Literatura académica sobre el tema "HSFA2"

ABSTRACT In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25°C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test inSaccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

ABSTRACT Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41°C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41°C even in the presence of LMB. The temperature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.

Plants have evolved mechanisms of stress tolerance responses to heat stress. However, little is known about metabolic responses to heat stress in trees. In this study, we exposed Populus tomentosa Carr. to control (25 °C) and heat stress (45 °C) treatments and analyzed the metabolic and transcriptomic effects. Heat stress increased the cellular concentration of H2O2 and the activities of antioxidant enzymes. The levels of proline, raffinose, and melibiose were increased by heat stress, whereas those of pyruvate, fumarate, and myo-inositol were decreased. The expression levels of most genes (PSB27, PSB28, LHCA5, PETB, and PETC) related to the light-harvesting complexes and photosynthetic electron transport system were downregulated by heat stress. Association analysis between key genes and altered metabolites indicated that glycolysis was enhanced, whereas the tricarboxylic acid (TCA) cycle was suppressed. The inositol phosphate; galactose; valine, leucine, and isoleucine; and arginine and proline metabolic pathways were significantly affected by heat stress. In addition, several transcription factors, including HSFA2, HSFA3, HSFA9, HSF4, MYB27, MYB4R1, and bZIP60 were upregulated, whereas WRKY13 and WRKY50 were downregulated by heat stress. Interestingly, under heat stress, the expression of DREB1, DREB2, DREB2E, and DREB5 was dramatically upregulated at 12 h. Our results suggest that proline, raffinose, melibiose, and several genes (e.g., PSB27, LHCA5, and PETB) and transcription factors (e.g., HSFAs and DREBs) are involved in the response to heat stress in P. tomentosa.

Heat Shock Factor A2 (HsfA2) is part of the Heat Shock Factor (HSF) network, and plays an essential role beyond heat shock in environmental stress responses and cellular homeostatic control. Arabidopsis thaliana cell cultures derived from wild type (WT) ecotype Col-0 and a knockout line deficient in the gene encoding HSFA2 (HSFA2 KO) were grown aboard the International Space Station (ISS) to ascertain whether the HSF network functions in the adaptation to the novel environment of spaceflight. Microarray gene expression data were analyzed using a two-part comparative approach. First, genes differentially expressed between the two environments (spaceflight to ground) were identified within the same genotype, which represented physiological adaptation to spaceflight. Second, gene expression profiles were compared between the two genotypes (HSFA2 KO to WT) within the same environment, which defined genes uniquely required by each genotype on the ground and in spaceflight-adapted states. Results showed that the endoplasmic reticulum (ER) stress and unfolded protein response (UPR) define the HSFA2 KO cells’ physiological state irrespective of the environment, and likely resulted from a deficiency in the chaperone-mediated protein folding machinery in the mutant. Results further suggested that additional to its universal stress response role, HsfA2 also has specific roles in the physiological adaptation to spaceflight through cell wall remodeling, signal perception and transduction, and starch biosynthesis. Disabling HsfA2 altered the physiological state of the cells, and impacted the mechanisms induced to adapt to spaceflight, and identified HsfA2-dependent genes that are important to the adaption of wild type cells to spaceflight. Collectively these data indicate a non-thermal role for the HSF network in spaceflight adaptation.

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the β-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5′ of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5′ of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.

HEAT SHOCK FACTOR A2 (HSFA2) is a regulator of multiple environmental stress responses required for stress acclimation. We analyzed HSFA2 co-regulated genes and identified 43 genes strongly co-regulated with HSFA2 during multiple stresses. Motif enrichment analysis revealed an over-representation of the site II element (SIIE) in the promoters of these genes. In a yeast 1-hybrid screen with the SIIE, we identified the closely related R2R3-MYB transcription factors TT2 and MYB5. We found overexpression of MYB5 or TT2 rendered plants heat stress tolerant. In contrast, tt2, myb5, and tt2/myb5 loss of function mutants showed heat stress hypersensitivity. Transient expression assays confirmed that MYB5 and TT2 can regulate the HSFA2 promoter together with the other members of the MBW complex, TT8 and TRANSPARENT TESTA GLABRA 1 (TTG1) and that the SIIE was involved in this regulation. Transcriptomic analysis revealed that TT2/MYB5 target promoters were enriched in SIIE. Overall, we report a new function of TT2 and MYB5 in stress resistance and a role in SIIE-mediated HSFA2 regulation.

Abstract The SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor regulates gene expression associated with multiple stress tolerances in plant roots. In this study, we investigated the mechanism responsible for the sensitivity of the stop1 mutant to low-oxygen stress in Arabidopsis. Transcriptomic analyses revealed that two genes involved in low-oxygen tolerance, namely GLUTAMATE DEHYDROGENASE 1 (GDH1) and GDH2, showed lower expression levels in the stop1 mutant than in the wild-type. Sensitivity of the gdh1gdh2 double-mutant to low-oxygen conditions was partly attributable to the low-oxygen sensitivity of the stop1 mutant. Two transcription factors, STOP2 and HEAT SHOCK FACTOR A2 (HsfA2), were expressed at lower levels in the stop1 mutant. An in planta complementation assay indicated that CaMV35S::STOP2 or CaMV35S::HsfA2 partially rescued the low-oxygen tolerance of the stop1 mutant, which was concomitant with recovered expression of genes regulating low-pH tolerance and genes encoding molecular chaperones. Prediction of cis-elements and in planta promoter assays revealed that STOP1 directly activated the expression of HsfA2. Similar STOP1-dependent low-oxygen sensitivity was detected in tobacco. Suppression of NtSTOP1 induced low-oxygen sensitivity, which was associated with lower expression levels of NtHsfA2 and NtGDHs compared with the wild-type. Our results indicated that STOP1 pleiotropically regulates low-oxygen tolerance by transcriptional regulation.

Les stress ont un impact majeur sur l'agriculture. Les études réalisées jusqu'à présent portent majoritairement sur des stress simples, appliqués indépendamment dans des conditions de culture idéales. Cependant, les combinaisons de stress sont la norme dans les champs et la réponse aux combinaisons de stress ne peut être extrapolée à partir de l'addition des réponses aux stress simples. Une analyse bioinformatique de banques de données de transcriptomique a permis d’identifier un gène (HSFA2) répondant a de multiple stress, isolés ou combinés. La présente thèse a pour but de découvrir les mécanismes contrôlant l'expression d’HSFA2. Deux stratégies ont alors été mise en oeuvre. Dans un premier temps, les facteurs capables de fixer une région promotrice d’HSFA2 ont été étudiés par système de « simple hybride levure ». Dans un second temps, une lignée transgénique biosenseur de l’activité d’HSFA2 a été produite et mutagénéisée. De nombreux mutants ont été sélectionnés sur la base d’une expression constitutive du biosenseur. La capacité des mutants sélectionnés à résister à une combinaison de stress chaleur et de stress lumière a ensuite été analysée. L’identification des mutations causales par séquençage du génome entier a permis, dans certains cas, de déterminer quels étaient les loci responsables des résistances observées. En particulier, deux mutations conférant une large résistance seront discutées
Stresses represent the major cause of yield loss in modern agriculture. Previously, the majority of studies concern simplified, single stress situations, whereas in the field, stresses are complex and most often occurring in combinations. However, multiple stress response cannot be extrapolated from single stress responses added together. Bioinformatic analysis of transcriptomic data banks allowed us to identify a gene (HSFA2) involved in multiple stress responses. The work presented here aimed at discovering the mechanisms controlling HSFA2 expression. Two strategies were used.First, factors able to bind a region of HSFA2 promoter were studied through a “yeast one hybrid” system. Secondly, a transgenic biosensor of HSFA2 activity line was produced and mutagenized. A great number of mutants were selected on the basis of showing a constitutive activation of the biosensor. Resistance potentials to a combination of heat and high light stresses were then determined. Causal mutations identification through whole genome sequencing allowed, in some cases, to determine which were the loci responsible for the resistance traits. In particular, two mutations confering broad spectrum stress resistance will be discussed

Le réchauffement climatique planétaire annoncé ne sera pas sans conséquence sur le métabolisme de la baie et en particulier sur la teneur en composés phénoliques. Les objectifs de cette thèse visent à mieux comprendre les processus de régulation associant le microclimat des baies et la synthèse des composés phénoliques. Par des approches moléculaires et biochimiques, ce travail a permis de mieux décrire les réponses spécifiques des baies de raisin (cultivar Cabernet-Sauvignon) aux facteurs rayonnement et température.L'analyse du transcriptome des baies exposées soit à un stress thermique soit à un stress lumineux a mis en lumière deux processus, une réponse rapide ayant lieu dès les premières heures de traitement et une réponse apparaissant au bout de plusieurs jours d’exposition aux stress. Cette étude a également permis de valider le système expérimental de découplage des effets du rayonnement et de la température.L'analyse des profils d’expression d’une vingtaine de gènes intervenant dans la voie de biosynthèse des flavonoïdes révèle des différences dans la réponse transcriptionnelle des gènes en fonction du stress et du stade développement auquel il a été appliqué. Néanmoins cette réponse n'est pas ou peu corrélée avec les variations des teneurs en anthocyanes et flavonols observées. Les teneurs en anthocyanes sont fortement réduites par l’action de la chaleur alors que les teneurs de certains flavonols augmentent sous l’influence du rayonnement. Au niveau des acides présents dans la pulpe des baies, l’acide malique voit sa teneur réduite sous l’effet de la chaleur ainsi que d’une forte intensité lumineuse. Les analyses montrent un impact important du stress thermique sur la teneur de la phénylalanine, de la tyrosine et de la lysine dans la pellicule.Parallèlement, l'initiation d’une étude du métabolome des pellicules de baies de raisin a été entreprise par UPLC-ESI-LTQ-Orbitrap™ et a permis d'acquérir des bases solides pour optimiser le protocole utilisé en vue d'analyses futures. Cette étude permet de dresser une liste préliminaire de métabolites d’intérêts.Enfin, le gène VvGOLS1 (Galactinol synthase 1) voit son expression stimulée dans les baies exposées au stress thermique, ce qui se traduit par une accumulation de galactinol, précurseur de la voie des RFOs. Un rôle de molécule «signal» est envisagé pour le galactinol. Un régulateur de la transcription de VvGOLS1 a également été identifié par des expériences d'expression transitoire en protoplastes. Il s'agit du facteur de transcription VvHsfA2, dont l'expression est également stimulée par le stress thermique. Dans ce contexte, la caractérisation des gènes de la famille des Hsfs (Heat Stress Factors) a été initée
Global warming will affect berry metabolism, and especially phenylpropanoïd contents. This PhD work aimed to acquire a better understanding on the cellular processus linking the microclimate and the phenolic synthesis. By molecular and biochemical approaches, we extended this study to detail specific responses taking place in berries under heat and light stress.Transcriptomic analysis of heat-stressed and light-stressed berries showed the existence of two processes that occur in exposed berries. The first one triggers a rapid and transient expression of genes within the first hours of treatment. The second one mobilizes a set of genes showing increase in their expression after several days of stress exposure. Furthermore, this study validated the experimental set used to discriminate the effects of light and temperature, respectively.Expression analysis of 20 genes involved in the flavonoid biosynthetic pathway revealed strong differences among the transcriptional responses, depending on the nature of stress and the developmental stage of the berry. However, expression patterns of genes involved in the biosynthesis of flavonoid could not fully explain the changes in anthocyanin and flavonol contents. This suggests that additional regulation processes such as post-traductional modifications of enzymes or metabolite degradation might take place in berries under abiotic stress. Anthocyanin content decreases under heat stress whereas flavonol content increases under high light. Malic acid increases in berry exposed to heat stress and high light. Moreover, heat-stressed berries showed an accumulation of phenylalanine, tyrosine and lysine in skin but not in pulp.In parallel, a metabolomic analysis was initiated on stress exposed berry skins by using UPLC-ESI-LTQ-Orbitrap™ technology. The first experiments revealed contrasted metabolite contents in berries according to the stress applied, and highlighted several metabolites of interest. The preliminary assays will help optimize this powerful tool for futures analysis.Finally, expression of VvGOLS1 (Galactinol synthase 1) was strongly induced in grape berries exposed to heat stress, in good agreement with the observed galactinol accumulation. Role of galactinol as a signaling molecule is discussed. Transient expression experiments revealed that VvGOLS1 expression is regulated at the transcriptional level through VvHsfA2 action. VvHsfA2 expression is also stimulated under heat stress. In this context, characterization of the grapevine heat stress factors was initiated

Un certain nombre d’études épidémiologiques font état d’une augmentation de l’infertilité masculine durant ces cinquante dernières années, en particulier dans les pays industrialisés, mais aussi d’une augmentation des malformations de l’appareil reproducteur masculin telles que la cryptorchidie (absence de migration des testicules dans les bourses) ou l’hypospadias (malformation du pénis), et des cancers testiculaires. Des données expérimentales suggèrent que ces anomalies du tractus génital mâle sont liées. Ces symptômes forment le syndrome de dysgénésie testiculaire. Les causes d’apparition ce syndrome semblent être d’origine environnementale. En effet, les évolutions relativement rapides de ce syndrome suggèrent des facteurs dynamiques, en lien avec le mode de vie ou l’environnement. Une des hypothèses est que, l’exposition durant la vie fœtale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens environnementaux, PEEs), serait responsable de l’augmentation de l’incidence de ces pathologies. Au banc des principaux accusés, les molécules qui possèdent des activités de type estrogénique ou antiandrogénique. A ce jour, les mécanismes d’action à l’origine du syndrome de dysgénésie testiculaire sont encore mal connus. Certaines études suggèrent des mécanismes de type épigenétique dans les effets à long terme des PEEs. L’objectif de notre travail était d’identifier et caractériser les mécanismes d’action de type épigenétique impliqué dans l’infertilité mâle. Pour cela, nous avons utilisé un modèle expérimental (rats nouveau-nés) reposant sur une exposition développementale à un estrogène (estradiol benzoate). Ce modèle induit chez le rat adulte un phénotype d’hypospermatogenèse liée à une à apoptose chronique des cellules germinales testiculaires. Nous montrons que ce phénotype est lié à l’altération de deux voies, impliquant en amont des effecteurs épigénétiques. La première voie implique la famille des miR-29s. Ainsi, nous observons une augmentation de l’expression des miR-29a, b, c qui provoque une diminution de deux de ses cibles: la protéine antiapoptotique MCL-1 et les enzymes de méthylation de l’ADN DNMTs. La chute des DNMTs entraine une hypométhylation globale (estimée à travers le gène Line-1) et spécifique du facteur de choc thermique HSF1. Ceci provoque une réexpression de ces facteurs entrainant l’apoptose des cellules germinales adultes. La deuxième voie implique le miR-18a. L’augmentation de son expression provoque une chute de l’expression de sa cible HSF2 qui régule la protéine de choc thermique HSP70/HSPA2. Le faible taux d’HSPA2 est une autre explication de l’apoptose des cellules germinales dans notre modèle. Nous montrons aussi que ce phénotype est irréversible lorsque l’exposition à lieu chez le nouveau-né alors qu’il est réversible quand l’exposition à lieu à l’âge adulte. Ces données suggèrent que l’exposition néonatale à l’estradiol benzoate induit une programmation développementale de l’hypospermatogenèse.Enfin, les anomalies tissulaires d’expression des miRNAs se retrouvent au niveau sanguin, suggérant leur utilisation potentielle comme biomarqueurs. Nous avons validé cet aspect chez l’homme en montrant que l’expression des miR29s et du miR-18a était plus élevée chez les patients oligo- ou azoospermiques que les chez patients normospermiques.En conclusion, nos résultats indiquent que l’hypospermatogenèse due à une apoptose chronique des cellules germinales observée chez l’animal adulte après exposition néonatale à l’EB met en jeu une modification d’expression de plusieurs effecteurs épigénétiques clés: miR-29s, miR-18a et DNMTs. De plus, les miR-29s et miR-18a pourraient être de nouveaux biomarqueurs circulants non invasifs de la stérilité masculine dans le contexte d’une oligo ou azoospermie chez l'homme
Epidemiological studies have reported an increase in male infertility over the past fifty years, especially in industrialized countries, but also an increase in malformations of the male reproductive tract such as cryptorchidism (no migration of the testes into the scrotum) and hypospadias (malformation of the penis), and testicular cancers. Experimental data suggest that these abnormalities of the male genital tract are related. These symptoms form the testicular dysgenesis syndrome. The causes of the occurrence of this syndrome appear to be environmental in origin. Indeed, the relatively rapid evolution of this syndrome suggests dynamic factors related to lifestyle or environment. One hypothesis is that exposure during fetal or neonatal life to compounds present in the environment can interfere with the hormonal system (environmental endocrine disruptors), would be responsible for the increased incidence of these pathologies. Bench of the main accused, molecules that have estrogenic or anti-androgenic activity types. To date, the mechanisms of action behind the testicular dysgenesis syndrome are poorly understood. Some studies suggest that epigenetic mechanisms are at playThe objective of our work was to identify and characterize the epigenetic mechanisms of action involved in male infertility induced by neonatal exposure to xenoestrogen. For this, we used an experimental model based on a developmental exposure to estrogen (estradiol benzoate). This model induced in adult rats a hypospermatogenesis phenotype due to chronic apoptosis of germ cells.We show that this phenotype is related to an alteration of two pathways, involving upstream effectors epigenetic. The first pathway involves the family of miR- 29s. Thus, we observe an up-regulation of miR -29a, b, c, which causes a decrease in two of his targets: the anti-apoptotic protein MCL- 1 and the enzymes of DNA methylation DNMTs. Falling DNMTs leads to a global hypomethylation (estimated through the Line -1 gene) and to specific hypomethylation of the heat shock factor, HSF1. This causes a re-expression of factors that induce apoptosis in adult germ cells. The second pathway involves up-regulation of miR -18a that causes a down-regulation of its target HSF2 which regulates the heat shock protein HSP70/HSPA2. The down-regulation of HSPA2 is another explanation of germ cell apoptosis in our model. We also show that this phenotype is irreversible when the estrogen exposure takes place in the newborn whereas it is reversible when exposure takes place in adulthood, suggesting that neonatal exposure to estradiol benzoate induced a developmental programming of hypospermatogenesis.Finally, abnormal tissue expressions of miRNAs are found in the blood, suggesting their potential use as biomarkers. We validated this aspect in humans showing that the expression of miR29s and miR-18a was higher in patients with decrease or no sperm counts compared to normal sperm count. In conclusion, our results indicate that hypospermatogenesis due to chronic germ cell apoptosis observed in adult animals after neonatal exposure to EB involves a change in expression of several key epigenetic effectors: miR-29, miR-18a and DNMTs. In addition, miR-29 and miR-18a could be new non invasive circulating biomarkers of men infertility

L'alcool (EtOH) est une des substances d'abus les plus consommées en France chez les adolescents comme chez les adultes. La consommation d'EtOH induit des déficits mnésiques en perturbant les phénomènes de plasticité synaptique de type potentialisation à long terme (PLT) et dépression à long terme (DLT) dépendants du récepteur NMDA (PLTNMDA et DLTNMDA), et qui constituent la base cellulaire des apprentissages et de la mémoire, notamment au niveau de l'hippocampe. La composition en sous-unités GluN2A et GluN2B du récepteur NMDA peut influencer l'induction de ces deux formes de plasticité synaptique selon le modèle théorique de Bienenstock et al. (1982). De plus, la plasticité synaptique est sous l'influence de mécanismes épigénétiques et/ou de facteurs de transcription. A l'heure actuelle, les mécanismes cellulaires qui sous-tendent la perturbation de la plasticité synaptique suite à la consommation d'EtOH demeurent mal compris. Durant ma thèse, j'ai testé l'hypothèse que les perturbations de la plasticité synaptique dépendante du NMDA impliqueraient une modulation des sous-unités GluN2A et GluN2B dans un modèle de binge drinking-like chez le rat jeune adulte et dans un modèle d'alcoolisation chronique chez des souris adultes. Afin de comprendre les mécanismes sous-tendant ces perturbations, j’ai étudié l'implication des facteurs épigénétiques et le rôle d'un facteur de transcription de la famille des heat shock factor, HSF2. Pour cela, j'ai utilisé la technique d'enregistrement de potentiel de champs somatique et dendritique (potentiel postsynaptique excitateur NMDA ; PPSE-NMDA) dans l'aire CA1 de tranches d'hippocampe. De manière intéressante, nos résultats montrent que les deux types d'alcoolisation, aigue et chronique, augmentent la sensibilité du PPSE-NMDA à un antagoniste de la sous-unité GluN2B alors que la sensibilité à l'antagoniste de la sous-unité GluN2A diminue. Chez le rat jeune adulte, ces modifications sont accompagnées d'une forte réduction de la DLTNMDA et d'un déficit d'apprentissage (test de reconnaissance de nouvel objet). Dans ce modèle, l'inhibition de l'activité des enzymes HDACs, responsables de la désacétylation des histones, prévient l'ensemble des effets de l’EtOH (sensibilité pharmacologique du PPSE-NMDA, DLTNMDA et apprentissage). Concernant HSF2 et avant toute alcoolisation, des souris adultes hsf-/- présentent une absence de DLTNMDA accompagnée d'une plus grande sensibilité du PPSE-NMDA à un antagoniste GluN2B comparé à des souris sauvages. L'exposition chronique à l'EtOH induit chez les souris sauvages, une abolition de la DLTNMDA accompagnée d'une augmentation de la sensibilité du PPSE-NMDA à un antagoniste GluN2B et à une diminution de la sensibilité de ce signal à un antagoniste GluN2A. En revanche, les souris hsf-/- ne présentent aucune de ces modifications. Ainsi, l'ensemble de mes travaux de thèse montre que quel que soit le type d'alcoolisation, aigue ou chronique, mais aussi l'espèce animale utilisée, rat ou souris, l'EtOH induit des adaptations du réseau hippocampique qui consiste en une augmentation de la sensibilité du PPSE-NMDA à un antagoniste GluN2B et en une diminution de sa sensibilité à un antagoniste GluN2A ; modifications qui accompagnent une abolition de la DLT. Cette réponse globale à l'EtOH mettrait en jeu des facteurs épigénétiques modulant l'état d'acétylation de l'ADN et des facteurs transcriptionnels de type heat shock
Alcohol (EtOH) remains one of the most consumed substances of abuse in France among adolescents and adults EtOH consumption, inducing learning deficits through disturbances of the NMDA-dependent form of synaptic plasticity (long term potentiation, LTP and long-term depression, LTD), the cellular signal responsible for learning and memory, notably into the hippocampus, involved in memory formation in mammals. Importantly, induction of NMDA-dependent synaptic plasticity relies on the subunit composition of the NMDA receptor (GluN2A and GluN2B) while mechanisms of genome regulation such as epigenetic or some transcription factors may have important role in determining the quality of synaptic plasticity signals. However, the molecular mechanisms by which EtOH disrupts NMDA-dependent synaptic plasticity are still unclear. During my thesis work, I tested the hypothesis that NMDA-dependent synaptic plasticity is disrupted by EtOH through the modulation of the involvement of GluN2B and GluN2A subunits of the receptor whatever the type of EtOH exposure, either acute in young adult (binge-drinking like model) rodents or chronic in adult rodents. I further tested the involvement of epigenetic and the role of HSF2, a transcription factor in the modifications induced by EtOH. Using pharmacological tools and field potential recordings in CA1 area of hippocampal slices from adolescent rats and adult mice, I found that both acute and chronic ethanol exposure increased field NMDA excitatory post synaptic potential (fNMDA-EPSP) sensitivity to a GluN2B antagonist while sensitivity to GluN2A antagonist was decreased. In adolescent rats, these modifications were accompanied with a lower LTD without affecting LTP and with memory impairment. Interestingly, inhibition of enzymes responsible for chromatin deacetylation (HDAC) in binge like adolescent rat model, prevents the EtOH effects in learning performance associated with a correction of the GluN2A/GluN2B balance and LTD. Concerning the role of HSF2, I found that before chronic EtOH consumption, fNMDA-EPSPs of HSF2 KO adult mice lack LTD and showed the opposite sensitivity to GluN2A and GluN2B antagonists compared to WT mice. Chronic EtOH exposure in HSF2 KO mice induced different adaptations than in WT animals. Altogether, my thesis work show that, 1) regardless the type of EtOH exposure, the hippocampus neuronal network adapt via changes in the balance between GluN2A and GluN2B subunits leading to LTD reduction and learning impairment; 2) these EtOH-induced changes in fNMDA-EPSPs involved epigenetic processes and 3) some transcription factors, affecting basal conditions of the role for GluN2A/GluN2B balance determines the capacity to respond to EtOH exposure

At the beginning of mitosis, chromosomes are condensed and segregated to facilitate correct alignment later in cytokinesis. Condensin is the pentameric enzyme responsible for this DNA compaction and is composed of two structural maintenance of chromosomes (SMC) subunits and three non-SMC subunits. Condensin mutations generate chromosomal abnormalities due to improper segregation, leading to genome instability and eventual malignant transformation of the cell. Cdc2 phosphorylation of the non-SMC subunits, CAP-G, CAP-D2, and CAP-H, has been demonstrated to be important for condensin supercoiling activity and function. While these subunits are thought to be phosphorylated by Cdc2, the exact sites have not yet been identified and characterized. The basis of this research was to determine the Cdc2 phosphorylation sites in the CAP-G subunit of the condensin enzyme and to characterize the functional significance of the sites in the regulation of condensin activity using site-directed mutagenesis and immunofluoresence microscopy. While DNA condensation represents a critical step early in mitosis, formation of the mitotic spindle represents a vital event leading to the division of a cell into two daughter cells in a process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a mitotic protein essential for cytokinesis that participates in formation of the mitotic spindle in a phosphorylation dependent manner. PRC1 possesses microtubule bundling properties. Loss of PRC1 leads to mis-segregation of chromosomes and abnormal cytokinesis. HSF2 is a transcription factor known to be important in development and differentiation. Previous research has determined that HSF2 plays a significant mechanistic role in the process of hsp70i gene bookmarking during mitosis. Bookmarking is an epigenetic phenomenon whereby certain gene promoters remain uncompacted, in contrast to the majoritiy of genomic DNA during mitosis. This lack of compaction allows quick reassembly to a transcriptionally competent in G1 of the cell cycle and ensures the ability of the cell to induce expression of the cytoprotective hsp70i protein. HSF2 and PRC1 were found to interact in a yeast-two hybrid screen. Given the importance of both of these proteins during mitosis, this study seeks to characterize the HSF2/PRC1 interaction and determine the potential role for PRC1 in hsp70i gene bookmarking.

The heat-shock response is one of the many complex physiological systems that organisms have developed in order to protect their cells against stress. This response is initiated by the binding of heat shock factor 1 (HSF1) to the promoters of genes containing heat-shock elements (HSEs,) which results in the expression of several proteins, among them the proteo-protective inducible heat-shock protein (hsp70i). Due to HSF1s critical role in this process, an active area of research is trying to understand of how HSF1 executes its function. Considering the rapidity with which the field of cell biology is expanding, in particular the sub-field of nuclear compartmentalization, this study seeks to understand how nuclear structure affects the function of HSF1. Specifically, this study investigates the potential role for the interaction between HSF1 and the translocated promoter region protein (Tpr,) a structural component of the nuclear pore, an interaction initially identified by yeast two-hybrid analysis, in the transcription of hsp70i. Due to Tprs location and its putative function in nucleo-cytoplasmic trafficking, this works seeks to answer to the question, Does Tpr play a role in the export of HSF1-driven mRNAs? In a similar vein, heat-shock transcription factor 2 (HSF2,) a less well-understood member of the heat-shock transcription factor family, also interacts with Tpr in the yeast two-hybrid assay. HSF2 has recently been shown to have an active role during mitosis, when the hsp70i gene is being bookmarked for potential expression that might be needed in early G1, when most genes are unable to be expressed. This body of work also seeks to answer the question of, Does the Tpr/HSF2 interaction have a role in positioning the gene in relation to the nuclear pore after mitosis? This study was performed using both novel and standard in vivo and in vitro molecular biology techniques. It ultimately aims to clarify the less understood, although much broader, subject of how does transcription occur in the three-dimensional space of the nucleus.

La phase de maturation des graines est caractérisée par l’ac-quisition successive de composantes qui constituent la qua-lité physiologique d’un lot de semences, à savoir la toléranceà la dessiccation (capacité à survivre au retrait total de l’eaucellulaire), la longévité (capacité de survivre à l'état sec pen-dant le stockage), la dormance ainsi que la vigueur germina-tive (capacité à germer de façon rapide et homogène quelquessoient les conditions de l’environnement). La production desemences à haute qualité germinative représente un enjeumajeur pour les semenciers car elle constitue un levier clefpour augmenter les rendements agricoles. Cependant, lesmécanismes régulant l’acquisition de la qualité germinativeet en particulier la longévité restent peu connus. Une étudepréalable d’un réseau de co-expression génique de facteursde transcription avait identifi é trois gènes candidats associésà la longévité chez Medicago truncatula :MtABL (ABA INSEN-SITIVE4-LIKE), MtABI5 (ABA INSENSITIVE5) et MtHSFA2.2(HEAT SHOCK FACTOR A2.2). L'objectif de cette thèse était devalider ces gènes et d’en comprendre leur fonction chez Medicagotruncatula et le pois par la caractérisation de mutantsd’insertion et EMS. ABL et ABI5 jouent un rôle dans la matu-ration en régulant positivement la longévité alors que celle-ci n’est pas affectée dans les mutants hsfa2.2. Des étudestranscriptomiques et biochimiques montrent que ABL et ABI5régulent de manière complexe la photosynthèse, la dégrada-tion de la chlorophylle et l’accumulation des oligosaccharide
Seed maturation is characterized by the acquisition ofthe various components that collectively constitute thephysiological quality or vigor of the seed: desiccation tolerance(DT, i.e. the capacity to survive complete drying), seedstorability or longevity (the capacity to remain alive duringstorage), dormancy, as well as fast and uniform germinationand seedling emergence under stressful conditions. Thesetraits are pivotal to ensure rapid and homogenous seedlingestablishment required for stable yield and are a majoreconomic challenge for the seed industry. Despite theiragronomic importance, the mechanisms regulating theiracquisition, including longevity, are still poorly understood. InMedicago truncatula, a gene co-expression network inferredthat transcription factors such asMtABL (ABA INSENSITIVE4-LIKE), MtABI5 (ABA INSENSITIVE5) and MtHSFA2.2 (HEATSHOCK FACTOR A2.2) are putative regulators of seedlongevity. The aim of this thesis was to characterize theirroles in Medicago truncatula and Pisum sativum using Tnt1insertion and EMS mutants. ABL and ABI5 are positiveregulators of longevity while defects in hsfa2.2 do not affectit. Transcriptomic and biochemical analyses show that ABLand ABI5 are involved in the regulation of photosynthesisassociated genes, chlorophyll loss and accumulation ofraffi nose family oligosaccharides (RFO). ABI5 is also involvedin the accumulation of stress proteins such as LEA proteins.By establishing a link between degreening, RFO contents andlongevity, our work offers new opportunities to tackle a

In this paper, a cluster-based hybrid security framework called HSFA for ad hoc networks is proposed and evaluated. The proposed security framework combines both specification and anomaly detection techniques to efficiently detect and prevent wide range of routing attacks. In the proposed hierarchical architecture, cluster nodes run a host specification-based intrusion detection system to detect specification violations attacks such as fabrication, replay, etc. While the cluster heads run an anomaly-based intrusion detection system to detect wormhole and rushing attacks. The proposed specification-based detection approach relies on a set of specifications automatically generated, while anomaly-detection uses statistical techniques. The proposed security framework provides an adaptive response against attacks to prevent damage to the network. The security framework is evaluated by simulation in presence of malicious nodes that can launch different attacks. Simulation results show that the proposed hybrid security framework performs significantly better than other existing mechanisms.