l'évaluer, une analyse par puce à ADN a été réalisée en comparant des cellules βTC-Tet traitées avec du GLP-1 à des cellules non-traitées. 376 gènes régulés ont été identifiés, dont RGS2, CREM, ICERI et DUSP14, augmentés significativement par le GLP-1. Nous avons confirmé que le GLP-1 augmente l'expression de ces gènes, aussi bien au niveau des transcripts que des protéines. De plus, nous avons montré que le GLP-1 induit leur expression par activation de la voie cAMP/PKA, et nécessite l'entrée de calcium extracellulaire. D'après leur fonction biologique, nous avons ensuite supposé que ces gènes pourraient agir comme régulateurs négatifs de la signalisation du GLP-l, et donc freiner son effet proliférateur. Pour vérifier notre hypothèse, des siRNAs contre ces gènes ont été développés, et leurs effets sur la prolifération des cellules β seront évalués ultérieurement. Abstract The pancreatic β-cell mass is a dynamic tissue which adapts to variations in metabolic demand in order to ensure normoglycemia. This adaptation occurs through a change in both insulin secretion and the total mass of ,β-cells. An absolute or relative loss of β-cells leads to type 1 and type 2 diabetes, respectively. The mechanisms that regulate the pancreatic β-cell mass and maintain the fully differentiated phenotype of the insulin-secreting β-cells are only poorly defined. Their identification is required to understand the progression of diabetes, but also to design strategies for the treatment of diabetes. Islet transplantation is a promising therapeutic approach for type 1 diabetes, but it is still limited by an early graft loss due to cytokine-induced apoptosis. In order to improve β-cell survival during islet transplantation, our first goal was to find novel blockers of FasL- and TNF-α-mediated cell death in the form of peptides. To that end, we screened two phage display libraries to select Fas DD- or TNFR1 DD-binding peptides. We identified six different small peptides. However, none of these peptides was able to prevent cells from FasL- or TNF-α-mediated apoptosis. Secondly, GLP-1 is a hormone that has been shown to stimulate insulin secretion and to be involved in β-cell proliferation, differentiation and inhibition of apoptosis. We hypothesized that GLP-1 plays a crucial role to control mass and function of β-cells. To evaluate this hypothesis, we performed a cDNA microarray analysis with GLP-1-treated βTC-Tet cells compared to untreated cells. We found 376 regulated genes, among these, RGS2, CREM, ICERI and DUSP14, which were significantly upregulated by GLP-1. We confirmed that both their mRNA and protein levels were strongly and rapidly increased after GLP-1 treatment. Moreover, we found that GLP-1 activates their expression mainly through the activation of the cAMP/PKA signaling pathway, and requires extracellular calcium entry. According to their biological function, we then hypothesized that these genes might act as negative regulators of the GLP-1 signaling. In particular, they might brake the effects of GLP-1 on β-cell proliferation. To verify this hypothesis, siRNAs against these genes were developed. The effect of these siRNAs on GLP-1-induced β-cell proliferation will be evaluated later.

Down-regulation of interferon signature in systemic lupus erythematosus patients by active immunization with interferon α-kinoid.

OBJECTIVE: We developed interferon-α-kinoid (IFN-K), a drug composed of inactivated IFNα coupled to a carrier protein, keyhole limpet hemocyanin. In human IFNα-transgenic mice, IFN-K induces polyclonal antibodies that neutralize all 13 subtypes of human IFNα. We also previously demonstrated that IFN-K slows disease progression in a mouse model of systemic lupus erythematosus (SLE). This study was undertaken to examine the safety, immunogenicity, and biologic effects of active immunization with IFN-K in patients with SLE. METHODS: We performed a randomized, double-blind, placebo-controlled, phase I/II dose-escalation study comparing 3 or 4 doses of 30 μg, 60 μg, 120 μg, or 240 μg of IFN-K or placebo in 28 women with mild to moderate SLE. RESULTS: IFN-K was well tolerated. Two SLE flares were reported as serious adverse events, one in the placebo group and the other in a patient who concomitantly stopped corticosteroids 2 days after the first IFN-K dose, due to mild fever not related to infection. Transcriptome analysis was used to separate patients at baseline into IFN signature-positive and -negative groups, based on the spontaneous expression of IFN-induced genes. IFN-K induced anti-IFNα antibodies in all immunized patients. Notably, significantly higher anti-IFNα titers were found in signature-positive patients than in signature-negative patients. In IFN signature-positive patients, IFN-K significantly reduced the expression of IFN-induced genes. The decrease in IFN score correlated with the anti-IFNα antibody titer. Serum complement C3 levels were significantly increased in patients with high anti-IFNα antibody titers. CONCLUSION: These results show that IFN-K is well tolerated, immunogenic, and significantly improves disease biomarkers in SLE patients, indicating that further studies of its clinical efficacy are warranted.

Regulation of the activity of DnaA and its role during the cell cycle of caulobacter crescentus

The initiation of chromosomal replication must be tightly regulated so that the genome is replicated only once per cell cycle. In most bacteria, DnaA binds to the origin of replication and initiates chromosomal replication. DnaA is a dual-function protein that also acts as an important transcription factor that regulates the expression of many genes in bacteria. Thus, understanding how this protein is regulated during the bacterial cell cycle is of major importance. The α-proteobacterium Caulobacter crescentus is an excellent model to study the bacterial cell cycle, mainly because it is possible to isolate synchronized cell cultures and because it initiates the replication of its chromosome once per cell cycle and at a specific time of the cell cycle. This latest feature is of special interest for the major aim of my thesis work, which focused on the temporal and spatial regulation of the activity of the essential DnaA protein in C. crescentus. In Escherichia coli, the Hda protein converts ATP-DnaA into ADP- DnaA by stimulating the ATPase activity of DnaA, to prevent over-initiation of chromosome replication. We propose that there exists a similar mechanism in C. crescentus, which is not only involved in the temporal control of chromosome replication, but also in the control of gene expression. First, we provided evidences indicating that the hydrolysis of the ATP bound to DnaA is essential for the viability of C. crescentus. Our results suggest that ATP-DnaA promotes the initiation of chromosome replication, since we found that cells over-expressing a DnaA protein with a mutated ATPase domain, DnaA(R357A), over-initiated chromosome replication, unlike cells expressing the wild-type DnaA protein at similar levels. By contrast, the DnaA(R357A) protein was less active than DnaA in promoting the transcription of three essential genes, suggesting that these may be more efficiently activated by ADP-DnaA than ATP-DnaA. We propose that the ATP-DnaA to ADP-DnaA switch down-regulates the initiation of DNA replication while activating the transcription of several essential genes involved in subsequent cell cycle events. Second, we studied the role of the HdaA protein, homologous to Hda, in promoting the ATP- DnaA to ADP-DnaA switch in C. crescentus. HdaA is essential for viability and its depletion in the cell leads to an over-replication of the chromosome, indicating that HdaA is a negative regulator of DNA replication. HdaA dynamically co-localizes with the replisome. In this work, we identified DnaN, the β-clamp of the DNA polymerase, as the replisome component that interacts directly with HdaA and that recruits HdaA to the replisome in live C. crescentus cells. We also showed that a mutant HdaA protein that cannot interact or co-localize with DnaN is not functional, indicating that HdaA is probably activated by DnaN. However, we found that another non-functional HdaA protein, mutated in the conserved Arginine finger of its AAA+ domain, was able to localize at the replisome, suggesting that the AAA+ domain of HdaA exerts its essential function after the recruitment of HdaA to the replisome. We propose that HdaA stimulates the ATPase activity of DnaA once DNA replication is ongoing, via its interaction with DnaN and the activity of the two conserved R fingers of DnaA and HdaA. Finally, we created different strains in which HdaA, DnaN or DnaA were over-produced. We observed that the over-production of HdaA seems to lead to a delay in chromosome replication, while the over-production of DnaN had an opposite effect. Our results also indicate that the over-production of DnaA may intensify the over-initiation phenotype of cells depleted for HdaA. We conclude that the dynamic interplay of HdaA and DnaN in the cell contributes to regulating the ATP-DnaA/ADP-DnaA ratio in the cell, to ensure once per cell cycle initiation of chromosomal replication in C. crescentus. Altogether, our work provided important information on the regulation of the activity of DnaA in C. crescentus. Since DnaA, HdaA and DnaN are well-conserved proteins, most of our findings are useful to understand how chromosome replication and gene expression are controlled by DnaA in many other bacterial species. - L'initiation de la réplication des chromosomes doit être précisément régulée de telle sorte que le génome ne soit répliqué qu'une seule fois par cycle cellulaire. Chez la plupart des bactéries, DnaA se lie à l'origine de réplication du chromosome et en initie sa réplication. DnaA est aussi un facteur de transcription qui régule l'expression de nombreux gènes bactériens. De ce fait, il est très important de comprendre comment DnaA est régulée au cours du cycle cellulaire bactérien. L'a-protéobactérie Caulobacter crescentus est un excellent modèle pour étudier le cycle cellulaire bactérien, essentiellement parce qu'il est aisé d'isoler des populations de cellules synchronisées à la même étape du cycle cellulaire et parce que cette bactérie n'initie la réplication de son chromosome qu'une seule fois et à un moment précis de son cycle. Cette dernière caractéristique est particulièrement pertinente pour l'objectif de mon travail doctoral, qui consistait à comprendre comment l'activité de la protéine essentielle DnaA est régulée dans l'espace et dans le temps chez C. crescentus. Chez Escherichia coli, la protéine Hda convertie DnaA-ATP en DnaA-ADP en stimulant l'activité ATPasique de DnaA, ce qui empêche la sur-initiation de la réplication du chromosome. Nous proposons qu'un mécanisme similaire existe chez C. crescentus. Il serait non seulement nécessaire au contrôle de la réplication du chromosome, mais aussi au contrôle de l'expression de certains gènes. Dans un premier temps, nous avons mis en évidence le fait que l'hydrolyse de l'ATP lié à DnaA est un processus essentiel à la viabilité de C. crescentus. Nos résultats suggèrent que DnaA-ATP initie la réplication du chromosome, comme nous avons observé que des cellules qui sur-expriment une protéine DnaA(R357A) mutée sans domaine ATPasique fonctionnel, sur-initie la réplication de leur chromosome, contrairement aux cellules qui sur-expriment la protéine DnaA sauvage à des niveaux équivalents. Au contraire, la protéine DnaA(R357A) était moins active que la protéine DnaA sauvage pour promouvoir la transcription de trois gènes essentiels, ce qui suggère que ces derniers sont peut-être plus efficacement activés par DnaA-ADP que DnaA-ATP. Nous proposons que la conversion de DnaA-ATP en DnaA-ADP réprime l'initiation de la réplication, tandis qu'elle active la transcription de plusieurs gènes impliqués dans des étapes plus tardives du cycle cellulaire. Dans un deuxième temps, nous avons étudié le rôle de la protéine HdaA, homologue à Hda, dans la conversion de DnaA-ATP en DnaA-ADP chez C. crescentus. Cette protéine est essentielle à la viabilité de C. crescentus et sa déplétion donne des cellules qui sur-initient la réplication de leur chromosome, suggérant que HdaA est un répresseur de la réplication du chromosome. HdaA co-localise de manière dynamique avec le réplisome. Lors de mon travail doctoral, nous avons démontré que DnaN, le β-clamp de l'ADN polymérase, est l'élément qui recrute HdaA au réplisome in vivo. Nous avons aussi montré qu'une protéine HdaA mutante qui ne peut pas interagir ou co-localiser avec DnaN, n'est pas fonctionnelle, ce qui suggère que HdaA est activée par DnaN. Nous avons néanmoins aussi isolé une autre protéine HdaA non fonctionnelle, dont une arginine conservée de son domaine AAA+ était mutée, mais qui pouvait toujours co-localiser avec le réplisome, ce qui suggère que le domaine AAA+ de HdaA est nécessaire après le recrutement de HdaA au réplisome. Nous proposons que HdaA stimule l'activité ATPasique de DnaA qu'une fois que la réplication a commencé, grâce à son interaction avec DnaN et aux deux arginines conservées des protéines HdaA et DnaA. Finalement, nous avons construit différentes souches sur-exprimant HdaA, DnaN ou DnaA. Nous avons observé que la sur-production de HdaA retarde la réplication du chromosome, tandis que la sur-production de DnaN a un effet opposé. Nos observations suggèrent aussi que la sur-expression de DnaA dans des cellules déplétées pour HdaA aggrave leur phénotype de sur-initiation. Nous en concluons que HdaA et DnaN collaborent étroitement et de manière dynamique pour réguler le rapport DnaA-ATP/DnaA-ADP dans la cellule, pour s'assurer que la réplication du chromosome ne soit initiée qu'une seule fois par cycle cellulaire chez C. crescentus. Globalement, notre travail a mis en évidence des informations importantes sur la régulation de l'activité de DnaA chez C. crescentus. Comme DnaA, HdaA et DnaN sont des protéines très conservées, la plupart de nos découvertes sont utiles pour mieux comprendre comment la réplication du chromosome bactérien et l'expression des gènes sont contrôlées par DnaA chez de nombreuses autres espèces bactériennes.

Salt tolerance and regulation of gas exchange and hormonal homeostasis by auxin-priming in wheat

The objective of this work was to assess the regulatory effects of auxin-priming on gas exchange and hormonal homeostasis in spring wheat subjected to saline conditions. Seeds of MH-97 (salt-intolerant) and Inqlab-91 (salt-tolerant) cultivars were subjected to 11 priming treatments (three hormones x three concentrations + two controls) and evaluated under saline (15 dS m-1) and nonsaline (2.84 dS m-1) conditions. The priming treatments consisted of: 5.71, 8.56, and 11.42 × 10-4 mol L-1 indoleacetic acid; 4.92, 7.38, and 9.84 × 10-4 mol L-1 indolebutyric acid; 4.89, 7.34, and 9.79 × 10-4 mol L-1 tryptophan; and a control with hydroprimed seeds. A negative control with nonprimed seeds was also evaluated. All priming agents diminished the effects of salinity on endogenous abscisic acid concentration in the salt-intolerant cultivar. Grain yield was positively correlated with net CO2 assimilation rate and endogenous indoleacetic acid concentration, and it was negatively correlated with abscisic acid and free polyamine concentrations. In general, the priming treatment with tryptophan at 4.89 × 10-4 mol L-1 was the most effective in minimizing yield losses and reductions in net CO2 assimilation rate, under salt stress conditions. Hormonal homeostasis increases net CO2 assimilation rate and confers tolerance to salinity on spring wheat.

Multilayered organization of jasmonate signalling in the regulation of root growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

PPARα is Required for PPARδ action in regulation of body weight and hepatic steatosis in mice.

NlmCategory="UNASSIGNED">Peroxisome proliferator activated receptors alpha (PPARα) and delta (PPARδ) belong to the nuclear receptor superfamily. PPARα is a target of well established lipid-lowering drugs. PPARδ (also known as PPARβ/δ) has been investigated as a promising antidiabetic drug target; however, the evidence in the literature on PPARδ effect on hepatic lipid metabolism is inconsistent. Mice conditionally expressing human PPARδ demonstrated pronounced weight loss and promoted hepatic steatosis when treated with GW501516 (PPARδ-agonist) when compared to wild type mice. This effect was completely absent in mice with either a dominant negative form of PPARδ or deletion of the DNA binding domain of PPARδ. This confirmed the absolute requirement for PPARδ in the physiological actions of GW501516 and confirmed the potential utility against the human form of this receptor. Surprisingly the genetic deletion of PPARα also abrogated the effect of GW501516 in terms of both weight loss and hepatic lipid accumulation. Also the levels of the PPARα endogenous agonist 16:0/18:1-GPC were shown to be modulated by PPARδ in wild type mice. Our results show that both PPARδ and PPARα receptors are essential for GW501516-driven adipose tissue reduction and subsequently hepatic steatosis, with PPARα working downstream of PPARδ.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

NLRP12 is a neutrophil-specific, negative regulator of in vitro cell migration but does not modulate LPS- or infection-induced NF-κB or ERK signalling.

NOD-like receptors (NLR) are a family of cytosolic pattern recognition receptors that include many key drivers of innate immune responses. NLRP12 is an emerging member of the NLR family that is closely related to the well-known inflammasome scaffold, NLRP3. Since its discovery, various functions have been proposed for NLRP12, including the positive regulation of dendritic cell (DC) and neutrophil migration and the inhibition of NF-κB and ERK signalling in DC and macrophages. We show here that NLRP12 is poorly expressed in murine macrophages and DC, but is strongly expressed in neutrophils. Using myeloid cells from WT and Nlrp12(-/)(-) mice, we show that, contrary to previous reports, NLRP12 does not suppress LPS- or infection-induced NF-κB or ERK activation in myeloid cells, and is not required for DC migration in vitro. Surprisingly, we found that Nlrp12 deficiency caused increased rather than decreased neutrophil migration towards the chemokine CXCL1 and the neutrophil parasite Leishmania major, revealing NLRP12 as a negative regulator of directed neutrophil migration under these conditions.