La cryptogéine, sécrétée par l’oomycète Phytophthora cryptogea, est un éliciteur protéique des réactions de défense qui active chez le tabac un ensemble d’événements de signalisation conduisant à la mise en place d’une mort cellulaire de type réponse hypersensible et d’une résistance systémique acquise. La caractérisation de la modulation de l’activité de kinases cytosoliques, dont SIPK et WIPK, par des événements de phosphorylation en réponse à la cryptogéine, traduit la place majeure que tiennent les modifications post-traductionnelles dans la cascade de signalisation induite dans les cellules de tabac en réponse à la cryptogéine. Il s’avère que la signalisation cellulaire induite par la cryptogéine, et impliquant ces protéines kinases, converge entre autre vers le noyau à travers la modulation de l’activité d’éléments nucléaires par phosphorylation. Dans ce contexte, d’importants travaux de purification/séquencage, visant à identifier les protéines nucléaires cibles de ces activités kinases, ont permis d’identifier deux isoformes redondantes d’histones désacétylases de type 2 nommés NtHD2a et NtHD2b qui sont rapidement phosphorylées en réponse à la cryptogéine dans les cellules de tabac.Ce travail de thèse s’inscrit dans l’étude du rôle de NtHD2a/b dans l’établissement du processus de mort cellulaire des cellules de tabac et de la RH in planta en réponse à la cryptogéine. Par des approches de pharmacologie ainsi que des approches de surexpression ou d’invalidation de l’expression de NtHD2a/b chez le tabac, nous avons d’une part confirmé l’implication de NtHD2a/b en tant que régulateurs négatifs de la mort cellulaire induite par la cryptogéine ou d’autres élicitines, et d’autre part mieux appréhendé les événements de la cascade de signalisation prépondérants dans l’établissement de cette mort cellulaire. Les mécanismes moléculaires sous-jacents à la mise en place de la mort cellulaire apparaissent complexes et semblent notamment impliquer la modulation de l’expression de gènes de défense, la synthèse de novo de protéines ainsi que l’activation de protéines kinases, dont notamment WIPK et SIPK.Des travaux relatifs à l’étude des événements de (dé)/acétylation dans les cellules de tabac traitées par la cryptogéine et invalidées dans l’expression de NtHD2a/b suggèrent le concours de modifications post-traductionnelles de protéines nucléaires telles que l’acétylation dans la mise en place de la mort cellulaire induite par la cryptogéine chez le tabac. / Cryptogein, which is secreted by the oomycete Phytophthora cryptogea, is a proteinaceous elicitor of plant defense reactions that activates a set of signaling events leading to the hypersensitive response and to systemic acquired resistance. Although the early cytosolic signaling events induced by cryptogein are well described, the only nuclear events characterized to date are the variations in free calcium concentrations and defense-related gene expression. The characterization of the activation of cytosolic protein kinases, including WIPK and SIPK, by phosphorylation in response to cryptogein highlights the key-role played by posttranslational modifications in cryptogein-induced signaling events in tobacco cells. In this context, purification/sequencing approaches revealed that two redundant isoforms of type-2 nuclear histone deacetylases, NtHD2a and NtHD2b, were rapidly phosphorylated in cryptogein-treated tobacco cells.This thesis work is part of a comprehensive study of the role of NtHD2a/b in the establishment of the cell death process in tobacco cells and of the hypersensitive response in planta, in response to cryptogein. By using a pharmacological approach and overexpression and RNA interference-based approaches, we confirmed the involvement of NtHD2a/b as negative regulators of elicitin-induced cell death and we achieved a better understanding of cell death signaling events. The molecular events that underly the cell death process appear particularly complex and seem to involve the modulation of defense-related gene expression, de novo protein synthesis and protein kinase activation such as WIPK and SIPK.The study of (de)/acetylation events in tobacco cells treated by cryptogein and invalidated in NtHD2a/b expression suggests a role for posttranslational modifications in cryptogein-induced cell death.

Depression and stimulant drug addiction each result in massive losses of health, productivity and human lives every year. Despite decades of research, current treatment regimes for depression are ineffective in approximately half of all patients. Therapy available to stimulant drug addicts is largely ineffective and moreover, dedicated treatments for drug dependence (including abuse of cocaine) are non-existent. Thus, there is a pressing need to further understanding of the molecular mechanisms underlying these disorders in order to develop novel, targeted therapeutic strategies. Chromatin remodeling, including changes in histone acetylation, has been proposed to play a role in both the etiology and treatment of depression and stimulant abuse. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate numerous cellular processes, including transcription, cell cycle progression and differentiation. Moreover, histone acetylation has been shown to regulate hippocampal neurogenesis, a cellular response associated with the pathogenesis and treatment of depression and stimulant abuse (Hsieh et al., 2004, Yamaguchi et al., 2004, Fischer et al., 2007). Ultimately, such basic cellular processes impact higher order function, namely cognition and emotion. Indeed, recent studies suggest that HDAC activity in selected forebrain regions, including ventral striatum and hippocampus, modulate stimulant- and antidepressantinduced behavior (Kumar et al., 2005, Tsankova et al., 2006a, Fischer et al., 2007). These reports highlight an association between chromatin remodeling and diverse behavioral changes, including changes induced by the pleiotropic HDAC inhibitor, sodium butyrate (SB), (Kumar et al., 2005, Tsankova et al., 2006a, Fischer et al., 2007). However, behavioral, brain-metabolic and molecular effects of SB treatment in the context of rodent models of depression, dopaminergic sensitization and repeated cocaine administration remained unclear. The work described in this thesis illustrates the potential for chromatin modifying drugs in mechanisms underlying the experimental pharmacology of depression and stimulant addiction. Specifically, the data presented here support the view that treatment with the short chain fatty acid, sodium butyrate enhances: (1) antidepressant-like behavioral effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine (2) locomotor sensitization induced by repeated administration of the dopamine D1/D5 receptor agonist SKF82958; and(3) brain metabolic activation upon repeated cocaine administration as evidenced by fMRI in awake rats. Furthermore, this report provides evidence that these treatment paradigms will result in chromatin modification changes associated with active transcription, in addition to increased mRNA levels of plasticity-associated genes, including brain-derived neurotrophic factor (BDNF) at key brain regions implicated in the pathogenesis of depression and stimulant addiction. To date, little is known regarding the underlying mechanisms of action mediating the enhancing effects of sodium butyrate on the various antidepressant- and stimulantrelated paradigms. Our findings underscore the potential of chromatin-modifying drugs to profoundly affect the behavioral response of an animal to antidepressant and stimulant drugs and warrants consideration in the context of developing novel therapeutic strategies.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. In this study we attempt to characterize novel chemical compounds identified as potential activators of the SMN2 gene. Additionally, we sought to determine the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 gene. We used quantitative PCR to determine the effects of novel compounds and shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that the compounds identified in multiple reporter high throughput screens increased SMN protein levels via transcriptional activation of the SMN2 gene. Other compounds identified in the same screen functioned post-transcriptionally, possibly stabilizing the SMN protein itself by decreasing degradation. Furthermore, we determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This collective work defines a quantitative RNA based protocol to determine mechanism of SMN reporter increase in response to any chosen treatment method. Additionally, this work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.