Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues. Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress conditions, these oligomers dissociate and/or undergo drastic conformational changes to facilitate their binding to misfolded substrate proteins in the cell. This interaction prevents the substrate from aggregating during stress. When physiological conditions are restored, the substrates are transferred to other ATP-dependent heat shock proteins for refolding. Thus sHSPs do not refold their substrates, but instead prevent them from aggregating and maintain them in a „folding-competent‟ state. The clientele of sHSPs includes proteins with a wide range of molecular masses, secondary structures and pIs. This promiscuity has led to sHSPs occupying key positions in the protein quality control network. As molecular chaperones that protect proteins, sHSPs prevent disease. Concomitantly, mutations in sHSPs have also been linked to various human diseases. Till date, high resolution crystal structures are available only for 3 sHSP oligomers. This insufficiency of structural information has hindered our understanding of the mechanism of chaperone function, the link between the oligomeric status and chaperone activity, identification of substrate binding sites and the role of the flexible terminal segments in mediating both the oligomerization and chaperone function. We undertook structural and functional characterization of plant and bacterial sHSPs in order to address some of these questions. Chapter 1 of this thesis gives an overview of the sHSP family, with special emphasis on the oligomeric assemblies of sHSPs of known structures. We highlight what we know about this family through mutational studies, what is as yet unknown, and why it is important to study this family. Chapter 2 describes our efforts at structural and functional characterization of 5 sHSPS in rice, each targeted to a different organelle. We probed the role played by the N-terminal region in mediating oligomer assembly and in the chaperone activity of the protein. Rice sHSPs displayed a wide range of hydrodynamic radii, from 4 nm to 14 nm, suggesting that their oligomeric assemblies are likely to be diverse. In chapter 3, we discuss our attempts at the structural characterization of a bacterial sHSP, Aggregation suppressing protein A, or AgsA from Salmonella enterica. We obtained a high resolution crystal structure of the dimer of the core sHSP domain. We compared this dimer with other known sHSP dimers, reported the deviations that we observed and analysed the structure to account for these differences. We used this dimer structure to successfully obtain solutions for low resolution X-ray diffraction data for oligomers of different truncated constructs of AgsA. We observed that a C-terminal truncated construct formed an octahedral 24¬mer (4.5 Å resolution), whereas a construct truncated at both termini formed a triangular bipyramidal 18-mer (7.7 Å resolution), an assembly hitherto unobserved for any sHSP. A similar 18-mer was obtained when the C-terminal truncated construct was incubated with a dipeptide prior to crystallisation (6.7 Å resolution). The cryo-EM map of the wild type protein (12 Å resolution) could be fitted with a different 18-mer. The low resolution of the data pre-empted an atomic-level description of the interfaces of the assemblies. However, our work highlights the structural plasticity of this protein and probes the sensitivity of the oligomeric assembly to minor differences in construct length.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The essential oil (EOs) extracted from plants Hesperozygis ringens and Lippia alba possess anesthetic and sedative properties and is an alternative to traditional anesthetics used in aquaculture for ease of handling and/or reduce stress. In this sense, the study aimed to investigate the effects of these EOs on the physiology of fish, through physiological, biochemical and endocrine indicators. In the article 1 was determined (a) the anesthetic activity of the EOs of H. ringens (EOHR) and L. alba (EOLA) and (b) its effects on silver catfish (Rhamdia quelen) after induction and recovery from anesthesia. Fish were subjected to one of the following treatments for each EO: basal group, control or anesthetized (150, 300 or 450 uL L-1 EO), evaluating the ventilatory rate (VR) during the induction period and thereafter transferred to anesthetics-free tanks for recovery from anesthesia. At 0, 15, 30, 60 and 240 min of recovery, samples of plasma and gills were collected to measure metabolic indicators and ionregulatory enzymes, respectively. In the article 2, the effects of prolonged exposure to low EOHR concentrations were studied on silver catfish. After 6 h of exposure to 0 (control), 30 or 50 uL L-1 EOHR added to water, it was analyzed: VR, metabolic indicators of stress in plasma, enzyme activity in liver, and expression of pituitary hormones (growth hormone - GH, prolactin - PRL and somatolactina - SL). In the manuscript, (a) evaluated the effectiveness of anesthesia EOLA on gilthead sea bream (Sparus aurata) and (b) we investigated the effects of 35 uL L-1 EOLA and 2-phenoxyethanol (2-PHE) on the stress response in gilthead sea bream undergoing persecution. After 4 h of exposure, the plasma was sampled (for the determination of cortisol, metabolites and osmolality), brain and pituitary (to evaluate the expression of endocrine indicators). In the article 1, anesthesia with EOs caused changes in some parameters measured in silver catfish, but did not prevent the restoration of most of the indicators assessed after 240 min of recovery. In the article 2, 50 uL L-1 EOHR led to an increase of glucose, lactate, protein and osmolality, as well as an increase in metabolic enzyme activity and reduced expression of GH and SL. In the manuscript, gilthead sea bream exposed to EOLA, stressed or not, exhibited higher levels of cortisol, glucose, lactate and osmolality. EOLA exposure added to the stress reduced the expression levels of CRH-BP (corticotropin releasing hormone bound to protein). PRL expression was reduced in the stressed control group and after exposure to EOLA and 2-PHE in fish not stressed. Higher expression of pro-opiomelanocortin (POMC) "a" and "b" were observed in fish stressed and exposed to EOLA and 2-PHE, respectively. We conclude that: (1) the EOLA is more efficient for silver catfish that EOHR in anesthesia concentrations; (2) for sedating the fish, it is recommended 30 uL EOHR L-1 (or less); (3) the EOLA was effective as an anesthetic for gilthead sea bream at 100-300 uL L-1, but for 4 h exposure, the 2-PHE was more effective in preventing the stress response. / Os óleos essenciais (OEs) extraídos das plantas Hesperozygis ringens e Lippia alba possuem propriedades anestésica e sedativa, constituindo uma alternativa aos anestésicos tradicionalmente usados em aquicultura para facilitar o manejo e/ou reduzir o estresse. Neste sentido, o estudo teve por objetivo investigar os efeitos desses OEs sobre a fisiologia de peixes, através de indicadores fisiológicos, bioquímicos e endócrinos. No artigo 1 determinou-se (a) a atividade anestésica dos OEs de H. ringens (OEHR) e L. alba (OELA) e (b) seus efeitos em jundiá (Rhamdia quelen) depois da indução e recuperação da anestesia. Os peixes foram submetidos a um dos seguintes tratamentos para cada OE: grupo basal, controle ou anestesiado (150, 300 ou 450 μL L-1 OE), avaliando-se a taxa ventilatória (TV) durante o período de indução e, posteriormente, transferidos para aquários sem anestésicos para recuperação da anestesia. Nos tempos 0, 15, 30, 60 e 240 min de recuperação foram realizadas amostragens de plasma e brânquias para medir indicadores metabólicos e enzimas ionorregulatórias, respectivamente. No artigo 2, os efeitos da exposição prolongada de jundiás a baixas concentrações do OEHR foram estudados. Após 6 h de exposição a 0 (controle), 30 ou 50 μL L-1 OEHR adicionado à água, analisou-se: TV, indicadores metabólicos e de estresse em plasma, atividade enzimática em fígado e expressão de hormônios hipofisários (hormônio do crescimento - GH, prolactina - PRL e somatolactina - SL). No manuscrito (a) avaliou-se a eficácia anestésica do OELA em dourada (Sparus aurata) e (b) investigaram-se os efeitos de 35 μL L-1 de OELA e 2-fenoxietanol (2-PHE) sobre a resposta ao estresse em douradas submetidos à perseguição. Após 4 h de exposição, foram amostrados plasma (para determinação dos níveis de cortisol, metabólitos e osmolalidade), cérebro e hipófise (para avaliar a expressão de indicadores endócrinos). No artigo 1, a anestesia com os OEs provocou alterações em alguns parâmetros medidos em jundiás, mas não impediu a restauração da maioria dos indicadores avaliados após 240 min de recuperação. No artigo 2, 50 μL L-1 do OEHR provocou a elevação dos níveis de glicose, lactato, proteína e osmolalidade, bem como aumento na atividade de enzimas metabólicas e redução na expressão do GH e SL. No manuscrito, douradas expostos ao OELA, estressados ou não, exibiram maiores níveis de cortisol, glicose, lactato e osmolalidade. A exposição ao OELA somado ao estresse reduziu os níveis de expressão de CRH-BP (hormônio liberador de corticotrofina ligado à proteína). A expressão de PRL foi reduzida no grupo controle estressado e após a exposição ao OELA e 2-PHE em peixes não estressados. Maiores expressões de pro-opiomelanocortina (POMC) a e b foram observadas em peixes estressados e expostos ao OELA e 2-PHE, respectivamente. Conclui-se que: (1) o OELA é mais eficiente para jundiás que o OEHR em concentrações para anestesia; (2) para sedar os peixes, recomenda-se 30 μL L-1 do OEHR (ou menos); (3) o OELA foi eficaz como anestésico para dourada entre 100-300 μL L-1, mas para 4 h de exposição o 2-PHE foi mais eficiente em prevenir a resposta ao estresse.

Les leucémies aiguës myéloïdes (LAM) représentent un groupe d’hémopathies malignes agressives, de pronostic sombre en dépit des traitements intensifs actuellement proposés. Malgré une grande hétérogénéité clinique et moléculaire, les cellules de LAM sont caractérisées par l’activation de voies de signalisation essentielles à leur prolifération et leur survie, comme par exemple celle du complexe mTORC1 (mammalian target of rapamycin complex 1). Cependant, l’utilisation clinique d’inhibiteurs tels que la rapamycine ou des inhibiteurs catalytiques s’est avérée décevante, ce qui suggère qu’il n’y a pas d’addiction oncogénique à mTORC1 dans les LAM. Au cours de ce travail, nous avons démontré que l’activation de mTORC1 est au contraire une condition nécessaire à l’induction de la mort cellulaire en réponse à l’activation d’AMPK (AMP-activated protein kinase), établissant une relation de létalité synthétique entre ces deux voies. Pour cela, nous avons utilisé un nouveau composé activateur spécifique d’AMPK, le GSK621. En invalidant la sous-unité catalytique AMPKα1 par ARN interférence ou par le système CRISPR/Cas9, nous avons démontré que les effets antileucémiques de ce composé sont bien dépendants de l’activation d’AMPK. Nous avons observé que ce composé favorise l’autophagie, et que ce processus est impliqué dans la mort des cellules leucémiques puisque l’inhibition des protéines ATG5 ou ATG7 a un effet protecteur sur les cellules leucémiques. Les effets antileucémiques du composé GSK621 ont été confirmés sur des cellules primaires, ainsi que sur un panel de vingt lignées de LAM, et dans un modèle murin de xénogreffe. De façon intéressante, l’activation d’AMPK pourrait également compromettre la survie des cellules souches leucémiques, comme en atteste l’atténuation du potentiel clonogénique en méthylcellulose de cellules murines transformées par MLL-ENL ou FLT3-ITD. Nous avons observé que le composé GSK 621 n’avait pas de toxicité envers les progéniteurs hématopoïétiques normaux, ouvrant ainsi une fenêtre thérapeutique intéressante. Comme l’activation d’AMPK conduit dans de nombreux modèles cellulaires à l’inhibition de mTORC1, et comme l’activation de mTORC1 est observée dans les cellules de LAM mais pas dans les progéniteurs hématopoïétiques normaux, nous avons proposé l’hypothèse que le niveau d’activation de mTORC1 déterminait les effets de l’activateur d’AMPK. Pour cela, nous avons inhibé mTORC1 dans les cellules leucémiques d’une part, et activé mTORC1 dans les progéniteurs normaux d’autre part. De façon inattendue, mTORC1 échappe au contrôle d’AMPK dans les LAM, et nous avons observé que l’activation de mTORC1 est une condition nécessaire et suffisante pour que le composé GSK621 entraîne la mort des cellules. Le substrat moléculaire de cette létalité synthétique est le facteur de transcription proapoptotique ATF4, dont la transcription est favorisée par mTORC1, et la traduction par AMPK via la phosphorylation d’eIF2A. Ces travaux proposent donc que malgré l’absence d’addiction oncogénique, l’activation de mTORC1 dans les LAM représente une opportunité thérapeutique originale via une relation de létalité synthétique avec l’activation d’AMPK. Ils constituent un rationnel au développement clinique d’activateurs d’AMPK dans les LAM, voire dans d’autres cancers ayant une activation constitutive de mTORC1. / Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis despite intensive treatments. Virtually all recurrent molecular alterations in AML functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as in clinical trials, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR/Cas9 and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, and blocking autophagy via shRNA-mediated knockdown of ATG5 or ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation between AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1, sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. The same synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. We further observed that the level of ATF4 protein is under a transcriptionnal control by mTORC1 and a translational control by AMPK (through eIF2A), and explains the synthetic lethal relationship between AMPK and mTORC1. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials.

Le diabète de type 2 et la dépression sont des problèmes majeurs de santé publique associés par un lien bidirectionnel. La dérégulation de l'axe hypothalamo-hypophyso-surrénalien (HPA), accompagnée par un taux élevé de glucocorticoïdes circulants, pourrait constituer un mécanisme commun à ces pathologies. L'axe HPA est régulé principalement au niveau de l'hypothalamus, siège de régulations nutritionnelles et émotionnelles. En ciblant les noyaux hypothalamiques, la néoglucogenèse intestinale (NGI) a des effets bénéfiques contre le développement du diabète de type 2 via la stimulation des nerfs vagal et spinal. Nous avons donc testé si la NGI, par sa communication avec l'hypothalamus, pourrait également réguler les comportements émotionnels et ainsi exercer des effets bénéfiques sur les maladies métaboliques et émotionnelles.L'absence de NGI provoque un dysfonctionnement de l'axe HPA et de son rétrocontrôle négatif (via des modifications moléculaires), caractérisés par une hypersécrétion de glucocorticoïdes et le développement d'une résistance aux glucocorticoïdes. Grâce à des études comportementales et moléculaires, nous montrons que les souris dépourvues de NGI développent des altérations phénotypiques et neurobiologiques caractéristiques d'un état anxio-dépressif. La restauration de la NGI par une perfusion de glucose portale rétablit les altérations neurobiologiques de l'axe HPA. L'induction de la NGI par un régime riche en protéines exerce des effets anxiolytiques et antidépresseurs. Ces données suggèrent que la NGI en ciblant l'hypothalamus, contrôle le métabolisme et, via l'axe HPA, les comportements émotionnels / Type 2 diabetes and major depressive disorder are major health concerns, which are highly comorbid. Hypothalamic-pituitary-adrenal (HPA) axis dysfunction, associated with elevated circulating levels of glucocorticoids, was suggested to be a common mechanism for those pathologies. The hypothalamus, which mainly regulates the HPA axis, is a key integrative center, playing a role in both metabolic and emotional processes. By targeting hypothalamic nuclei, intestinal gluconeogenesis (IGN) exerts beneficial effects against the development of type 2 diabetes through the stimulation of the vagal and spinal nerves. We therefore evaluated whether IGN, via the hypothalamus, may represent a putative common regulator of metabolic and emotional disorders.In the absence of IGN, mice exhibited HPA axis dysregulation along with decreased glucocorticoid-mediated negative feedback (due to molecular modifications), highlighted by hypercortisolism and glucocorticoid resistance. Using behavioral and molecular studies, we demonstrated that mice lacking IGN displayed phenotypic and neurobiological hallmarks of anxiety/depression-like state. Rescuing IGN by portal glucose infusion reversed neurobiological alterations of the HPA axis. Induction of IGN by a protein-enriched diet had anxiolytic and antidepressant effects. Together, these data raise the possibility that IGN by targeting hypothalamus, controls metabolism and, via the HPA axis, emotional behavior

L’endosymbiose trophique établie entre un hôte Cnidaire et ses symbiotes Dinoflagellés photosynthétiques est à l’origine du succès évolutif des écosystèmes coralliens. Les symbiotes sont internalisés par un mécanisme de phagocytose et maintenus dans les cellules du gastroderme de l'hôte. La symbiose est régie par un dialogue moléculaire intime entre les deux partenaires, interrompu lors de perturbations environnementales ou anthropiques, responsables du déclin mondial des récifs coralliens. Les objectifs de mon projet de recherche sont de définir les acteurs moléculaires localisés à l’interface symbiotique chez l’anémone de mer, Anemonia viridis. Premièrement, nous avons étudié les mécanismes cellulaires impliqués dans différents types de rupture de la symbiose et mis en évidence des phénomènes d’apoptose, nécrose et symbiophagie. Parallèlement, nous avons caractérisé chez l’anémone les gènes npc1 et npc2, impliqués chez les vertébrés dans le transport endosomal de stérols, et dont l’expression est modulée par l’état symbiotique. Nous avons pu montrer que le gène npc2d est issus d’une duplication et vraisemblablement d’une sub-fonctionnalisation et que les protéines NPC1 et NPC2 sont exprimées au voisinage des symbiotes. Nous proposons donc que la protéine NPC2-d soit utilisée comme marqueur de l’état de santé des Anthozoaires symbiotiques et que la protéine NPC1 soit un marqueur de la membrane périsymbiotique. Nous avons également développé un protocole afin d’identifier les protéines associées à l’interface symbiotique entre les deux partenaires. A terme, les cibles identifiées permettront une meilleure compréhension des mécanismes qui régulent la relation symbiotique. / The trophic endosymbiosis interaction between a cnidarian host and its photosynthetic dinoflagellatessymbionts form the basis of coral reef ecosystems. Cnidarians host their symbionts in gastrodermis cells, in a phagocytosis-derived vacuole. Establishment and maintenance of the symbiotic interaction depend on an intimate molecular communication between the two partners. However, environmental and/or anthropogenic disturbances can lead to the breakdown of the symbiotic association, which is responsible for the worldwide decline of coral reefs. The main objectives of my research project are to improve the knowledge regarding symbiosis maintenance and disruption mechanisms, but also to define the molecular key players involved at the symbiotic interface in the sea anemone, Anemonia viridis. First, we have described the cellular mechanisms involved in the different types of symbiosis breackdown. Meanwhile, the characterization of npc1 and npc2 genes (involved in endosomal sterol transport), showed a duplication and a sub-functionalization of the npc2d gene. Both NPC1 and NPC2 proteins are expressed around symbionts. We therefore suggest that the duplicated protein NPC2-d is a biomarker of symbiosis health and that NPC1 protein is a marker of the perisymbiotic membrane. We then developed a protocol to characterize the proteome of the symbiotic interface between the two symbiotic partners. The newly-identified symbiotic key players will increase the general knowledge on the symbiotic interaction and its regulation during both stable and bleaching conditions.

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Les théories évolutives du vieillissement et la théorie du «disposable soma» en particulier ont été la base théorique d'une avance récente de recherche sur le vieillissement animal. Pourtant, leur hypothèse centrale sur la physiologie de l'entretien et de la réparation cellulaires n'a pas été testée empiriquement. Dans cette thèse, j'ai analysé la physiologie du vieillissement de Escherichia coli sous restriction de carbone, en tant que système modèle pour valider empiriquement les théories évolutives du vieillissement. Les outils microfluidiques sont utilisés pour isoler de larges populations de cellules isolées de E. coli et pour obtenir une restriction carbonée homogène. Malgré le partage de la même génétique et des conditions environnementales, les cellules individuelles de la population présentent des variations significatives de la durée de vie et de cause de décès. Les distributions de durée de vie présentent des caractéristiques typiques du processus de vieillissement, souvent observées en études démographiques animales et humaines. Le taux de vieillissement peut être modifié par des mutations de la réponse générale au stress. Comme la longévité induite par la restriction calorique, la réponse générale au stress prolonge la durée de vie d'E.coli en atténuant l'effet du vieillissement au détriment des besoins immédiats des cellules. Un modèle quantitatif de ce compromis physiologique est construit et correctement prédit des observations expérimentales. En conclusion, je confirme la théorie du «disposable soma» du vieillissement avec les détails physiologiques du vieillissement de E.coli en famine. / The evolutionary theories of aging and the disposable soma theory in particular, have been the theoretical basis for a recent surge of animal aging research. Yet their central assumption about the physiology of cellular maintenance and repair has not been empirically tested. In this thesis, I analysed the physiology of E.coli aging under carbon starvation, as a model system to empirically validate evolutionary theories of aging. Microfluidic tools are used to isolate large populations of isogenic single E.coli cells, and to achieve homogenous carbon starvation. Despite sharing the same genetical background and environmental conditions, individual cells in the population exhibit significant variations in lifespans and causes of death. Distributions of lifespans exhibit typical features of the aging process, often seen in animal and human demographic studies. The rate of aging can be altered by mutations of the general stress response pathway. Resembling caloric restriction induced longevity, the general stress response pathway extends starvation lifespans of E.coli by attenuating the effect of aging at the expense of immediate needs of the cells. A quantitative model of this physiological trade-off is constructed and correctly predicted experimental observations. As a conclusion, I substantiate the disposable soma theory of aging with the physiological details of E.coli aging in starvation.

This thesis deals with the characterization of bacterial cells capable of polyhydroxyalkanoates (PHA) accumulation. The dissertation thesis is written in the form of a discussed published publications which are attached to the thesis as appendixes. The work develops a study of the current topic of the protective functions of PHA and clarifies protective mechanisms against selected stressors. Firstly, we focused on the protective effects of PHA granules against UV radiation and osmotic stress, specifically hypotonic conditions. In the case of UV exposition, the cells protected themselves by scattering UV radiation on the intracellular granules protecting especially nucleoid. When exposed to osmotic stress, the amorphous state of PHA granules is very important since it is capable of stabilization of cell membranes under hypertonic stress, afterwards, bacterial cells can maintain their integrity during the subsequent hypotonic challenge. In general, the amorphous state of PHA granules is key to ensure the proper biological functions of PHA whether as storage or protective polymer. Therefore, in the next part of this work, we focused on the core of the stabilization mechanism that protects native PHA granules from crystallization and thus the intracellular polymer maintains in a thermodynamically unfavorable amorphous phase state. Based on experimental work, we applied selected stresses because we proposed a new model of stabilization of the amorphous state of PHA granules in vivo. It consists of two mechanisms, where small volumes of PHA granules reduce the rates of crystallization and at the same time the water present in the granules plays the role of a low molecular plasticizer. Due to the metabolic apparatus of bacterial cells, PHA are simultaneously synthesized and degraded which leads to an increment of intracellular concentration of monomers that also figure in the protective effect of PHA. In this context, we aimed at the description of the mechanism of cryoprotective effects of 3-hydroxybutyrate, the monomer of the most common of PHA, poly(3-hydroxybutyrate). Hence, we constructed an equilibrium and non-equilibrium phase diagram of the 3HB-water system to prove that 3HB is a very effective cryoprotectant. This fundamental understanding of the protective properties of PHA monomers could be also used in the food industry or cryopreservation of biological samples.