Cancer cells survive the harsh oxygen and nutrient deprivation of the tumour microenvironment through the selection of apoptosis-resistant and glycolytic clones (Cairns et al., 2011; Graeber et al., 1996). In particular, the integrated stress response (ISR) has been shown to be pivotal in cancer cell survival in vivo and the resistance of cancer cells to therapy (Harding et al., 2003). In recent years, it has become apparent that increased autophagy is one mechanism by which the ISR can confer resistance to stress (Kroemer et al., 2010). ATF4 is a major transcriptional effector of the integrated stress response in severe hypoxia (<0.01% O₂). ATF4 is a well-established regulator of genes involved in oxidative stress, amino acid synthesis and uptake, lipid metabolism, protein folding, metastasis, and angiogenesis. Recent work has demonstrated an important role of ATF4 in promoting resistance to severe hypoxia through the transcriptional upregulation of MAP1LC3B and ATG5, essential components of the autophagy machinery (Rouschop et al., 2009b; Rzyski et al., 2010). In this work, the author describes several novel ATF4 target genes, and examines their role in the regulation of autophagy and the resistance of cancer cells to severe hypoxia. In the first part of this thesis, the author shows that three BH3-only members of the BCL-2 family of proteins--HRK, PUMA, and NOXA--are upregulated in response to severe hypoxia in an ATF4-dependent manner. In particular, the author shows that the poorly described BH3-only protein HRK is a direct target of transcriptional activation by ATF4, and that HRK induces autophagy in severe hypoxia, thereby providing the first evidence that the integrated stress response can transcriptionally trigger the autophagy process. In contrast to the previously described role of HRK in apoptosis, this thesis demonstrates that HRK can play a pro-survival role in the context of breast cancer cells. In the latter part of this thesis, the author identifies the essential autophagy gene ULK1 as an ISR target. The author shows that ULK1 expression in severe hypoxia is transcriptionally upregulated through direct activation by ATF4. The author identifies ULK1 as a crucial regulator of autophagy and mitophagy in both normoxia and severe hypoxia and shows that ULK1 plays a pivotal role in cancer cell survival. Furthermore, it is shown that human breast cancer patients with high levels of ULK1 relapse earlier than those with low levels of ULK1, thereby identifying ULK1 as a potential target for cancer therapy.

On solid surfaces wild strains of Saccharomyces cerevisiae form biofilm-like, structured colonies enabling them to survive long-term in hostile environments in the wild. However, the molecular mechanisms underlying the spatio-temporal development of colonies and their resistance to hostile conditions are still largely unknown. In this study, we analyzed the effect of the HAC1 gene on the colony morphology of wild strains of S. cerevisiae. The transcription factor Hac1p activates the unfolded protein response (UPR), which leads to activation of the expression of genes encoding components of the protein secretory pathway, and genes involved in stress responses in the endoplasmic reticulum (ER). The impact of HAC1 deletion is significant even under non-stress conditions and causes a radical reduction of structured colony architecture in hac1∆ strains derived from two wild S. cerevisiae strains (PORT and BR-F-Flo11p-GFP) and one laboratory ΣSh strain forming semi-fluffy or fluffy colonies. The hac1∆ strains exhibit a decreased vegetative growth rate, reduced cell attachment to the agar and an ineffective cell-cell adhesion resulting in decreased flocculation. The hac1∆ strains derived from BR-F-Flo11p-GFP contain a low level of Flo11p surface adhesin which is considered very important for the proper...

La protéostase cellulaire représente l'ensemble des mécanismes régulant la production, le repliement, le transport et la dégradation des protéines dans la cellule afin de maintenir son homéostasie. La protéostase cellulaire est fréquemment altérée dans les cellules tumorales, pouvant induire une accumulation de protéines mal repliées. En réponse à cette accumulation, la cellule met en place une réponse physiologique adaptative appelée "Unfolded Protein Response" (UPR). Dans la 1ère partie de l'étude nous avons montré que le sorafénib, i.e. le traitement de référence du carcinome hépatocellulaire (CHC) avancé, altérait la protéostase tumorale et inhibait l'initiation de la traduction des protéines. Nous avons cherché des outils permettant de mesurer l'altération de la protéostase tumorale chez les patients en s'intéressant à la régulation des marqueurs tumoraux sériques par la protéostase cellulaire. Dans la deuxième partie de l'étude, nous avons exploré un potentiel rôle de l'UPR dans la tumorigénèse des carcinomes à cellules rénales (RCC) post-transplantation. L'incidence des RCC est largement augmentée chez les patients transplantés en comparaison à la population générale. Bien que la carcinogénèse du RCC soit multifactorielle, la prise chronique de traitements immunosuppresseurs tels que la ciclosporine (CsA) semble impliquée dans ce processus. Nous avons montré in vitro que la CsA pouvait altérer la protéostase tumorale et induire l'UPR. Cette induction semble liée à l'agressivité des RCC dans ce contexte / Cellular proteostasis is the process regulating the production, folding, trafficking and degradation of proteins within the cell in order to maintain its homeostasis. Cellular proteostasis is frequently altered in tumor cells, leading to an accumulation of unfolded proteins. In response to this accumulation, the cell activates an adaptive physiological response called "Unfolded Protein Response" (UPR). In the first part of the study we showed that sorafenib, i.e. the standard of care for advanced hepatocellular carcinoma (HCC), altered tumor proteostasis and inhibited the initiation of protein translation. We looked for tools to measure the alteration of tumor proteostasis in patients by focusing on the regulation of serum tumor markers by cellular proteostasis. In the second part of the study, we explored a potential role of UPR in tumorigenesis of post-transplant renal cell carcinoma (RCC). The incidence of RCC is greatly increased in transplant patients compared to the general population. Although carcinogenesis of RCC is multifactorial, chronic intake of immunosuppressive drugs such as ciclosporin (CsA) appears to be involved in this process. We showed in vitro that CsA alters tumor proteostasis and induce UPR. This induction seemed linked to the aggressiveness of the RCC in this context

The unfolded protein response (UPR) is activated by protein misfolding stress in the endoplasmic reticulum (ER). The UPR is a transcriptional program that aims to maintain ER folding capacity, where imbalances between protein load and processing ability is termed ER stress. Signal transduction of the UPR begins with 3 ER-resident transmembrane sensors: PERK, IRE1 and ATF6. All sensors initiate downstream signaling cascades which culminate in improved protein folding, transcriptional upregulation of genes encoding ER chaperones, and mechanisms to reduce translational and transcriptional ER load, therefore re-establishing ER homeostasis. The signaling cascades of each sensor are distinct but cooperative, and involve a significant amount of crosstalk, feedback and overlap. Indeed, there are many pathological and physiological conditions have an effect on ER protein burden, and therefore on activation of the UPR. Increases in protein load in professional secretory cells, hypoxic conditions in a tumor mass, obesity all induce cause changes in the ER folding environment. Although we understand how the UPR contributes to relieve ER stress under acute conditions (e.g. pharmacological treatment) much less is understood about the contributions to physiological processes and chronic stress conditions. Our overall goal was to understand how the UPR is activated during physiological settings, the mechanisms it uses to maintain folding capacity under these setting and the specific components responsible for adapting the response to various stresses. We first decided to understand a chronic stress from a transgenic approach. By creating a knockout mouse, the genetic deletion functions as a stress and we can understand its physiological role. By compounding two genetic deletions in UPR components (ATF6α and p58IPK) we provide evidence for the developmental role these components play. Homozygous deletion ATF6α bears no gross histological phenotype yet causes synthetic lethality when combined with p58IPK deletion. This also reveals that the UPR is able to adapt to genetic impairment of protein folding in vivo. Next, to better understand these chronic states, we established an experimentally tractable chronic stress treatment in vivo. Our treatment suppressed ATF6α dependent chaperone expression through an mRNA degradative mechanism, which led to long term changes in UPR expression. We determined that chronic conditions can change the sensitivity of the UPR to ER stress, potentially as an adaptive consequence. We also showed that sensitivity to ER stress can be changed during chronic stress. Finally we simulated the UPR in a computational ordinary differential equation (ODE) model in order to determine how various stresses and component interactions determine the output of the UPR. We built a series of equations to describe the UPR signaling network, entrained it on experimental data and refined it through the use of transgenic knockout cells. Our model was robust enough to recreate experimental measurements of UPR components when tested in parallel with knockout cells. We found that stress sensitivity is dependent on the crosstalk and negative feedback connections of the UPR. This study has enhanced our understanding of activation of the UPR under non-acute settings. It demonstrates that the UPR is a signaling hub with a broad output range that is capable of handling a variable degree of insults because of the intrinsic properties of the signaling network. This provides a better understanding for the contributions of the UPR to physiological stresses and certain chronic diseases.

Fetal alcohol spectrum disorders (FASD) are caused by alcohol exposure during pregnancy and is the leading cause of mental retardation. Alcohol exposure during development results in the loss of neurons in the developing brain. The underlying molecular mechanisms are unclear and there currently is no cure for FASD. Ethanol-induced neuronal death is accompanied by neuroinflammation. Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. Using a third trimester equivalent mouse model of ethanol exposure, we found that treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3β, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3β. Further, we determined whether knocking out of MCP-1/CCR2 ameliorates neonatal alcohol exposure-induced long-lasting behavioral deficits in adolescent and adult mice. C57BL/6 and MCP-1-/-/CCR2-/- mice were exposed to alcohol (5 g/kg) by subcutaneously injection on PD4. A series of behavioral tests including Open Field (PD 35-36 and PD 70-71), Rotor-Rod (PD 38 and PD 73), Balance Beam (PD 40 and PD75) and Morris Water Maze (PD 42 and PD77) were performed in the adolescence and adulthood. We found that MCP-1-/-/CCR2-/- mice were resistant to neonatal alcohol exposure-induced deficits in motor function in the Rotor-Rod and Balance Beam tests; MCP-1 and CCR2 deficiency also protected mice against neonatal ethanol exposure induced long lasting deficits in learning and memory in the Morris Water Maze testing. Collectively, these results suggest that MCP-1/CCR2 signaling plays an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain and also plays an important role in developmental alcohol exposure induced long-lasting behavioral deficits in adolescence and adulthood.

Equine Herpesvirus type 1 (EHV-1) is a worldwide threat to the health of horses. It can cause mild respiratory disease, abortions and deaths of newborn foals as well as a potentially fatal neurologic disorder known as Equine Herpesvirus Myeloencephalopathy (EHM). The virus is maintained in populations by stress-induced periodic reactivation of virus in long-term latently infected horses and transmission of the reactivated virus to susceptible individuals. In horses, peripheral blood leukocytes (PBLs) are thought to be an important site for EHV-1 latent genomes. Since the Unfolded Protein Response (UPR) is a cellular response to a variety of stressors that has been linked to reactivation of herpes simplex virus in humans, a virus closely related to EHV-1, I tested the hypothesis that latent EHV-1 relies on the UPR as a pluripotent stress sensor and uses it to reactivate lytic gene expression. Since little work has been done in defining the UPR in horses, I first successfully developed a quantitative real-time polymerase chain reaction (RT-qPCR) assay to detect and quantitate transcripts for selected UPR genes in equine dermal (E.Derm) cells and PBLs. Activation of the UPR was achieved in both cell types using thapsigargin and a difference in gene expression after activation of the UPR in two equine cell types was found. A nested PCR assay to detect and distinguish latent EHV-1 and EHV-4 was evaluated and the sensitivity of the technique to detect EHV-1 was determined. I discovered that the nested PCR technique was not sensitive enough to detect the estimated one latent viral genome in 50,000 PBLs. Lytic EHV-1 infection was characterized by single step growth curve in E.Derm cells and consistent detection of temporal EHV-1 gene expression by RT-qPCR was achieved. The relationship between EHV-1 gene expression and UPR gene expression during lytic infection was investigated. While EHV-1 infection had no effect on UPR gene expression, activation of the UPR appeared to decrease the expression of EHV-1 genes temporarily and reversibly during the first 4 h after infection. Finally, detection of EHV-1 in PBLs from horses presumed to be latently infected by co-cultivation with E. Derm cells permissive to EHV-1 infection was attempted. To detect viral DNA, PBLs were stimulated with thapsigargin or interleukin 2 (IL-2) which was previously reported to induce reactivation of latent EHV-1. I was not able to reproduce previously published experiments of reactivation in vitro of latent EHV-1 by stimulation with IL-2, and virus reactivation did not occur after stimulation of PBLs with thapsigargin. In summary, a RT-qPCR assay to measure the expression of equine UPR genes was developed and activation of the UPR by treatment of E.Derm cells and PBLs with thapsigargin was successfully achieved. A difference in gene expression after activation of the UPR in two equine cell types was found. In contrast to what has been reported for other alphaherpesviruses, there appears to be no, or only little, interaction between the UPR and EHV-1 during viral infection. Detection of latent EHV-1 genomes in PBLs was not achieved by using a nested PCR, as this technique was not sensitive enough to detect the estimated one latent viral genome in 50,000 PBLs. Finally, latent EHV-1 was not detected in presumed latently infected PBLs or reactivated by triggering the UPR in equine PBLs.

Luman (CREB3, LZIP) is a basic leucine zipper transcription factor involved in regulation of the unfolded protein response (UPR), dendritic cell maturation, and cell migration. But despite reported expression in primary sensory neurons, little is known about its role in the nervous system. Luman mRNA from rat sensory neurons was amplified and its coding sequence was determined. The rat Luman cDNA contains a full-length open reading frame encoding 387 amino acids, and the recombinant protein generated from this clone activated transcription from UPR elements. Quantitative RT-PCR revealed rat Luman transcripts in a variety of rat tissues with the highest levels in nervous system tissue. In situ hybridization confirmed the findings and demonstrated that the Luman mRNA hybridization signal localizes to neurons and satellite glial cells in dorsal root ganglia (DRG), the cytoplasm of hepatocytes in liver, and the hippocampal pyramidal cell layers in CA1 and CA3 and the granular cell layer of the dentate gyrus. Luman protein localizes with axonal endoplasmic reticulum (ER) components along the axon length within the sciatic nerve and is activated by sciatic nerve injury. Adult sensory axons also contain Luman mRNA which is translated within the axon and transported to the cell body via the importin-mediated retrograde transport system in response to nerve injury. Further, creation of an N-terminal, C-terminal dual fluorescence-tagged Luman adenoviral construct allowed visualization of the cleavage and retrograde translocation of the N-terminal portion of Luman to the nucleus in real time in vivo and in vitro. Neuronal or subcellular axonal knockdown of Luman significantly impaired the intrinsic ability of injury-conditioned, but not naïve, sensory neurons to extend the regeneration-associated elongating form of neurites. Sciatic nerve crush injury also induced activation of the UPR in axotomized DRGs, including genes linked to cholesterol biosynthesis. Knockdown of Luman decreased the activation of UPR and cholesterol biosynthesis, and axotomy-inducted increases in neurite outgrowth, which could be largely rescued with either mild UPR inducer treatment or cholesterol supplementation. Together these findings provide novel insights linking remote injury-associated axonal ER responses to the regenerative growth capacity of adult sensory neurons via axonal activation and synthesis of Luman and reveal a role for the UPR in regulation of axotomy-induced neurite outgrowth that is critically dependent on Luman.

Abiotic stress is highly detrimental to crop productivity worldwide. Research is key to meeting the challenges of modern agriculture in a sustainable and positive fashion. This thesis contributes to our understanding of plant stress responses by examining two molecular aspects of abiotic stress. The first part of the work focused on the Unfolded Protein Response (UPR), a general stress response mechanism triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. The study was conducted with the model plant Arabidopsis and shed new light on key players in the pathway. An unconventional alternative splicing mechanism, similar to the one identified in other higher eukaryotes, was found to parallel the activation of an ER-resident chaperone. The data suggest that this event is important to alleviate cellular stress in response to adverse environmental conditions such as heat. Further understanding of this pathway will help to develop a more complete view of the mechanisms involved in this response. The second part of the work investigated the interaction between nitrogen limitation and drought at the transcriptional level. A genome-wide transcript profiling experiment was performed to provide a comprehensive view of the response to nitrogen and water limitation in corn. The main finding was the demonstration of a clear synergistic effect between both stresses, an effect that was unexpectedly as important as either stress applied alone. This study adds to our current knowledge of abiotic stress response in plants and should provide the groundwork necessary to build future strategies for crop enhancement.

Endoplasmic reticulum (ER) stress has been implicated as a causative factor in the development of pancreatic beta-cell dysfunction and death resulting in type 2 diabetes. This thesis examined the role of ATF6beta in the ER stress response of beta-cells. Using an ATF6beta-specific antibody, expression of full-length ATF6beta was detected in various insulinoma cell lines and rodent islets and the induction of the active form (ATF6beta-p60) under ER stress conditions. Knock-down of ATF6beta in INS-1 832/13 cells did not affect mRNA induction of known ER stress response genes in response to tunicamycin-induced ER stress, however it increased the susceptibility of beta-cells to apoptosis. Conversely, overexpression of ATF6beta-p60 reduced the apoptotic phenotype. Microarray results suggest ATF6beta functions to induce expression of adaptive genes also regulated by ATF6alpha, but also several specific targets genes. These findings have increased our understanding of the role of ATF6beta in the ER stress response of beta-cells.

Endoplasmic reticulum (ER) stress has been implicated as a causative factor in the development of pancreatic beta-cell dysfunction and death resulting in type 2 diabetes. This thesis examined the role of ATF6beta in the ER stress response of beta-cells. Using an ATF6beta-specific antibody, expression of full-length ATF6beta was detected in various insulinoma cell lines and rodent islets and the induction of the active form (ATF6beta-p60) under ER stress conditions. Knock-down of ATF6beta in INS-1 832/13 cells did not affect mRNA induction of known ER stress response genes in response to tunicamycin-induced ER stress, however it increased the susceptibility of beta-cells to apoptosis. Conversely, overexpression of ATF6beta-p60 reduced the apoptotic phenotype. Microarray results suggest ATF6beta functions to induce expression of adaptive genes also regulated by ATF6alpha, but also several specific targets genes. These findings have increased our understanding of the role of ATF6beta in the ER stress response of beta-cells.