The failure to balance protein synthesis, folding, and degradation in the endoplasmic reticulum (ER) leads to the accumulation of unfolded proteins, leading to ER stress. Cells respond to this stress by activating a response signaling pathway known as the Unfolded Protein Response (UPR). One of the branches of the UPR induces the phosphorylation of eIF2α (Eukaryotic Initiation Factor 2) to attenuate global protein synthesis, allowing for a chance to clear misfolded and unfolded proteins. This phosphorylation of eIF2α is opposed by a phosphatase, containing a catalytic subunit, Protein Phosphatase 1, and a scaffolding protein, either GADD34 or CReP. Inhibition of eIF2α phosphatases has shown to promote survival in cell types by prolonging the effects of the UPR. This research focuses on understanding the gene expression patterns and localization of UPR specific genes with the presence of constant ER stress. Zebrafish are an ideal model for this research because they are a good mimic of what happens in humans and provide the ability to study gene expression and localization patterns at different stages during ER stress and its recovery. The eIF2α phosphatases were shown to have a protective effect on apoptosis when overexpressed in acute ER stress but were shown to have a protective effect on apoptosis when knocked out in chronic ER stress. We sought to determine the flow of gene expression of these phosphatases as well as other UPR specific genes, such as BiP and CHOP, to determine the contradictory effects of acute versus chronic ER-stress induced apoptosis. We studied the changes in gene expression for these genes in zebrafish embryos by isolating RNA and performing RT-qPCR after the induction of ER stress with pharmacological drugs across multiple time points. There was increased gene upregulation and mRNA localization to the fin epidermis and eye of GADD34, CReP, and BiP in acute ER stress from 2 hours to 6 hours, and these genes steadily declined in chronic ER stress from 24 hours to 48 hours. CHOP is a late-phase pro-apoptotic protein whose gene expression was upregulated in chronic ER stress from 12 hours to 48 hours. This data was also supported by mRNA localization studies performed by conducting whole mount in-situ hybridization on zebrafish embryos treated with ER stress inducers for 4 hours and 24 hours. Our results indicate that all UPR genes examined are affected by ER stress and their expression patterns are dependent on the time length of ER stress induction, allowing us to get a more in-depth working model of this branch of the UPR signaling pathway in zebrafish.

No description available.

Fibroproliferative disorders are the leading cause of morbidity and mortality worldwide, with one specific group of fibroproliferative disorders being interstitial lung diseases (ILD). Idiopathic pulmonary fibrosis is the most common ILD; however its pathogenesis is not entirely understood. What is known is that there is repetitive cellular injury preceding the fibrotic remodeling in the lungs that contributes to the irreversible deposition of extracellular matrix (ECM) proteins. Myofibroblasts that accumulate at the site of injury are thought to be the key drivers of ECM deposition and are often associated in the disease. Although it is poorly understood how these immune cells differentiate in the lung, one hypothesis suggests the role of alternatively activated profibrotic macrophages in this process. The data presented in this thesis suggest that there are a presence of UPR and macrophage proteins in the lungs of IPF patients and the UPR may be necessary in the polarization of alternatively activated macrophages. / Thesis / Master of Science (MSc)

No description available.

Les maladies à expansion de polyglutamine sont un ensemble de troubles neurodégénératives héréditaires se développant lorsqu’il y a répétitions de trinucléotides CAG dans les gènes causatifs au-delà d’un certain seuil. L’expansion des répétitions de trinucléotides CAG entraîne des désordres neurologiques héréditaires précoces, dont de multiples formes d’ataxie spinocérébelleuse (SCA). Parmi celles-ci, le type le plus commun et dominant est l’ataxie spinocérébelleuse de type 3 (SCA3), aussi connue sous le nom de la maladie de Machado-Joseph (MMJ). Ce dernier est un désordre neurologique progressif autosomique dominant. Le gène causatif de MMJ est ATXN3 (ATAXINE-3). Plusieurs études récentes suggèrent une association entre ce gène et la modulation du stress du réticulum endoplasmique (RE). Lors de ce travail de maîtrise, des souches transgéniques de C. elegans exprimant les formes sauvage et mutante du gène ATXN3 humain ont été générées. Les résultats suggèrent des phénotypes importants chez la souche transgénique mutante associés à la pathologie humaine: défaut de motilité, longévité réduite et profil neurodégénératif considérable. Ceci dit, ces résultats nous ont poussé à vouloir déterminer si l’utilisation des composés chimiques, connus en tant que modulateurs du stress du RE et possédant des rôles neuroprotecteurs, sont capables de restaurer les phénotypes notés. Les composés utilisés, c’est-à-dire le Bleu de Méthylène, le Salubrinal et le Guanabenz, ont démontré une capacité de corriger les phénotypes rapportés dans la souche transgénique mutante. De plus, ces composés ont aussi été en mesure de prévenir une augmentation du niveau du stress oxydatif et de la réponse au stress du RE exhibé chez les vers mutants. Par le développement de nouveaux modèles C. elegans pour la MMJ, où il y a expression du gène ATXN3 complet dans les motoneurones, il a été possible de trouver qu’une modulation chimique du stress du RE peut réduire considérablement la neurodégénérescence et par conséquent, être une possible nouvelle approche thérapeutique pour traiter cette pathologie. / Polyglutamine expansion diseases are a class of dominantly inherited neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders such as multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited spinocerebellar ataxia is the type 3 (SCA3) also known as Machado-Joseph disease (MJD), an autosomal dominant, progressive neurological disorder. The gene causing MJD is ATXN3 (ATAXIN-3): MJD is caused by an abnormal CAG trinucleotide repeat expansion in the ATXN3 gene. Several recent studies have shown that this gene is associated with endoplasmic reticulum (ER) stress. In this study, we generated transgenic C. elegans strains expressing wild type or mutant human ATXN3 genes and tested them for recovery of locomotor phenotype, lifespan and neurodegeneration phenotypes upon treatment with compounds known to modulate ER stress and having neuroprotective roles. We observed differences between both transgenic lines and found that the motility defects, the reduced lifespan and the neurodegeneration can be rescued by methylene blue, guanabenz and salubrinal. These compounds were also able to prevent the oxidative stress and the ER stress response induced by mutant transgenic worms. We introduce novel C. elegans models for MJD based on the expression of full-length ATXN3 in GABAergic motor neurons. Using these models we discovered that chemical modulation of the ER unfolded protein response reduced neurodegeneration and could be a new therapeutic approach for the treatment of MJD.

Plusieurs expériences et études cliniques ont démontré que l’activation du système rénine-angiotensine (RAS) peut induire l’hypertension, un facteur de risque majeur pour les maladies cardiovasculaires et rénales. L’angiotensinogène (Agt) est l’unique substrat du RAS. Cependant, il n’a pas encore été démontré si l’activation du RAS intrarénal peut à elle seule induire des dommages rénaux, indépendamment de l’hypertension systémique, et ainsi jouer un rôle prépondérant dans la progression de la néphropathie diabétique. Afin d’explorer le rôle du RAS intrarénal dans les dommages rénaux, un diabète a été induit par l’injection de streptozotocin chez des souris transgéniques (Tg) surexprimant l’Agt de rat dans les cellules des tubules proximaux du rein (RPTC). Les souris Tg diabétiques ont été traitées soit avec des inhibiteurs du RAS (perindopril et losartan), de l’insuline ou une combinaison des deux pour 4 semaines avant d’être euthanasiées. Pour une autre étude, des souris Tg non-diabétiques ont été traitées soit avec des inhibiteurs du RAS, l’hydralazine (vasodilatateur) ou l’apocynine (inhibiteur de la NADPH oxydase) pour une période de 8 semaines avant l’euthanasie. Des souris non-Tg ont été utilisées comme contrôles. Des cellules immortalisées de tubule proximal de rat (IRPTC) transfectées de manière stable avec un plasmide contenant l’Agt ou un plasmide contrôle ont été employées comme modèle in vitro. Nos résultats ont démontré que les souris Tg présentaient une augmentation significative de la pression systolique, l’albuminurie, l’apoptose des RPTC et l’expression de gènes pro-apoptotiques par rapport aux souris non-Tg. Les mêmes changements ont été observés chez les souris Tg diabétiques par rapport aux souris non-Tg diabétiques. L’insuline et/ou les inhibiteurs du RAS ont permis d’atténuer ces changements, sauf l’hypertension qui n’était réduite que par les inhibiteurs du RAS. Chez les IRPTC transfectées avec l’Agt in vitro, les hautes concentrations de glucose augmentent l’apoptose et l’activité de la caspase-3 par rapport aux cellules contrôles et l’insuline et/ou les inhibiteurs du RAS empêchent ces augmentations. En plus des changements physiologiques, les RPTC des souris Tg présentent aussi une augmentation significative de la production des espèces réactive de l’oxygène (ROS) et de l’activité de la NADPH oxydase, ainsi qu’une augmentation de l’expression du facteur de croissance transformant-beta 1 (TGF-β1), de l’inhibiteur activateur du plasminogène de type 1 (PAI-1), des protéines de la matrice extracellulaire, du collagène de type IV et de la sousunité p47 de la NADPH oxydase. Le traitement des souris Tg avec l’apocynine et le perindopril a permis d’améliorer tous ces changements, sauf l’hypertension qui n’était pas corrigée par l’apocynine. D’autre part, l’hydralazine a prévenu l’hypertension, sans modifier l’albuminurie, l’apoptose des RPTC ou l’expression des gènes pro-apoptotiques. Ces résultats montrent bien que l’activation du RAS intrarénal et l’hyperglycémie agissent de concert pour induire l’albuminurie et l’apoptose des RPTC, indépendamment de l’hypertension systémique. La génération des ROS via l’activation de la NADPH oxydase induit en partie l’action du RAS intrarénal sur l’apoptose des RPTC, la fibrose tubulo-interstitielle et l’albuminurie chez les souris Tg. D’autre part, une expérience en cours a tenté d’encore mieux délimiter les effets de l’activation du RAS intrarénal, tout en éliminant la néphrotoxicité du STZ. Pour cette étude, les souris Tg surexprimant l’Agt de rat dans leurs RPTC ont été croisées aux souris Ins2Akita, un modèle spontané de diabète de type I, afin de générer des souris Akita-rAgt-Tg. Les résultats préliminaires indiquent que le RAS intrarénal est activé dans les souris Akita et que la combinaison avec l’hyperglycémie induit du stress du réticulum endoplasmique (ER) dans les RPTC in vivo. Le stress du ER contribue à l’apoptose des RPTC observée dans le diabète, à tout le moins dans le modèle Akita. Le traitement avec des inhibiteurs du RAS permet d’atténuer certains des dommanges rénaux observés dans les souris Akita-rAgt-Tg. / Experimental and clinical studies have shown that renin-angiotensin system (RAS)activation may lead to hypertension, a major cardiovascular and renal risk factor. Angiotensinogen (Agt) is the sole substrate of the RAS. However, it is unclear whether intrarenal RAS activation alone could induce kidney injury independently of systemic hypertension and play an important role in the progression of diabetic nephropathy (DN). To explore the role of intrarenal RAS in kidney injury, transgenic (Tg) mice overexpressing rat Agt in their renal proximal tubular cells (RPTCs) were rendered diabetic by streptozotocin (STZ). Diabetic Tg mice were treated with RAS blockers (perindopril and losartan), insulin or a combination of both and then euthanized after 4 weeks of treatment. In a separate study, non-diabetic Tg mice were treated with RAS blockers or hydralazine (a vasodilator) or apocynin (an NADPH oxidase inhibitor) and then euthanized after 8 weeks of treatment. Non-Tg littermates served as controls in both studies. Immortalized rat proximal tubule cells (IRPTCs) stably transfected with Agt cDNA or control plasmid were used in the experiments as an in vitro model. Our results showed that non-diabetic Tg mice displayed a significant increase in systolic blood pressure (SBP), albuminuria, RPTC apoptosis, and proapoptotic gene expression. Diabetic Tg mice had a further increase of albuminuria, RPTC apoptosis, and proapoptotic gene expression, though the SBP of the diabetic Tg mice was similar to that of non-diabetic Tg mice. RAS blockers and/or insulin treatments markedly attenuated these changes, except that insulin had no impact on hypertension. In vitro, high-glucose melieu significantly increased apoptosis and caspase-3 activity in Agt stable transfectants compared to control cells, and these changes were attenuated by insulin and/or RAS blockers. Furthermore, non-diabetic Tg mice showed significantly elevated reactive oxygen species (ROS) production and NADPH oxidase activity, as well as enhanced expression of transforming growth factor-beta 1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), extracellular matrix proteins, collagen type IV, and NADPH oxidase subunit p47 in their RPTC. Treatment with apocynin and perindopril ameliorated these changes, but apocynin had no effect on SBP. In contrast, hydralazine prevented hypertension but not albuminuria, RPTC apoptosis, or proapoptotic gene expression. These data indicate that intrarenal RAS activation and hyperglycemia act in concert to induce albuminuria and RPTC apoptosis independent of systemic hypertension. ROS generation via NADPH oxidase activation mediates, at least in part, intrarenal RAS action on RPTC apoptosis, tubulointerstitial fibrosis and albuminuria in Tg mice. On the other hand, in an on-going experiment, to avoid the nephro-toxic effects of STZ and further delineate the effects of intrarenal RAS activation, Tg mice overexpressing rat Agt in their RPTCs were crossbred with Ins2Akita mice, a spontaneous type I diabetes model, to generate Akita-rAgt-Tg mice. Preliminary data indicated that hyperglycaemia and intrarenal RAS activation induced endoplasmic reticulum (ER) stress in RPTC in vivo, and the ER stress pathway contributed to RPTC apoptosis in diabetes, at least in the Akita model. RAS blockade was effective in attenuating some parameters of renal injury in AkitarAgt-Tg mice.

No description available.

<p><b>Background and aims:</b> Type 2 diabetes mellitus is connected with elevated glucose levels, which cause impaired glucose-stimulated insulin secretion (GSIS) and degeneration of β-cells. Mechanisms for such glucotoxic effects were explored in the present study.</p><p><b>Materials and methods:</b> INS-1E cells were cultured for 5 days in 5.5, 11, 20 or 27 mM glucose in the presence or absence of AMPK-agonist AICAR. GSIS was determined from INS-1E cells and islets obtained from type 2 diabetes and control donors. Human islets and INS-1E cells were functionally characterized (GSIS) and protein profiled (SELDI-TOF MS). Glucose-induced <i>de novo</i> synthesis of fatty acyls (HR-MAS NMR spectroscopy), fatty acid composition (GC-MS), triglyceride content and specific proteins (Western blotting) were determined in INS-1E cells.</p><p><b>Results:</b> Impaired GSIS was observed from INS-1E cells exposed to chronic hyperglycaemia and islets isolated from type 2 diabetics compared to INS-1E cells cultured at normal glucose levels and control islets, respectively. Several glucose-regulated proteins were found when type 2 diabetes and control islets or mitochondria from INS-1E cells cultured at different glucose concentrations were protein profiled. Glucose induced lipid <i>de novo</i> synthesis of both saturated and unsaturated fatty acids in specific proportions. Glucose-induced impairment of function and mass was reverted by inclusion of AICAR, which lowered levels of pro-apoptotic protein CHOP but left triglyceride content unaffected.</p><p><b>Conclusions:</b> Impaired GSIS and increased apoptosis observed in β-cells after prolonged exposure to elevated glucose concentrations involved accumulation of lipid species in specific proportions, AMPK-inactivation, ER-stress activation and complex, coordinated changes in expression patterns of mitochondrial and human islet proteins.</p>

<b>Background and aims:</b> Type 2 diabetes mellitus is connected with elevated glucose levels, which cause impaired glucose-stimulated insulin secretion (GSIS) and degeneration of β-cells. Mechanisms for such glucotoxic effects were explored in the present study. <b>Materials and methods:</b> INS-1E cells were cultured for 5 days in 5.5, 11, 20 or 27 mM glucose in the presence or absence of AMPK-agonist AICAR. GSIS was determined from INS-1E cells and islets obtained from type 2 diabetes and control donors. Human islets and INS-1E cells were functionally characterized (GSIS) and protein profiled (SELDI-TOF MS). Glucose-induced de novo synthesis of fatty acyls (HR-MAS NMR spectroscopy), fatty acid composition (GC-MS), triglyceride content and specific proteins (Western blotting) were determined in INS-1E cells. <b>Results:</b> Impaired GSIS was observed from INS-1E cells exposed to chronic hyperglycaemia and islets isolated from type 2 diabetics compared to INS-1E cells cultured at normal glucose levels and control islets, respectively. Several glucose-regulated proteins were found when type 2 diabetes and control islets or mitochondria from INS-1E cells cultured at different glucose concentrations were protein profiled. Glucose induced lipid de novo synthesis of both saturated and unsaturated fatty acids in specific proportions. Glucose-induced impairment of function and mass was reverted by inclusion of AICAR, which lowered levels of pro-apoptotic protein CHOP but left triglyceride content unaffected. <b>Conclusions:</b> Impaired GSIS and increased apoptosis observed in β-cells after prolonged exposure to elevated glucose concentrations involved accumulation of lipid species in specific proportions, AMPK-inactivation, ER-stress activation and complex, coordinated changes in expression patterns of mitochondrial and human islet proteins.

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.