Plant development: should I stop or should I grow?

Plant growth is tightly controlled through the integration of environmental cues with the physiological status of the seedling. A recent study now proposes a model explaining how the plant hormone ethylene triggers opposite growth responses depending on the light environment.

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

Multifocal epithelial tumors and field cancerization from loss of mesenchymal CSL signaling.

It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes. In human skin samples, stromal fields adjacent to multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.

Motility tests for drugs preventing PolyQ aggregation in recombinant Caenorhabditis elegans.

The pathological formation of proteinaceous aggregates that accumulate into the brain cells of patients are hallmarks of neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis and the heterogeneous group of polyglutamine (polyQ) diseases. In the polyQ diseases, the most upstream events of the pathogenic cascade are the misfolding and aggregation of proteins, such as huntingtin in Huntington's disease, that contain expanded stretch of glutamine residues above 35--‐40 repeats. This expanded polyQ stretch triggers the misfolding and aggregation of cytotoxic polyQ proteins in the neurons that cause cell death through different processes, like apoptosis, excessive inflammation, formation of free radicals, eventually leading to neuronal loss and neurodegeneration. This study focuses on the cellular network of chaperone proteins that can prevent protein aggregation by binding misfolding intermediates and may, as in the case of HSP70, actively unfold misfolded proteins into refoldable non--‐toxic ones (Hinault et al., 2010; Sharma et al., 2011). The chaperones can also collaborate with the proteasome to convert stable harmful proteins into harmless amino acids. Thus, the chaperone proteins that are the most important cellular factors of prevention and curing of protein misfolding, are negatively affected by aging (Morley et al., 2002) and fail to act properly in the neurons of aged persons, which eventually may lead to neurodegenerative pathologies. The general aim of this research was to identify least toxic drugs that can upregulate the expression of chaperone genes in cells suffering from polyQ--‐ mediated protein aggregation and degeneration. The specific aim of this study was to observe the effect of ten drugs on polyQ aggregation in a recombinant nematode Caenorhabditis elegans expressing a chimeric protein containing a sequence of 35 glutamines (Q35) fused to the green fluorescent protein in muscle cells, which causes an age--‐ and temperature--‐ dependent phenotype of accelerated paralysis. The drugs were selected after having proven their causing the overexpression of chaperone proteins in a previous wide screening of 2000 drugs on the moss plant Physcomitrella patens. The screening that we performed in this study was on these ten drugs. It suggested that piroxicam and anisindione were good reducers of polyglutamine disease mediated paralysis. A hypothesis can be made that they may act as good enhancers of the heat shock response, which causes the overexpression of many HSP chaperones and thus reduce motility impairment of polyQ disease expressing nematodes. Piroxicam was found to have the greatest effect on reducing polyQ35 proteins aggregates mediated paralysis in a dose--‐dependent manner but was also found to either have a toxic effect on wild type C.elegans, either to change its natural motility behavior, eventually reducing its motility in both cases. Chloroform should be preferred over DMSO as a drug solvent as it appears to be less toxic to C.elegans.

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

Role of AP-1 family members in lymphomagenesis

Constitutive activation of the nuclear factor-KB (NF-KB) transcriptional pathway is the main characteristic of the activated B-cell-like (ABC) subtype of diffuse large B- cell lymphoma (DLBCL). This has been attributed to oncogenic mutations in the CARMA1, CD79A/B, MyD88 or RNF31 signaling proteins, which control NF-kB activation at different levels. Since several of these mutations lead to a state that mimics chronically antigen receptor-stimulated B-cells, and since the antigen receptor also triggers other transcription pathways, these might be important in ABC DLBCL malignancy too. In this study we analyzed whether abnormal expression and activity of members of the AP-1 transcription factor family could contribute to the pathogenesis of ABC DLBCL. Here, we identified activation of Jun as well as ATF members of the dimeric AP-1 transcription factor family, as a hallmark of ABC but not of germinal center B- cell-like (GCB) DLBCL cell lines. ABC DLBCL cell lines harbored an upregulated expression of c-Jun, JunB, JunD and ATF3 proteins. We could show that the upregulation of c-Jun, JunB and ATF3 was dependent on constitutive BCR and MyD88 signaling. Since AP-1 transcription factors need to dimerize to be active, Jun binding partners were investigated and we could demonstrate the presence of several ATF/Jun heterodimers (including c-Jun/ATF2, c-Jun/ATF3, c-Jun/ATF7, JunB/ATF2, JunB/ATF3, JunB/ATF7, JunD/ATF2, JunD/ATF3 and JunD/ATF7 heterodimers). The disruption of ATF/Jun heterodimers by A-Fos, a dominant negative form of Jun members, was toxic to ABC but not to GCB DLBCL cell lines. Finally, ATF3 immunohistochemistry on DLBCL patient samples revealed that samples classified as non-GCB had more intense and preferentially nuclear staining of ATF3, which could be of diagnostic relevance since the histological classification of the ABC and GCB DLBCL subtypes is difficult in clinical practice. In conclusion, we could show that ABC DLBCL are not only addicted to NF-KB signaling, but also to signaling by some members of the AP-1 transcription factor family. Thus, the AP-1 pathway might be a promising therapeutic target for the treatment of ABC DLBCL. Additionally, monitoring ATF3 levels could improve the diagnosis of ABC DLBCL by IHC. -- L'activation constitutive du facteur de transcription NF-KB est l'une des caractéristiques principales des lymphomes B du type ABC-DLBCL. Cette addiction est dépendante de mutations oncogéniques de CARMA1, CD79A/B, MyD88 et RNF31 qui contrôlent NF-KB à différents niveaux. Etant donné que la plupart de ces mutations mène à un état d'activation chronique du récepteur des cellules B (BCR) et que le BCR active d'autres voies de signalisation, d'autres facteurs de transcription pourraient être impliqués dans la lymphomagénèse des ABC-DLBCL. Dans cette étude, nous nous sommes demandé si les membres de la famille du facteur de transcription AP-1 contribuaient à la pathogénèse de ce type de lymphome. Dans des lignées cellulaires de lymphomes du type ABC-DLBCL en comparaison avec le type GCB-DLBCL, nous avons pu identifier une activation anormale de plusieurs membres de la famille Jun et ATF, deux sous-familles du facteur de transcription AP-1. Les lignées cellulaires dérivées de lymphomes du type ABC-DLBCL surexpriment les facteurs de transcription c-Jun, JunB, JunD et ATF3. Leur surexpression dépend de l'activation constitutive de la voie du BCR et de MyD88. Etant donné qu'AP-1 requiert la formation de dimères pour être actif, nous nous sommes intéressés aux partenaires d'interactions de c-Jun, JunB et JunD et avons pu montrer la formation de plusieurs hétérodimères Jun/ATF (incluant les hétérodimères c-Jun/ATF2, c-Jun/ATF3, c-Jun/ATF7, JunB/ATF2, JunB/ATF3, JunB/ATF7, JunD/ATF2, JunD/ATF3 et JunD/ATF7). Lorsque l'on empêche la formation de ces hétérodimères avec A-Fos, un dominant négatif des membres Jun, la survie des lignées cellulaires du type ABC-DLBCL est diminuée, tandis que les lignées cellulaires GCB-DLBCL ne sont pas affectées. Pour finir, des immunohistochimies (IHC) pour ATF3 sur des échantillons de patients classifiés comme GCB et non-GCB ont pu montrer une coloration d'ATF3 nucléaire et beaucoup plus intense que les échantillons du type GCB. Ainsi, ATF3 pourrait être potentiellement utile en clinique pour différencier le sous type non-GCB des GCB. En conclusion, nous avons pu montrer que les lymphomes du type ABC- DLBCL ne présentent pas uniquement une addiction à NF-KB, mais également de certains membres de la famille de facteur de transcription AP-1. Par conséquent, AP- 1 pourrait être une cible thérapeutique prometteuse pour le développement de futures stratégies. En outre, la détermination des niveaux d'ATF3 par IHC pourraient améliorer le diagnostic des patients du type ABC DLBCL.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Role of glutamate in neuron-glia metabolic coupling.

The coupling between synaptic activity and glucose utilization (neurometabolic coupling) is a central physiologic principle of brain function that has provided the basis for 2-deoxyglucose-based functional imaging with positron emission tomography. Approximately 10 y ago we provided experimental evidence that indicated a central role of glutamate signaling on astrocytes in neurometabolic coupling. The basic mechanism in neurometabolic coupling is the glutamate-stimulated aerobic glycolysis in astrocytes, such that the sodium-coupled reuptake of glutamate by astrocytes and the ensuing activation of the Na(+)-K(+) ATPase triggers glucose uptake and its glycolytic processing, which results in the release of lactate from astrocytes. Lactate can then contribute to the activity-dependent fueling of the neuronal energy demands associated with synaptic transmission. Analyses of this coupling have been extended in vivo and have defined the methods of coupling for inhibitory neurotransmission as well as its spatial extent in relation to the propagation of metabolic signals within the astrocytic syncytium. On the basis of a large body of experimental evidence, we proposed an operational model, "the astrocyte-neuron lactate shuttle." A series of results obtained by independent laboratories have provided further support for this model. This body of evidence provides a molecular and cellular basis for interpreting data that are obtained with functional brain imaging studies.

The role of the δ-opioid receptor in skin homeostasis and wound healing

Au regard des agressions environnementales constantes que la peau doit endurer, l'équilibre fragile entre l'expression et la répression des gènes épidermiques, nécessaire à la différentiation et la prolifération des kératinocytes, pourrait facilement être perturbé en l'absence des mécanismes de stabilisation robustes. La présence d'un système neuroendocrinien local est donc importante afin de coordonner une réponse aux éventuelles irritations. En effet, l'expression de plusieurs neurohormones, des neurotransmetteurs et des neuropeptides, y compris des dérivés pro-opiomélanocortine comme la ß-endorphine et [Met5]-enképhaline, ainsi que l'expression du récepteur 8-opioïde (DOR) a été démontré dans la peau. Cependant, les mécanismes moléculaires par lesquels ils modulent la fonction des kératinocytes sont mal connus. Le présent travail démontre que la voie de signalisation DOR active spécifiquement la voie ERK 1/2 MAPK dans les lignées cellulaires de kératinocytes humains, inhibant la prolifération des cellules et entraîne une diminution de l'épaisseur épidermique dans un modèle organotypique de peau. De plus, l'expression de DOR retarde nettement l'induction de la kératine 10 (KRT 10) et la kératine 1 (KRT 1) dans une modèle 2D de différentiation in vitro, et supprime l'induction de KRT 10 dans un modèle organotypique de peau. Ceci est accompagné de la dérégulation de l'involucrine (IVL), la loricrine (LOR) et la fïlaggrin (FLG), résultant en une induction nettement réduite de leur expression lors de l'initiation de la différentiation in vitro. De plus, POU2F3 a été identifié comme un facteur de transcription régulant les gènes de différentiation des kératinocytes modulés par DOR. Il a été démontré que la régulation négative de POU2F3 via la voie DOR-ERK affecte les principaux aspects de la fonction des kératinocytes. Toutefois, il est évident que des facteurs supplémentaires influencent la fonctionnalité de la voie DOR elle-même. Le calcium et le contact cellule-cellule augmentent la quantité des récepteurs à la surface cellulaire des kératinocytes. Les kératinocytes dont les récepteurs sont internalisés ne répondent pas de la même manière que ceux possédant des récepteurs fonctionnels localisée à la membrane. Ce travail suggère que lors de signaux intrinsèques ou extrinsèques spécifiques, les kératinocytes sont capable de répondre via le système opioïdergique neuro-epidermique. Cette réponse doit être spatialement et temporairement contrôlée afin d'éviter un déséquilibre de l'homéostasie épidermique et un retard de cicatrisation. La compréhension de ce processus très complexe pourrait permettre à terme le développement de meilleurs traitements des affections cutanées pathologiques. En complément des études précédentes sur des souris DOR-défïcientes, ces données suggèrent que l'activation de DOR dans les kératinocytes humains influence la morphogenèse et l'homéostasie de l'épiderme, et pourrait jouer un rôle lors du processus de cicatrisation. - In view of the constant environmental assaults that the skin must endure, the delicate balance of an eloquent sequence of epidermal gene expression and repression, that is required for appropriate differentiation and proliferation of keratinocytes, might easily become derailed in the absence of robust stabilizing mechanisms. The presence of a local neuroendocrine system is thereby important to coordinate a response towards irritations. In fact, the expression of several neurohormones, neurotransmitters, and neuropeptides, including proopiomelanocortin derivatives, such as ß- endorphin and [Met5]-enkephalin has been shown in skin, as well as expression of the 6-opioid receptor (DOR). However, there is currently a lack of understanding of the molecular mechanisms by which their signalling modulates keratinocyte function. The present work demonstrates that DOR signalling specifically activates the ERK 1/2 MAPK pathway in human keratinocyte cell lines. This activation inhibits cell proliferation, resulting in decreased epidermal thickness in an organotypic skin model. Furthermore, DOR expression markedly delays induction of keratin intermediate filament Keratin 10 (KRT 10) and KRT 1 during in vitro differentiation, and abolishes the induction of KRT 10 in the organotypic skin model. This is accompanied by deregulation of involucrin (IVL), loricrin (LOR), and filaggrin (FLG), illustrated by a markedly reduced induction of their expression upon initiation of differentiation in vitro. Additionally, POU2F3 was identified as a transcription factor mediating the DOR induced regulation of keratinocyte differentiation related genes. It was revealed that DOR-mediated ERK-dependent downregulation of this factor affects key aspects of keratinocyte function. However, it is evident that additional triggers influence the functionality of the DOR itself. Calcium at concentrations above 0.1 mM and cell-cell contact both enhance the presence of receptor molecules on the keratinocytes cell surface. Keratinocytes with internalized receptor do not respond to DOR ligands in the same way as keratinocytes with a functional membrane localized receptor.

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Roles of astrocytic sodium dynamics in the neurometabolic coupling : a study by cellular imaging

RESUME LARGE PUBLIC Le système nerveux central est principalement composé de deux types de cellules :les neurones et les cellules gliales. Ces dernières, bien que l'emportant en nombre sur les neurones, ont longtemps été considérées comme des cellules sans intérêts par les neuroscientifiques. Hors, les connaissances modernes à leurs sujets indiquent qu'elles participent à la plupart des tâches physiologiques du cerveau. Plus particulièrement, elles prennent part aux processus énergétiques cérébraux. Ceux-ci, en plus d'être vitaux, sont particulièrement intrigants puisque le cerveau représente seulement 2 % de la masse corporelle mais consomme environ 25 % du glucose (substrat énergétique) corporel. Les astrocytes, un type de cellules gliales, jouent un rôle primordial dans cette formidable utilisation de glucose par le cerveau. En effet, l'activité neuronale (transmission de l'influx nerveux) est accompagnée d'une augmentation de la capture de glucose, issu de la circulation sanguine, par les astrocytes. Ce phénomène est appelé le «couplage neurométabolique » entre neurones et astrocytes. L'ion sodium fait partie des mécanismes cellulaires entrant en fonction lors de ces processus. Ainsi, dans le cadre de cette thèse, les aspects dynamiques de la régulation du sodium astrocytaire et leurs implications dans le couplage neurométabolique ont été étudiés par des techniques d'imagerie cellulaires. Ces études ont démontré que les mitochondries, machineries cellulaires convertissant l'énergie contenue dans le glucose, participent à la régulation du sodium astrocytaire. De plus, ce travail de thèse a permis de découvrir que les astrocytes sont capables de se transmettre, sous forme de vagues de sodium se propageant de cellules en cellules, un message donnant l'ordre d'accroître leur consommation d'énergie. Cette voie de signalisation leur permettrait de fournir de l'énergie aux neurones suite à leur activation. RESUME Le glutamate libéré dans la fente synaptique pendant l'activité neuronale, est éliminé par les astrocytes environnants. Le glutamate est co-transporté avec des ions sodiques, induisant une augmentation intracellulaire de sodium (Na+i) dans les astrocytes. Cette élévation de Na+i déclenche une cascade de mécanismes moléculaires qui aboutissent à la production de substrats énergétiques pouvant être utilisés par les neurones. Durant cette thèse, la mesure simultanée du sodium mitochondrial (Na+mit) et cytosolique par des techniques d'imagerie utilisant des sondes fluorescentes spécifiques, a indiqué que les variations de Na+i induites par le transport du glutamate sont transmises aux mitochondries. De plus, les voies d'entrée et de sortie du sodium mitochondrial ont été identifiées. L'échangeur de Na+ et de Ca2+ mitochondrial semble jouer un rôle primordial dans l'influx de Na+mit, alors que l'efflux de Na+mit est pris en charge par l'échangeur de Na+ et de H+ mitochondrial. L'étude du Na+mit a nécessité l'utilisation d'un système de photoactivation. Les sources de lumière ultraviolette (UV) classiques utilisées à cet effet (lasers, lampes à flash) ayant plusieurs désavantages, une alternative efficace et peu coûteuse a été développée. Il s'agit d'un système compact utilisant une diode électroluminescente (LED) à haute puissance et de longueur d'onde de 365nm. En plus de leurs rôles dans le couplage neurométabolique, les astrocytes participent à la signalisation multicellulaire en transmettant des vagues intercellulaires de calcium. Ce travail de thèse démontre également que des vagues intercellulaires de sodium peuvent être évoquées en parallèle à ces vagues calciques. Le glutamate, suite à sa libération par un mécanisme dépendent du calcium, est réabsorbé par les transporteurs au glutamate. Ce mécanisme a pour conséquence la génération de vagues sodiques se propageant de cellules en cellules. De plus, ces vagues sodiques sont corrélées spatialement avec une consommation accrue de glucose par les astrocytes. En conclusion, ce travail de thèse a permis de montrer que le signal sodique astrocytaire, déclenché en réponse au glutamate, se propage à la fois de façon intracellulaire aux mitochondries et de façon intercellulaire. Ces résultats suggèrent que les astrocytes fonctionnent comme un réseau de cellules nécessaire au couplage énergétique concerté entre neurones et astrocytes et que le sodium est un élément clé dans les mécanismes de signalisations cellulaires sous-jacents. SUMMARY Glutamate, released in the synaptic cleft during neuronal activity, is removed by surrounding astrocytes. Glutamate is taken-up with Na+ ions by specific transporters, inducing an intracellular Na+ (Na+i) elevation in astrocytes which triggers a cascade of molecular mechanisms that provides metabolic substrates to neurons. Thus, astrocytic Na+i homeostasis represents a key component of the so-called neurometabolic coupling. In this context, the first part of this thesis work was aimed at investigating whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Simultaneous monitoring of both mitochondrial Na+ (Na+mit) and cytosolic Na+ changes with fluorescent dyes revealed that glutamate-evoked cytosolic Na+ elevations are indeed transmitted to mitochondria. The mitochondrial Na+/Ca2+ exchangers have a prominent role in the regulation of Na+mit influx pathway, and Na+mit extrusion appears to be mediated by Na+/H+ exchangers. To demonstrate the implication of Na+/Ca2+ exchangers, this study has required the technical development of an UV-flash photolysis system. Because light sources for flash photolysis have to be powerful and in the near UV range, the use of UV lasers or flash lamps is usually required. As an alternative to these UV sources that have several drawbaks, we developped a compact, efficient and lowcost flash photolysis system which employs a high power 365nm light emitting diode. In addition to their role in neurometabolic coupling, astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. The third part of this thesis show that intercellular Na+ waves can be evoked in parallel to Ca2+ waves. Glutamate released by a Ca2+ wave-dependent mechanism is taken up by glutamate transporters, resulting in a regenerative propagation of cytosolic Na+ increases. Na+ waves in turn lead to a spatially correlated increase in glucose uptake. In conclusion, the present thesis demonstrates that glutamate-induced Na+ changes occurring in the cytosol of astrocytes propagate to both the mitochondrial matrix and the astrocytic network. These results furthermore support the view that astrocytic Na+ is a signal coupled to the brain energy metabolism.

Vitamin C supplementation in the critically ill patient.

PURPOSE OF REVIEW: Vitamin C is not only an essential nutrient involved in many anabolic pathways, but also an important player of the endogenous antioxidant defense. Low plasma levels are very common in critical care patients and may reflect severe deficiency states. RECENT FINDINGS: Vitamin C scavenges reactive oxygen species such as superoxide and peroxynitrite in plasma and cells (preventing damage to proteins, lipids and DNA), prevents occludin dephosphorylation and loosening of the tight junctions. Ascorbate improves microcirculatory flow impairment by inhibiting tumor-necrosis-factor-induced intracellular adhesion molecule expression, which triggers leukocyte stickiness and slugging. Clinical trials in sepsis, trauma and major burns testing high-dose vitamin C show clinical benefit. Restoration of normal plasma levels in inflammatory patients requires the administration of 3 g/day for several days, which is 30 times the daily recommended dose. SUMMARY: The recent research on the modulation of oxidative stress and endothelial protection offer interesting therapeutic perspectives, based on the biochemical evidence, with limited or even absent side-effects.

On the retinal toxicity of intraocular glucocorticoids.

Corticosteroids are hormones involved in many physiological responses such as stress, immune modulation, protein catabolism and water homeostasis. The subfamily of glucocorticoids is used systemically in the treatment of inflammatory diseases or allergic reactions. In the eye, glucocorticoides are used to treat macular edema, inflammation and neovascularization. The most commonly used glucocorticoid is triamcinolone acetonide (TA). The pharmaceutical formulation of TA is not adapted for intravitreal administration but has been selected by ophthalmologists because its very low intraocular solubility provides sustained effect. Visual benefits of intraocular TA do not clearly correlate with morpho-anatomical improvements, suggesting potential toxicity. We therefore studied, non-common, but deleterious effects of glucocorticoids on the retina. We found that the intravitreal administration of TA is beneficial in the treatment of neovascularization because it triggers cell death of endothelial cells of neovessels by a caspase-independent mechanism. However, this treatment is toxic for the retina because it induces a non-apoptotic, caspase-independent cell death related to paraptosis, mostly in the retinal pigmented epithelium cells and the Müller cells.

Geoepidemiology of autoimmune hemolytic anemia.

Autoantibodies against red blood cell antigens are considered the diagnostic hallmark of AIHA: Direct antiglobulin test (DAT) completed by cytofluorometry and specific diagnostic monoclonal antibodies (mAbs) allow for a better understanding of autoimmune hemolytic anemia (AIHA) triggers. Once B-cell tolerance checkpoints are bypassed, the patient loses self-tolerance, if the AIHA is not also caused by an possible variety of secondary pathogenic events such as viral, neoplastic and underlying autoimmune entities, such as SLE or post-transplantation drawbacks; treatment of underlying diseases in secondary AIHA guides ways to curative AIHA treatment. The acute phase of AIHA, often lethal in former times, if readily diagnosed, must be treated using plasma exchange, extracorporeal immunoadsorption and/or RBC transfusion with donor RBCs devoid of the auto-antibody target antigen. Genotyping blood groups (www.bloodgen.com) and narrowing down the blood type subspecificities with diagnostic mAbs help to define the triggering autoantigen and to select well compatible donor RBC concentrates, which thus escape recognition by the autoantibodies.