33 research outputs found
Aurintricarboxylic acid prevents GLUR2 mRNA down-regulation and delayed neurodegeneration in hippocampal CA1 neurons of gerbil after global ischemia
Aurintricarboxylic acid (ATA), an inhibitor of endonuclease activity and other proteinânucleic acid interactions, blocks apoptosis in several cell types and prevents delayed death of hippocampal pyramidal CA1 neurons induced by transient global ischemia. Global ischemia in rats and gerbils induces down-regulation of GluR2 mRNA and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced Ca(2+) influx in CA1 before neurodegeneration. This result and neuroprotection by antagonists of AMPA receptors suggests that formation of AMPA receptors lacking GluR2, and therefore Ca(2+) permeable, leads to excessive Ca(2+) influx in response to endogenous glutamate; the resulting delayed neuronal death in CA1 exhibits many characteristics of apoptosis. In this study, we examined the effects of ATA on expression of mRNAs encoding glutamate receptor subunits in gerbil hippocampus after global ischemia. Administration of ATA by injection into the right cerebral ventricle 1 h before (but not 6 h after) bilateral carotid occlusion prevented the ischemia-induced decrease in GluR2 mRNA expression and the delayed neurodegeneration. These findings suggest that ATA is neuroprotective in ischemia by blocking the transcriptional changes leading to down-regulation of GluR2, rather than by simply blocking endonucleases, which presumably act later after Ca(2+) influx initiates apoptosis. Maintaining formation of Ca(2+) impermeable, GluR2 containing AMPA receptors could prevent delayed death of CA1 neurons after transient global ischemia, and block of GluR2 down-regulation may provide a further strategy for neuroprotection
Etude des consequences comportementales et histologiques d'une ischemie cerebrale globale chez le rat. Application a la recherche de l'activite anti-ischemique de differents agents pharmacologiques
INIST T 76865 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc
Coordination of the cell cycle and DNA damage checkpoint in adaptation to DNA damage
Les dommages Ă l'ADN constituent une grave menace pour l'intĂ©gritĂ© du gĂ©nome. Lorsque l'ADN d'une cellule est endommagĂ©, le point de contrĂŽle des dommages Ă l'ADN les dĂ©tecte et arrĂȘte le cycle cellulaire, laissant ainsi du temps pendant lequel la cellule peut rĂ©parer son ADN. Toutefois, si les dommages ne sont pas rĂ©parĂ©s aprĂšs plusieurs heures, chez la levure Saccharomyces cerevisiae, les cellules finissent par reprendre leur cycle cellulaire malgrĂ© la persistance du dommage. Ce processus, appelĂ© adaptation aux dommages de l'ADN, semble agir comme une derniĂšre tentative de survie des cellules, bien qu'il favorise les mutations et l'instabilitĂ© gĂ©nomique dans les cellules filles. Chez Saccharomyces cerevisiae, la polo-kinase Cdc5PLK1, impliquĂ©e par ailleurs dans de nombreux processus liĂ©s au cycle cellulaire, est critique pour l'adaptation. Cependant, ses cibles spĂ©cifiques de l'adaptation ne sont pas encore clairement dĂ©finies, et son rĂŽle exact dans ce processus reste incertain. Ces travaux de thĂšse font Ă©tat de nos avancĂ©es sur les rĂ©gulations transcriptionnelles et post-traductionnelles de Cdc5 en rĂ©ponse Ă un dommage Ă lâADN de type dĂ©stabilisation tĂ©lomĂ©rique. En particulier, nous sommes parvenus Ă identifier des rĂ©sidus de Cdc5 dont la phosphorylation module lâefficacitĂ© de lâadaptation et avons mis en Ă©vidence que le checkpoint de dommages Ă lâADN provoque une rĂ©gulation nĂ©gative de lâexpression de Cdc5 via lâaction de Mec1, Rad53 et Ndd1, retardant lâadaptation. Ces travaux ont menĂ© Ă la rĂ©daction dâun article dĂ©posĂ© sur bioRxiv et en cours de soumission. Par ailleurs, Cdc5 cible de nombreux substrats au cours dâun cycle cellulaire normal. Identifier les substrats spĂ©cifiques de lâadaptation permettrait de comprendre son rĂŽle au sein de ce processus. Nous avons entrepris un crible Ă grande Ă©chelle en utilisant une mĂ©thode basĂ©e sur lâinsertion alĂ©atoire dâun transposon dans le gĂ©nome entier et de façon unique (Saturated Analyses Transposons Assay in Yeast). De ce crible sont ressortis un certain nombre de gĂšnes potentiellement impliquĂ©s dans lâadaptation et que nous avons analysĂ©, Ă la fois Ă une Ă©chelle globale par Gene Ontology, et Ă une Ă©chelle assez fine, en repĂ©rant les gĂšnes enrichis et en sâintĂ©ressant particuliĂšrement aux gĂšnes inclus dans un mĂȘme complexe afin de gagner en robustesse. Dans les trois expĂ©riences indĂ©pendantes menĂ©es pour ce crible, nous voyons ressortir un certain nombre de complexes, notamment MRX, qui est impliquĂ© dans les voies de rĂ©paration de lâADN, la gestion du stress rĂ©plicatif et qui participe Ă lâactivation du checkpoint de dommages Ă lâADN. En plus des analyses bio-informatiques, lâimplication potentielle des gĂšnes du complexe MRX a Ă©tĂ© testĂ©e expĂ©rimentalement. Cdc5 joue un rĂŽle essentiel dans lâadaptation au dommage Ă lâADN, mais son rĂŽle dans ce processus reste mal dĂ©fini. Nous avons mis en Ă©vidence que Cdc5 est fortement phosphorylĂ©e et que ses sites de phosphorylation rĂ©gulent lâefficacitĂ© de lâadaptation, et est aussi rĂ©gulĂ©e au niveau de sa quantitĂ©. Dâautre part, nous avons rĂ©alisĂ© une caractĂ©risation approfondie des gĂšnes candidats dĂ©tectĂ©s par nos cribles, ce qui aide Ă rĂ©vĂ©ler les mĂ©canismes molĂ©culaires de lâadaptation.DNA damage is a serious threat to genome integrity. When a cell's DNA is damaged, the DNA damage checkpoint detects it and stops the cell cycle, allowing time for the cell to repair its DNA. However, if the damage is not repaired after several hours, in the yeast Saccharomyces cerevisiae, the cells eventually resume their cell cycle despite the persistence of the damage. This process, called adaptation to DNA damage, appears to act as a last-ditch attempt at cell survival, although it promotes mutations and genomic instability in the daughter cells. In Saccharomyces cerevisiae, the Cdc5PLK1 polo-kinase, otherwise involved in many cell cycle-related processes, is critical for adaptation. However, its specific targets in adaptation are not yet clearly defined, and its exact role in this process remains unclear. In this thesis, we report on the transcriptional and post-translational regulation of Cdc5 in response to telomere destabilization-type DNA damage. In particular, we identified Cdc5 residues whose phosphorylation modulates adaptation efficiency and demonstrated that the DNA damage checkpoint down-regulates Cdc5 expression via the action of Mec1, Rad53 and Ndd1, delaying adaptation. This work has led to the writing of a paper filed on bioRxiv and currently being submitted. Moreover, Cdc5 targets many substrates during a normal cell cycle. Identifying the specific substrates of adaptation would help to understand its role in this process. We undertook a large-scale screen using a method based on random insertion of a transposon in the whole genome in a unique way (Saturated Analyses Transposons Assay in Yeast). From this screen, a certain number of genes potentially involved in adaptation emerged and we analyzed them, both on a global scale using Gene Ontology, and on a fairly fine scale, by identifying enriched genes and by focusing on genes included in the same complex in order to gain robustness. In the three independent experiments conducted for this screen, we see a number of complexes stand out, notably MRX, which is involved in DNA repair pathways, replicative stress management and participates in DNA damage checkpoint activation. In addition to the bioinformatics analyses, the potential involvement of the MRX complex genes was tested experimentally. Cdc5 plays an essential role in adaptation to DNA damage, but its role in this process remains poorly defined. We have shown that Cdc5 is highly phosphorylated and that its phosphorylation sites regulate the efficiency of the adaptation, and is also regulated in its quantity. On the other hand, we performed an extensive characterization of the candidate genes detected by our screens, which helps to reveal the molecular mechanisms of adaptation
Fonctions et régulations de la polo kinase Cdc5 dans l'adaptation aux dommages à l'ADN chez Saccharomyces cerevisiae
DNA damage is a serious threat to genome integrity. When a cell's DNA is damaged, the DNA damage checkpoint detects it and stops the cell cycle, allowing time for the cell to repair its DNA. However, if the damage is not repaired after several hours, in the yeast Saccharomyces cerevisiae, the cells eventually resume their cell cycle despite the persistence of the damage. This process, called adaptation to DNA damage, appears to act as a last-ditch attempt at cell survival, although it promotes mutations and genomic instability in the daughter cells. In Saccharomyces cerevisiae, the Cdc5PLK1 polo-kinase, otherwise involved in many cell cycle-related processes, is critical for adaptation. However, its specific targets in adaptation are not yet clearly defined, and its exact role in this process remains unclear. In this thesis, we report on the transcriptional and post-translational regulation of Cdc5 in response to telomere destabilization-type DNA damage. In particular, we identified Cdc5 residues whose phosphorylation modulates adaptation efficiency and demonstrated that the DNA damage checkpoint down-regulates Cdc5 expression via the action of Mec1, Rad53 and Ndd1, delaying adaptation. This work has led to the writing of a paper filed on bioRxiv and currently being submitted. Moreover, Cdc5 targets many substrates during a normal cell cycle. Identifying the specific substrates of adaptation would help to understand its role in this process. We undertook a large-scale screen using a method based on random insertion of a transposon in the whole genome in a unique way (Saturated Analyses Transposons Assay in Yeast). From this screen, a certain number of genes potentially involved in adaptation emerged and we analyzed them, both on a global scale using Gene Ontology, and on a fairly fine scale, by identifying enriched genes and by focusing on genes included in the same complex in order to gain robustness. In the three independent experiments conducted for this screen, we see a number of complexes stand out, notably MRX, which is involved in DNA repair pathways, replicative stress management and participates in DNA damage checkpoint activation. In addition to the bioinformatics analyses, the potential involvement of the MRX complex genes was tested experimentally. Cdc5 plays an essential role in adaptation to DNA damage, but its role in this process remains poorly defined. We have shown that Cdc5 is highly phosphorylated and that its phosphorylation sites regulate the efficiency of the adaptation, and is also regulated in its quantity. On the other hand, we performed an extensive characterization of the candidate genes detected by our screens, which helps to reveal the molecular mechanisms of adaptation.Les dommages Ă l'ADN constituent une grave menace pour l'intĂ©gritĂ© du gĂ©nome. Lorsque l'ADN d'une cellule est endommagĂ©, le point de contrĂŽle des dommages Ă l'ADN les dĂ©tecte et arrĂȘte le cycle cellulaire, laissant ainsi du temps pendant lequel la cellule peut rĂ©parer son ADN. Toutefois, si les dommages ne sont pas rĂ©parĂ©s aprĂšs plusieurs heures, chez la levure Saccharomyces cerevisiae, les cellules finissent par reprendre leur cycle cellulaire malgrĂ© la persistance du dommage. Ce processus, appelĂ© adaptation aux dommages de l'ADN, semble agir comme une derniĂšre tentative de survie des cellules, bien qu'il favorise les mutations et l'instabilitĂ© gĂ©nomique dans les cellules filles. Chez Saccharomyces cerevisiae, la polo-kinase Cdc5PLK1, impliquĂ©e par ailleurs dans de nombreux processus liĂ©s au cycle cellulaire, est critique pour l'adaptation. Cependant, ses cibles spĂ©cifiques de l'adaptation ne sont pas encore clairement dĂ©finies, et son rĂŽle exact dans ce processus reste incertain. Ces travaux de thĂšse font Ă©tat de nos avancĂ©es sur les rĂ©gulations transcriptionnelles et post-traductionnelles de Cdc5 en rĂ©ponse Ă un dommage Ă lâADN de type dĂ©stabilisation tĂ©lomĂ©rique. En particulier, nous sommes parvenus Ă identifier des rĂ©sidus de Cdc5 dont la phosphorylation module lâefficacitĂ© de lâadaptation et avons mis en Ă©vidence que le checkpoint de dommages Ă lâADN provoque une rĂ©gulation nĂ©gative de lâexpression de Cdc5 via lâaction de Mec1, Rad53 et Ndd1, retardant lâadaptation. Ces travaux ont menĂ© Ă la rĂ©daction dâun article dĂ©posĂ© sur bioRxiv et en cours de soumission. Par ailleurs, Cdc5 cible de nombreux substrats au cours dâun cycle cellulaire normal. Identifier les substrats spĂ©cifiques de lâadaptation permettrait de comprendre son rĂŽle au sein de ce processus. Nous avons entrepris un crible Ă grande Ă©chelle en utilisant une mĂ©thode basĂ©e sur lâinsertion alĂ©atoire dâun transposon dans le gĂ©nome entier et de façon unique (Saturated Analyses Transposons Assay in Yeast). De ce crible sont ressortis un certain nombre de gĂšnes potentiellement impliquĂ©s dans lâadaptation et que nous avons analysĂ©, Ă la fois Ă une Ă©chelle globale par Gene Ontology, et Ă une Ă©chelle assez fine, en repĂ©rant les gĂšnes enrichis et en sâintĂ©ressant particuliĂšrement aux gĂšnes inclus dans un mĂȘme complexe afin de gagner en robustesse. Dans les trois expĂ©riences indĂ©pendantes menĂ©es pour ce crible, nous voyons ressortir un certain nombre de complexes, notamment MRX, qui est impliquĂ© dans les voies de rĂ©paration de lâADN, la gestion du stress rĂ©plicatif et qui participe Ă lâactivation du checkpoint de dommages Ă lâADN. En plus des analyses bio-informatiques, lâimplication potentielle des gĂšnes du complexe MRX a Ă©tĂ© testĂ©e expĂ©rimentalement. Cdc5 joue un rĂŽle essentiel dans lâadaptation au dommage Ă lâADN, mais son rĂŽle dans ce processus reste mal dĂ©fini. Nous avons mis en Ă©vidence que Cdc5 est fortement phosphorylĂ©e et que ses sites de phosphorylation rĂ©gulent lâefficacitĂ© de lâadaptation, et est aussi rĂ©gulĂ©e au niveau de sa quantitĂ©. Dâautre part, nous avons rĂ©alisĂ© une caractĂ©risation approfondie des gĂšnes candidats dĂ©tectĂ©s par nos cribles, ce qui aide Ă rĂ©vĂ©ler les mĂ©canismes molĂ©culaires de lâadaptation
Proposed Malewa dam in Kenya: Adequate adaptations to original design
Malewa dam in Kenya was first studied in the 1990s at preliminary design stage, through a study funded by Japanese Government who entrusted JICA with it. At that time, its main purpose was water supply to Nakuru, Naivasha and Gilgil cities in the Rift Valley. Thirty years later, its design was reviewed within the frame of a feasibility study funded by World Bank to account for revised needs -now excluding Nakuru- also taking into account increased awareness of environmental impact on downstream Lake Naivasha (Ramsar zone), management of the water scarcity, climate change trends, reliability issue, quality of water, sedimentation, as well as growing concern with operation and maintenance costs. Among studied solutions, focused on supply by gravity for economic reasons, and as ground water poses a serious health problem in this area due to a high fluoride content detrimental to human consumption, the construction of Malewa dam was confirmed to be the best solution after a decision making process, subject to some changes to the basic design, such as using the compensation flow to generate hydro-power to pump water to a WTP nearby the dam, mixing water with groundwater, building check dams and implementing water management and compensation measures
Projet « IsÚre amont » : 16 Champs d'Inondation ContrÎlée (CIC) le long de l'IsÚre
Le projet « IsĂšre amont » est portĂ© en tant que maĂźtre dâouvrage par le Symbhi (Syndicat Mixte des Bassins Hydrauliques de lâIsĂšre). Son but est de prĂ©venir les crues de lâIsĂšre et de protĂ©ger les zones urbanisĂ©es de la vallĂ©e du GrĂ©sivaudan entre Pontcharra et Grenoble.
La mesure phare de ce projet est la mise en place de zones dâexpansion de crue Ă grande Ă©chelle : tous les Ă©coulements au-delĂ de la crue trentennale sont stockĂ©s dans 16 champs dâinondation contrĂŽlĂ©e, ou « CIC ». Le stockage dans ces zones est optimisĂ© pour recevoir entre 1 et 2 mĂštres dâeau pour la crue bicentennale. Les CIC permettront dâĂ©crĂȘter le dĂ©bit de lâIsĂšre, de 1890 m3/s Ă Pontcharra, Ă lâentrĂ©e du systĂšme en amont, Ă 1240 m3/s Ă lâaval Ă Grenoble.
Les champs dâinondation contrĂŽlĂ©e sont alimentĂ©s par des dĂ©versoirs, ou des vannes-clapets. Leur vidange est assurĂ©e par les fossĂ©s et canaux de drainage agricole. Aucune intervention humaine nâest ainsi nĂ©cessaire, et un systĂšme de tĂ©lĂ©surveillance sera mis en place pour le suivi de lâexploitation du dispositif.
Toutes les zones naturelles et agricoles de la vallĂ©e du GrĂ©sivaudan participeront au stockage, soit plus de 3 500 ha, ce qui en fait lâun des projets de plus grande ampleur en cours de rĂ©alisation Ă lâĂ©chelon national.
Les travaux ont dĂ©marrĂ© dĂ©but 2012 pour une durĂ©e dâenviron 9 Ă 10 ans, et un montant global Ă terme de 135 millions dâeuros HT
The Polo kinase Cdc5 is regulated at multiple levels in the adaptation response to telomere dysfunction
International audienceAbstract Telomere dysfunction activates the DNA damage checkpoint to induce a cell cycle arrest. After an extended period of time, however, cells can bypass the arrest and undergo cell division despite the persistence of the initial damage, a process called adaptation to DNA damage. The Polo kinase Cdc5 in Saccharomyces cerevisiae is essential for adaptation and for many other cell cycle processes. How the regulation of Cdc5 in response to telomere dysfunction relates to adaptation is not clear. Here, we report that Cdc5 protein level decreases after telomere dysfunction in a Mec1-, Rad53- and Ndd1-dependent manner. This regulation of Cdc5 is important to maintain long-term cell cycle arrest but not for the initial checkpoint arrest. We find that both Cdc5 and the adaptation-deficient mutant protein Cdc5-ad are heavily phosphorylated and several phosphorylation sites modulate adaptation efficiency. The PP2A phosphatases are involved in Cdc5-ad phosphorylation status and contribute to adaptation mechanisms. We finally propose that Cdc5 orchestrates multiple cell cycle pathways to promote adaptation
Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii
International audienceChlamydomonas reinhardtii is a model unicellular organism for basic or biotechnological research, such as the production of high-value molecules or biofuels thanks to its photosynthetic ability. To enable rapid construction and optimization of multiple designs and strains, our team and collaborators have developed a versatile Chlamydomonas Modular Cloning toolkit comprising 119 biobricks. Having the ability to use a wide range of selectable markers is an important benefit for forward and reverse genetics in Chlamydomonas. We report here the development of a new selectable marker based on the resistance to the antibiotic blasticidin S, using the Bacillus cereus blasticidin S deaminase (BSR) gene. The optimal concentration of blasticidin S for effective selection was determined in both liquid and solid media and tested for multiple laboratory strains. In addition, we have shown that our new selectable marker does not interfere with other common antibiotic resistances: zeocin, hygromycin, kanamycin, paromomycin, and spectinomycin. The blasticidin resistance biobrick has been added to the Chlamydomonas Modular Cloning toolkit and is now available to the entire scientific community