Background: Vascular stiffening develops with both hypertension and aging, and is a strong predictor of end-organ damage. Excessive deposition of collagen by vascular smooth muscle cells (VSMCs) can lead to decreased compliance of vessels such as the aorta. The IRE1α arm of the unfolded protein response is activated in cells with a secretory phenotype due to its role in augmenting protein folding capacity. We hypothesize that by a similar mechanism, VSMCs transitioning to a collagen-secreting phenotype in response to TGF-β1 require the activation of IRE1. Inhibition of this pathway is hypothesized to reduce collagen secretion and hence prevent the development of fibrosis in the aorta. Methods: Collagen deposition by VSMCs in vitro was measured using immunoblotting and a Picrosirius Red-based colorimetric assay. Western blot and qRT-PCR were used to assess the expression of ER stress markers. Ex vivo culture of aortic rings was also performed to determine the effect of 4µ8c on TGF-β1-induced vascular stiffening. 12-14 week old male spontaneously hypertensive rats were divided into three treatment groups: 1) No treatment, 2) L-NAME (50 mg/L), and 3) L-NAME and the IRE1α inhibitor 4µ8c (2.5 mg/kg/day i.p.). Aortic compliance after 18 days of treatment was measured ex vivo using a wire myograph to construct tension-diameter curves. Results: Inhibition of IRE1α endonuclease activity by 4µ8c reduced collagen production in VSMCs stimulated with TGF-β1 or Ang II. A decrease in the expression of the collagen-associated chaperones PDI, GRP78 and GRP94 was observed. Aortic rings treated with TGF-β1 developed vascular stiffening, which was improved by co-treatment with 4µ8c. SHRs treated with L-NAME for 18 days developed aortic stiffening, which was prevented by daily injections of 4µ8c. Conclusions: Our data suggest that inhibition of the IRE1α pathway can reduce vascular stiffening and fibrosis by disrupting the collagen biosynthesis pathway in VSMCs. / Thesis / Master of Science (MSc)

No description available.

In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This triggers a series of signaling pathways known as the unfolded protein response (UPR). The response helps to both enhance protein folding capacity and initiate mis-folded protein degradation, reducing E.R. stress. Alternatively, misfolded proteins are degraded and nutrients are recycled through autophagy. Thus, E.R. homeostasis depends on both UPR and autophagy. However, if E.R. stress is not resolved, UPR and autophagy can also cause apoptosis by mechanisms that are not fully understood. In chicken embryo fibroblasts, gamma-aminobutyric acid receptor-associated protein or GABARAP (a protein involved in autophagy) can promote apoptosis in conditions of prolonged starvation (Maynard et al. 2015). In these conditions, the down-regulation of GABARAP by shRNA/RNA interference reduces the expression of the pro-apoptotic CHOP (CAAT-enhancer-binding protein homologous protein) transcription factor (a marker of E.R. stress) and enhances cell survival. This suggests that elevated levels of autophagy compromises E.R. homeostasis and promotes the expression of CHOP in UPR lethal pathways. While GABARAP induction and processing/activation has been linked to the expression of CHOP upon prolonged starvation (Maynard et al. 2015), nothing is known about the pathway mediating CHOP expression and the relationship with other pathways of the UPR in cells with GABARAP mis-expression. Understanding these pathways will allow us to determine if GABARAP is a general determinant of E.R. stress or acts specifically on the expression of CHOP to control cell survival. Elucidating mechanisms which are involved in E.R. stress and the cellular transition between pro-survival to pro-apoptotic roles can allow understanding of processes associated with several pathological conditions like cancer and neuro-degenerative diseases. Additionally, establishing a role for GABARAP tumor suppressor in the control of the UPR and cell fate is also important. / Thesis / Bachelor of Science (BSc) / In response to starvation, mis-folded proteins accumulate in the endoplasmic reticulum (E.R.) causing E.R. stress. This activates both the Unfolded Protein Response (UPR) and Autophagy as both processes help to reduce E.R. stress. GABARAP, a protein involved in autophagy, has been shown to be involved in the promotion of apoptosis in conditions of prolonged starvation as its downregulation reduces apoptosis and CHOP expression (Maynard et al. 2015). However, how GABARAP regulates apoptosis remains unknown. Here, we investigate if GABARAP mis-expression affects multiple pathways in the UPR relieving global E.R. stress or if its specifically involved in blocking CHOP expression.

Die Unfolded Protein Response (UPR) ist ein in Eukaryoten konservierter Signalweg, der durch Akkumulation von un-/fehlgefalteten Proteinen im endoplasmatischen Retikulum (ER) aktiviert wird um die Proteinhomöostase zu gewährleisten. In <i>Saccharomyces cerevisiae</i> wird die UPR durch den ER-Stresssensor Ire1p und den bZIP Transkriptionsfaktor Hac1p, oder XBP1 in höheren Eukaryoten, reguliert. In dieser Arbeit konnten die Homologe der zentralen UPR-Regulatoren im biotrophen Pilz <i>Ustilago maydis</i> charakterisiert und die UPR als essenzieller Koordinator der pathogenen Entwicklung identifiziert werden. Die Komplementation der <i>∆HAC1</i> Mutante durch <i>cib1</i> (Homolog von <i>HAC1</i>) und umfassende Expressionsanalysen zeigten, dass die Regulationsmechanismen der UPR in <i>U. maydis</i> weitestgehend konserviert sind, das Spektrum der regulierten Zielgene jedoch sekretierte Virulenzfaktoren beinhaltet die für die pathogene Entwicklung notwendig sind. So konnten durch <i>in silico</i> Vorhersage möglicher Cib1 Bindestellen (UPRE) mit pit1/pit2 und tin1-1 drei bereits charakterisierte Effektorgene als direkt regulierte UPR-Zielgene identifiziert werden. Die gezielte Deletion des vorhergesagten UPREs führt zu einer Aufhebung der ER-Stress induzierten und Cib1 abhängigen Expression von pit2 und verringert die Virulenz signifikant. Darüber hinaus konnte gezeigt werden, dass eine funktionelle UPR sowohl notwendig für eine verstärkte Expression wie auch für die korrekte Prozessierung des Pit2-Effektors innerhalb des ERs ist. Im Gegensatz zur Bäckerhefe <i>S. cerevisiae</i> und den filamentösen Ascomyceten <i>Aspergillus niger</i> und <i>Trichoderma reseei</i> kodiert die ungespleißte XBP1 mRNA in höheren Eukaryoten für einen negativen Regulator der UPR. Mit den vorliegenden Untersuchungen in <i>U. maydis</i> konnte erstmals für niedere Eukaryoten gezeigt werden, dass die ungespleißte cib1 mRNA für einen negativen Regulator kodiert, der darüber hinaus eine bislang unbeschriebene und vermutlich konservierte Funktion in der Antwort auf ER-Stress besitzt. Die genaue Kontrolle der UPR-Aktivität ist Voraussetzung für die korrekte Ausführung der verschiedenen Schritte innerhalb der pathogenen Entwicklung von <i>U. maydis</i>. Während eine vorzeitige UPR-Aktivierung zur Inhibition des zur Pflanzeninfektion notwendigen filamentösen Wachstums führt, ist die gezielte Aktivierung der UPR nach erfolgreicher Penetration der Pflanzenoberfläche und ihre andauernde Aktivität während des Wachstums <i>in planta</i> notwendig für die pathogene Entwicklung. Die direkte Interaktion zwischen Cib1 und dem Entwicklungsregulator Clp1 während dieser Entwicklungsphase führt zur Stabilisierung von Clp1 und der Modulation der Cib1 abhängigen Genexpression. Auf diese Weise wird die Proliferation <i>in planta</i> ermöglicht und eine erhöhte ER-Stressresistenz vermittelt. Zusammenfassend zeigen die gewonnenen Ergebnisse, dass die UPR in <i>U. maydis</i> als Kontrollpunkt dient, um die zelluläre Physiologie, den Entwicklungsverlauf und die Sekretion von Effektoren aufeinander abzustimmen.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically devastating pathogen affecting the global swine industry. Since the emergence of the virus in the late 1980s, vaccination strategies aimed to control the virus have not been very effective. Current commercial vaccines are generally protective against homologous or closely-related strains but ineffective at conferring heterologous protection against genetically-diverse strains of the virus. Consequently, emergence of variant and sometime more pathogenic strains of PRRSV continues in global swine herds. As such, there is a need for better understanding of the molecular mechanisms involved in the replication of the virus. In order to better understand the molecular mechanisms of host responses to PRRSV replication, we first sought to evaluate the ability of the virus to induce stress granules (SGs) during PRRSV infection. SGs are intracellular, cytoplasmic aggregates of RNA-binding proteins (RBPs) and mRNA. Formation of SGs is observed upon cellular stress and ultimately function to arrest cellular translation to promote cellular survival until the stress has been remedied. Indeed, several viruses have been shown to modulate the SG pathways to facilitate viral replication and even suppress the host's immune response. However, it is currently unknown whether PRRSV modulates the SG response. First, we used confocal microscopy and fluorescent in situ hybridization (FISH) to determine the distribution of known SG marker proteins and cellular mRNAs. Our findings revealed that PRRSV induces a potent SG response at late time points post-infection, and that SGs were closely associated with viral replication complexes (VRCs). Subsequently, we demonstrated that SGs are dispensable for viral replication, as short hairpin RNA (shRNA)-mediated knockdown of critical SG components (G3BP1 and G3BP2) did not affect viral replication. Interestingly, we found that the PRRSV-induced SGs are formed in a PERK-dependent manner. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Further investigation into the PERK signaling pathway revealed that PRRSV induces a significant amount of ER stress upon the cell during viral infection, and that exogenous stress significantly impaired the ability of the virus to replicate in MARC145 cells. We also showed that PRRSV potently induces all three signaling branches of the UPR, including PERK. While PERK knockdown had no effect on cell viability or viral replication, it significantly upregulated the mRNA expression of interferon-β and interferon stimulated genes (ISGs). The results from our studies suggest a critical role for PERK in regulating the host innate immune response to PRRSV infection. Only with a better understanding of the underlying molecular mechanisms of PRRSV replication will we be able to rationally design more effective vaccines against the virus. / Doctor of Philosophy / Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically-devastating disease in the global swine industry. Annually, PRRSV is estimated to cause more than $600 million in economic losses to the swine industry in the United States alone. Current commercial vaccines against the virus are not effective against the diverse field strains largely due to the extreme heterogeneity of the virus. PRRSV is also able to potently suppress several aspects of the host's immune response and therefore establish a persistent infection. The underlying mechanisms of PRRSV-mediated immune suppression are not well understood. Therefore, in this dissertation we decided to investigate the molecular mechanisms of host responses to PRRSV infection. We first investigated the ability of the virus to induce stress granules (SGs). SGs are important intracellular regulatory components that modulate many aspects of the host's cellular processes, and have even been shown to play roles in regulating viral replication and controlling immune responses to viral infection. We demonstrate that PRRSV not only induces SGs, but that the PRRSV-induced SGs are closely associated with viral replication complexes (VRCs) within infected cells. The PRRSV-induced SGs were dispensable for viral replication. PRRSV-induced SGs were previously shown to form in a PERK dependent manner. Therefore, in the second part of this dissertation research, we decided to investigate the PERK signaling pathway during PRRSV infection. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Our results showed that PRRSV potently induces ER stress and all three signaling branches of the UPR, including PERK. Furthermore, we revealed that PERK may play an important role in regulating the type I interferon response to PRRSV infection. The results from our studies will aid in understanding the underlying molecular mechanism of PRRSV replication which will help rationally design the next generation of more effective vaccines against this devastating swine pathogen.

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.