Axonal protein synthesis is rapidly regulated by extrinsic cues during neural wiring but the full landscape of proteomic changes and their translational control mechanisms remain unknown. The ability to investigate the nascent proteome on subcellular compartments has been hampered by the low sensitivity of existing methodology on quantity-limited samples combined with the difficulty of obtaining sufficient amounts of pure material. By combining pulsed Stable Isotope Labelling by Amino acids in Cell culture (pSILAC) with Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), I have established an approach to characterize the nascent proteome from quantity-limited somaless retinal axons (~2μg) on an unparalleled rapid time-scale (5 min). The results show that a surprisingly large number of proteins (>350) is translated constitutively in axons, many of which are linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) each show a signature set of up/down newly synthesised protein (NSP) changes (>100) within 5 min. Remarkably, conversion of Netrin-1-induced responses from repulsion to attraction triggers opposite translational regulation for 73% of a common subset corresponding to >100 NSPs. Further, I show that pharmacological increase in cAMP, known to induce chemoattractive response, also leads to rapid and wide-scale remodelling of the nascent axonal proteome (~100 NSP changes). I find that the cAMP-elicited NSP changes underlie the attractive turning but are distinct from those induced by the physiological chemoattractant Netrin-1, suggesting that the same type of chemotropic response can be mediated by different protein synthesis-dependent mechanisms. Finally, I show that Sema3A, but not Slit1, triggers a physiological and non-canonical PERK-eIF2α-eIF2B signalling pathway required in neural wiring to elicit the rapid (< 15 min) local translation control of a specific subset of NSPs. Collectively my findings lead to the general conclusion that guidance molecules rapidly induce cue-specific remodelling of the nascent axonal proteome via distinct regulatory mechanisms.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

En esta tesis se estudian los efectos farmacológicos de derivados lipídicos frente el glioma y el Alzheimer. Los beneficios de este tipo de fármacos, basados en la terapia lipídica de membrana, están asociados con la modulación de la composición y las propiedades fisicoquímicas de membrana. En concreto, el ácido 2-hidroxioleico (2OHOA) es un potente fármaco antitumoral que fue diseñado para regular la composición y la estructura de la membrana lipídica así como la función de importantes proteínas de membrana. Por otro lado, el ácido 2-hidroxiaraquidónico (2OHARA), el ácido 2-hidroxieicosapentaenóico (2OHEPA), y el ácido 2-hidroxidocosahexanóico (2OHDHA) son derivados lipídicos hidroxilados que fueron diseñados en nuestro grupo de investigación para el tratamiento del Alzheimer. Este trabajo se ha basado en el estudio del funcionamiento de estos derivados de ácidos grasos hidroxilados en la modulación de las vías de señalización de la UPR (respuesta a las proteínas mal plegadas) y de la autofagia en células de glioma y células neuronales. / In this thesis, the pharmacological effects of lipid derivatives against glioma and Alzheimer's Disease are studied. The benefits of this type of drugs, which are based on the lipid membrane therapy, are associated with the modulation of the composition and physicochemical properties of membranes. 2-Hydroxyoleic acid (2OHOA) is a potent antitumor drug designed to regulate membrane lipid composition and structure and the function of important membrane proteins. In addition, 2- hydroxyarachidonic acid (2OHARA; LP204A1), 2-hydroxyeicosapentaenoic acid (2OHEPA; LP205A1), and 2-hydroxydocosahexanoic acid (2OHDHA; LP226A1) are new hydroxy derivated lipids designed in our group for the treatment of Alzheimer’s Disease. The main goal of this work was to study how these synthetic hydroxy derivates modulate the unfolded protein response and the autophagy pathways in glioma cells and neuron-like cells for Alzheimer’s Disease.

We have identified a novel Cyclin, called Cyclin O, which is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli. We have focused on the study of Cyclin O&#945; and Cyclin O&#946;, alternatively spliced products of the gene. Upon treatment with different stress stimuli, transfected Cyclin O&#945; accumulates in dense aggregations in the cytoplasm compatible with being Stress Granules (SGs). Furthermore, we have seen that Cyclin O&#946; and a point mutant of the N-terminal part of the protein constitutively localize to the SGs. Although both alpha and beta isoforms are proapoptotic, only Cyclin O&#945; can bind and activate Cdk2. On the other hand, we have demonstrated that Cyclin O is upregulated by Endoplasmic Reticulum (ER) stress and is necessary for ER stress-induced apoptosis. Cyclin O activates specifically the PERK pathway and interacts with the PERK inhibitor protein p58IPK. Moreover, Cyclin O participates in the activation of other eIF2&#945; kinases. We have also observed that a pool of Cyclin O is located in active mitochondria, suggesting a function of the protein linked to oxidative metabolism.Hemos identificado una nueva Ciclina, llamada Ciclina O, que es capaz de unirse y activar Cdk2 en respuesta a estímulos apoptóticos intrínsecos. Nos hemos centrado en el estudio de la Ciclina O&#945; y la Ciclina O&#946;, productos de splicing alternativo del gen. En respuesta a diferentes tipos de estrés, la Ciclina O&#945; se acumula en agregaciones citoplásmicas densas que podrían corresponder a Gránulos de Estrés (SGs). Además, hemos visto que la Ciclina O&#946; y un mutante puntual de la parte N-terminal de la proteína se localizan constitutivamente en los SGs. Aunque las dos isoformas alfa y beta son proapoptóticas, solo la Ciclina O&#945; es capaz de unirse y activar Cdk2. Por otro lado, hemos demostrado que los niveles de Ciclina O se incrementan en respuesta al estrés de Retículo Endoplásmico (RE) y que esta proteína es necesaria para la inducción de apoptosis dependiente de estrés de RE. La Ciclina O activa específicamente la vía de PERK e interacciona con la proteína inhibidora de PERK p58IPK. Además, la Ciclina O participa en la activación de otras quinasas de eIF2&#945;. La Ciclina O se localiza en mitocondrias activas, lo que sugiere una función de la proteína ligada al metabolismo oxidativo.

The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible. / Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą]

Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą] / The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible.

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.

Le rein natif et le greffon rénal peuvent être soumis à de multiples agressions conduisant à la détérioration progressive du parenchyme. Ces agressions peuvent être spécifiques (stress toxique, immunologique) et/ou non spécifiques (stress ischémique) et vont engendrer des réponses pouvant entraîner à la fois une diminution de la consommation d’énergie, une augmentation des apports afin de maintenir l’homéostasie tissulaire et la survie mais aussi une réaction inflammatoire et l’apoptose pouvant conduire à la fibrose. Parmi celles-ci, on peut nommer les voies HIF1α, mTOR, le stress du réticulum endoplasmique (RE), l’autophagie, l’activation de l’immunité innée et acquise. La réponse adaptative qui suit le stress du RE, la réponse UPR (Unfolded protein response), est une voie adaptative dont les implications sont actuellement encore peu connues dans le domaine de la pathologie rénale. Celle-ci se compose de trois effecteurs principaux : Perk, Ire1 et ATF6. A l’aide de deux modèles de stress toxique (ciclosporine) et ischémique (carence en glucose) sur deux modèles cellulaires distincts (cellulaires endothéliales artérielles et cellules tubulaires rénales), et dans des modèles in vivo, nous avons montré que le stress du RE était impliqué à la fois dans l’apparition de modifications phénotypiques endothéliales évocatrices de transition endothélio-mésenchymateuse induites par la ciclosporine et à la fois dans l’induction de réponses inflammatoire (régulation de NF-κB par Ire1) et angiogénique (régulation distincte de VEGF, bFGF et angiogénine par Perk et Ire1) induites par la carence en glucose. La réponse UPR semble modulée de façon subtile au cours de ces stress car les trois effecteurs n’engendrent pas des réponses identiques. Ces travaux apportent ainsi une meilleure compréhension des mécanismes d’adaptation au cours de stress variés, montrent que le stress du RE est impliqué dans ces réponses adaptatives et que la réponse peut être différente selon les effecteurs de la réponse UPR. Cette meilleure compréhension pourra permettre de valider des biomarqueurs précoces et des modulateurs de la réponse UPR afin de prévenir la dégradation du parenchyme rénal. / Native and grafted kidneys are stressed by multiple specific or non-specific insults leading to progressive structural deterioration. Responses to these insults are adaptive and preserve cell survival but may also promote inflammation, fibrosis and apoptosis. The most important of these adaptive pathways are HIF1α pathway, mTOR pathway, autophagy, unfolded protein response (UPR). The consequences of the UPR in kidney injuries are not well known. The objective of this study is to delineate the mechanisms and consequences of the activation of the UPR in response to toxic (cyclosporine) and ischemic (glucose starvation) stresses in two distinct cellular models (arterial endothelial cells and renal tubular cells). Here, we showed that UPR was engaged in cyclosporine-induced endothelial phenotypic changes, glucose starvation-induced inflammatory and angiogenic responses: NF-κB regulation by Ire1; distinct VEGF, bFGF and angiogenin regulation by Perk and Ire1. UPR is subtly modulated since its transducers do not induce identical processes. In conclusion these comprehensive works, we demonstrate the UPR is implicated in stress-induced adaptive pathways with different downstream responses according to the effector. Renal tissue degradation could be prevented by discovering and validating early biomarker and UPR modulators.

The endoplasmic reticulum is the primary organelle in the cell which has the responsibility of properly folding proteins belonging to the secretory pathway. Secretory proteins are essential for a variety of functions within the body like metabolism, growth and survival. Hence, proper folding of the proteins in the ER is absolutely essential to maintain cellular and body function. The environment of the ER is substantially different from that of the cytoplasm and is primed essentially to provide the optimum conditions to fold newly synthesized polypeptides following translation by the ribosomes in the cytoplasm and on the surface of the ER. In order for secretory proteins to fold properly, ER homeostasis must be maintained. ER homeostasis is defined by the dynamic balance between the ER protein load and the ER capacity to process this load. The optimum environment of the ER, or ER homeostasis, can be perturbed by pathological processes such as hypoxia, glucose deprivation, viral infections, environmental toxins, inflammatory cytokines, and mutant protein expression, as well as by physiological processes such as aging. Disruption of ER homeostasis causes accumulation of unfolded and misfolded proteins in the ER. This condition is referred to as ER stress. Cells cope with ER stress by activating the unfolded protein response (UPR). The UPR is initiated by three ER transmembrane proteins: Inositol requiring 1 (IRE1), PKR-like ER kinase, and activating transcription factor 6 (ATF6). These three master regulators sense and interpret protein folding conditions in the ER and translate this information across the ER membrane to activate downstream effectors, spliced XBP1, phosphorylated eIF2α and ATF4, and cleaved active ATF6 respectively. These effectors have two distinct outputs, homeostatic and apoptotic. Homeostatic outputs are adaptive responses that function to attenuate ER stress and restore ER homeostasis. These responses include the attenuation of protein translation to reduce ER workload and prevent further accumulation of unfolded proteins, upregulation of molecular chaperones and protein processing enzymes to enhance the ER folding activity, and the increase in ER-associated degradation (ERAD) components to promote clearance of unfolded proteins. When ER stress reaches a point where the cells cannot tolerate the load of unfolded proteins any more, apoptosis sets in. One of the major secretory proteins in mammals, vascular endothelial growth factor VEGF, is essential for either normal or pathological angiogenesis (blood vessel development). VEGFA is the primary member of this family which is expressed in all endothelial cells and is responsible for sprouting and invasion of blood vessels into the interstitium and thus helps in supplying nutrients and oxygen to growing cells. Recent studies have indicated that cells suffering from insufficient blood supply experience ER stress. The ER needs energy and oxygen for the folding process, thus nutrient deprivation (low ATP production) and hypoxia caused by insufficient blood supply leads to inefficient protein folding and ER stress in cells, especially in cancer cells that grow and spread rapidly. This condition also occurs in the development of the mammalian placenta. The placenta is an essential tissue characterized by a lot of blood vessels. It is responsible for the exchange of nutrients and growth factors between maternal and fetal blood vessels and hence is essential for survival of the embryo. Nutrient deprivation and hypoxia stimulate the production of VEGFA and other angiogenic factors, leading to protection against ischaemic injury in both cancer cells as well as the developing placenta. In this dissertation, we report that the three master regulators of the UPR, IRE1α, PERK and ATF6α, mediate transcriptional regulation of VEGFA under ER stress in cancer cells. Inactivation of any of the three master regulators leads to attenuation of VEGFA expression under ER stress. We show that IRE1α is able to regulate VEGFA through its downstream transcription factor XBP1 which activates the VEGFA promoter. IRE1α mediated VEGFA regulation is also essential for normal development of labyrinthine trophoblast cells in the placenta. ATF6α also regulates VEGFA via its promoter. PERK is able to activate VEGFA by preferential activation of its downstream effector, ATF4, which binds intron 1 of the VEGFA gene. Thus our work reveals a twopronged differential regulatory action of the UPR sensors on VEGFA gene expression. This work suggests that a fully active UPR is essential for VEGFA upregulation under ER stress. All three regulators are required in cancer cells for normal VEGFA expression. This tight regulation of VEGFA by the UPR presents a wonderful opportunity for therapeutic intervention into angiogenic growth of tumors.