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1
Kraupner, Nicolas. "Conception d'outils pharmacologiques pour comprendre le rôle de l'Insulin Degrading Enzyme (IDE) dans la gestion du stress protéotoxique." Electronic Thesis or Diss., Université de Lille (2022-....), 2022. http://www.theses.fr/2022ULILS039.
Abstract:
L'Insulin Degrading Enzyme (IDE) est une métalloprotéase à zinc ubiquitaire retrouvée dans les compartiments extracellulaires et intracellulaires. IDE est impliquée dans la dégradation de peptides physiologiquement importants tels que l'insuline et d'autres peptides amyloïdogènes. Cependant, elle est remarquablement conservée dans les espèces et les tissus ne produisant pas ces peptides substrats. Cette observation suggère un rôle important d'IDE, non complétement identifié, et pas seulement corrélé à son activité catalytique. IDE est une enzyme pour laquelle on continue de découvrir de nouvelles implications biologiques et sa caractérisation est nécessaire pour mieux comprendre ces nouveaux rôles physiologiques ou pathologiques. En particulier, ces dernières années des études ont mis en évidence le lien entre IDE et le stress du réticulum endoplasmique (ER) notamment dans la voie ubiquitine-protéasome et la voie de l'Unfolded Protein Response (UPR). L'unité a récemment breveté l'utilisation d'une première série d'inhibiteurs d'IDE dont le chef de file est le BDM 44768 pour booster des cytotoxiques, notamment les inhibiteurs du protéasome tels que le carfilzomib, un des traitements de référence du myélome multiple.Basé sur cette série chimique du BDM 44768 et guidé par la structure cristallographique de nos composés dans IDE, plusieurs modulations ont été réalisées sur quatre différentes parties du pharmacophore ainsi que l'élaboration d'une série macrocyclique. Ces pharmacomodulations ont permis l'obtention de plusieurs molécules puissantes avec une activité de l'ordre du nanomolaire et possédant des propriétés pharmacocinétiques qui pourraient permettre leurs utilisations dans des modèles in vivo.Dans le but d'explorer les fonctions et l'implication d'IDE dans différents processus cellulaires cette thèse a également permis la conception et la synthèse de différents outils chimiques d'exploration. Premièrement, suite aux différents modes de liaison de nos molécules dans IDE, deux séries de sondes PROTAC ont été synthétisées. Leurs évaluations biologiques ont révélé qu'elles n'induisent pas la dégradation d'IDE mais de deux autres protéines, une protéine homologue à IDE, la pitrilysine, ainsi que la DPP3, une dipeptidyl peptidase. Pour terminer, deux sondes fluorescentes, qui restent à être optimisées, ont été conçues et synthétisées afin de pouvoir suivre la localisation d'IDE au sein de la cellule et plus particulièrement dans le réticulum endoplasmique dans le but de corréler cette localisation au cours du temps avec les effets sur les protéines de l'UPR et le stress réticulaire.Ainsi, lors de cette thèse de précieux résultats pour concevoir de futurs outils chimiques permettant l'étude d'IDE et l'élucidation des différents rôles de cette protéine ont été obtenus. De plus, plusieurs petites molécules puissantes, modulant l'activité d'IDE, ont été synthétisées dans le but de répondre aux différents besoins thérapeutiques associés à cette cibleInsulin Degrading Enzyme (IDE) is a ubiquitous zinc metalloprotease found in extracellular and intracellular compartments. IDE is involved in the degradation of physiologically important peptides such as insulin and other amyloidogenic peptides. However, it is remarkably conserved in species and tissues that do not produce these substrate peptides. This observation suggests an important role for IDE, not fully identified, and not only correlated with its catalytic activity. IDE is an enzyme for which new biological implications continue to be discovered and its characterization is necessary to better understand these new physiological or pathological roles. In particular, in the last few years studies have highlighted the link between IDE and endoplasmic reticulum (ER) stress, notably in the ubiquitin-proteasome pathway and the Unfolded Protein Response (UPR) pathway.The unit has recently patented the use of a first series of IDE inhibitors, with the BDM 44768 as lead compound, to boost cytotoxics, including proteasome inhibitors such as carfilzomib, one of the gold standard treatments for multiple myeloma.Based on this chemical series of BDM 44768 and guided by the crystallographic structure of our compounds in IDE, several modulations were performed on four different parts of the pharmacophore as well as the development of a macrocyclic series. These pharmacomodulations resulted in several potent molecules with nanomolar activity and pharmacokinetic properties that could allow their use in in vivo models.In order to explore the functions and involvement of IDEs in different cellular processes, this thesis also allowed the design and synthesis of different chemical exploration tools. First, following the different binding modes of our molecules in IDE, two series of PROTAC probes were synthesized. Their biological evaluations revealed that they do not induce the degradation of IDE but of two other proteins, a protein homologous to IDE, pitrilysin, and DPP3, a dipeptidyl peptidase. Finally, two fluorescent probes, that still need to be optimized, were designed and synthesized in order to be able to follow the localization of IDE within the cell and more particularly in the endoplasmic reticulum with the aim of correlating this localization over time with the effects on the UPR proteins and reticular stress.Thus, during this thesis, valuable results to design future chemical tools to study IDE and to elucidate the different roles of this protein were obtained. In addition, several potent small molecules modulating the activity of IDE were synthesized in order to address the different therapeutic needs associated with this target
2
Johnson, Charlotte. "Targeting endoplasmic reticulum stress and autophagy in cancer." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/84379/.
Abstract:
Mammalian/mechanistic target of mTOR complex 1 (mTORC1) regulates multiple cellular processes, including de novo protein synthesis, autophagy and apoptosis. mTORC1 overactivation occurs in a range of cancers and benign tumour dispositions as a result of mutations which increase mitogenic stimulus or cause malfunction of the tuberous sclerosis complex, the prime regulator of mTORC1 activity. mTORC1 overactivation results in elevated endoplasmic reticulum (ER) stress which, at low levels, elicits a pro-survival response. However, prolonged or excessive ER stress causes cell death. The present study utilised clinically relevant drug combinations to simultaneously enhance levels of ER stress and inhibit compensatory survival pathways in in vitro models of mTORC1 overactivity in order to cause non-genotoxic cell death. The main drugs used in this study were nelfinavir, an ER stress-inducer, chloroquine, an autophagy inhibitor, and bortezomib, a proteasome inhibitor. The key findings of this study include identification of drug combinations nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib to induce significant and selective cell death in mTORC1-driven cells, as measured by flow cytometry with DRAQ7 staining and western blot analysis for cleavage of apoptotic markers. Cell death is likely mediated through ER stress signalling, as shown by increased ER stress markers at both the level of mRNA and protein. Of interest, this study found cell death as a result of combined treatment with nelfinavir was not dependent on proteasome inhibition by nelfinavir, or autophagy inhibition by chloroquine. Additionally, nelfinavir-chloroquine-mediated cell death was completely rescued by inhibition of the vacuolar ATPase by bafilomycin-A1. In conclusion, mTORC1 overactive cells have higher basal levels of ER stress which can be manipulated with drug treatment beyond a survivable threshold, whereas cells capable of reducing mTORC1 signalling are able to survive. This study ascertained a combination of nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib, to cause effective cytotoxicity in mTORC1-driven cells.
3
Chan, Cheuk-wing Wilson. "ER stress in the pathogenesis of osteochondrodysplasia." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085192.
4
Zachariah, Matshediso. "High selenium induces endothelial dysfunction via endoplasmic reticulum stress." Thesis, University of Surrey, 2017. http://epubs.surrey.ac.uk/845246/.
Abstract:
Selenium (Se) is associated with insulin resistance and may affect endothelial function thereby increasing the risk of type 2 diabetes and associated cardiovascular disease (CVD). However, the molecular mechanisms involved are not clear. The endoplasmic-reticulum (ER) stress response is a mechanism involved in apoptosis induced by high Se in some cancer cells and, also in the pathogenesis of insulin resistance and endothelial dysfunction (ED). Thus, we hypothesised that high Se status causes ED through ER stress response. Endothelial cells (HUVECs) and EA.hy926 cell lines were treated with selenite (0.5-10 µM) for 24 hours in the presence or absence of the ER chemical chaperone, 4-phenylbutryic acid (PBA). ER stress markers were investigated using qPCR and western-blot technique. Endothelial function was assessed by the Griess assay, flow cytometry, Matrigel® and colourimetric assays. Data were expressed as S.E.M (p < 0.05) vs. control. High Se concentration (5-10 µM) compared to physiological concentration (0.5–2.0 µM) enhanced mRNA expression of ER-stress markers:- activating transcription factor-4 (ATF4), CAAA/enhanced-binding homologous protein (CHOP) and X-binding box-1 (XBP-1). In addition, high selenite concentration reduced nitric oxide production and angiogenic capacity in endothelial cells. Moreover, high selenite treatment significantly (p < 0.05) increased production of reactive oxygen species (ROS) and induced apoptosis through caspase-3/7 activity. Interestingly, PBA completely reversed all the effects of high selenite on endothelial function, indicating the involvement of the ER-stress response. High Se treatment caused endothelial dysfunction through the activation of the ER-stress response. This thesis additionally warns the public to be aware of the risks of the use of Se supplements as a prophylactic agent against oxidative-stress disease.
5
Voyias, Philip D. "Regulation of endoplasmic reticulum stress in adipose tissue metabolism." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/74256/.
Abstract:
Obesity is the most significant risk factor for developing type II diabetes mellitus (T2DM). Obesity induces adipocyte endoplasmic reticulum (ER) stress, prior to onset of insulin resistance. A pathological inability of white adipose tissue (WAT) to expand to accommodate excess energy is predominantly due to impaired adipogenesis. The research hypothesis was that ER stress in human WAT is important in inducing WAT dysfunction and subsequent insulin resistance and T2DM. The aims of this study were to elucidate interactions of ER stress in human WAT and to characterise the role of ER stress in human adipogenesis. Abdominal SAT biopsies and anthropometry were collected from T2DM subjects before and after bariatric surgery and non-diabetic subjects. Preadipocytes were extracted from human WAT and differentiated into adipocytes. Lipogenesis, lipolysis, glucose uptake, insulin sensitivity, and ER stress and adipogenesis gene and protein expression were assessed in control cells and with ER stress inducers, inhibitors or siRNA. The results of this study found both restrictive and malabsorptive bariatric interventions are effective weight loss interventions for obese T2DM patients and result in significantly improved glucose and insulin levels six months after surgery. WAT health is better following restrictive procedures as shown by lower and better regulation of ER stress markers. Adipogenesis in primary human preadipocytes is influenced by adiposity and WAT depot and the IRE1-XBP1s UPR is essential in human adipogenesis. XBP1s plays a vital role upstream of CEBP and PPAR in human adipogenesis and it is necessary for mediating the action of insulin. Wnt10b plays an inhibitory role in human adipogenesis and acts independently of XBP1s. Collectively these findings suggest that WAT function is key for metabolic health and can be impaired by ER stress; however regulated adipogenesis may serve to improve WAT function and therefore improve metabolic health.
6
Furmanik, Malgorzata. "The role of endoplasmic reticulum stress in vascular calcification." Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/the-role-of-endoplasmic-reticulum-stress-in-vascular-calcification(a0138614-e3d8-42ef-9cbf-02a01f6e6eaf).html.
Abstract:
Vascular calcification (VC) is a health problem common in ageing populations, diabetes and chronic kidney disease. It leads to vascular stiffening and heart failure. VC is a regulated process mediated by vascular smooth muscle cells (VSMCs), with similarities to developmental osteogenesis. The exact molecular events responsible for triggering it are unknown. The endoplasmic reticulum (ER) is involved in folding of proteins. ER stress occurs as a result of unfolded protein accumulation and has been implicated in osteoblast differentiation and bone mineralization. Therefore, I hypothesized that ER stress signalling regulates osteogenic differentiation and calcification of VSMCs. I showed that calcification of human aortas was associated with changes in ER stress marker expression. Warfarin and TNFα, which are both established inducers of vascular calcification, increased expression of ER stress markers in VSMCs. ER stress modelled in human primary VSMCs in vitro increased their calcification and was shown to modulate expression of a number of bone related genes, such as BMP-2, Runx2, Osterix, ALP, BSP and OPG in VSMCs in vitro. I also demonstrated that ER stress activated features characteristic of a secretory phenotype in VSMCs, such as downregulation of SMC markers and components of TGFβ signalling related to contractile differentiation, as well as BMP-2. Taken together these results suggested that ER stress can induce changes that lead to osteogenic differentiation. To further explore the relationship between ER stress and osteogenic differentiation of VSMCs Osterix and ALP were studied in more detail. ALP activity was upregulated by ER stress, but did not change when VSMCs calcified. Promoter analysis showed that ALP might be regulated by ER stress via indirect mechanisms and potential regulators of ALP transcription were identified using proteomic analysis.
7
Darling, Nicola Jane. "Regulation of ER stress-induced cell death by the ERK1/2 signalling pathway." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708709.
8
Preston, Amanda Miriam Clinical School St Vincent's Hospital Faculty of Medicine UNSW. "The role of endoplasmic reticulum stress in beta-cell lipoapoptosis." Publisher:University of New South Wales. Clinical School - St Vincent's Hospital, 2008. http://handle.unsw.edu.au/1959.4/41231.
Abstract:
Beta-cell failure is a key step in the progression from metabolic disorder to overt type 2 diabetes (T2D). This failure is characterised by both secretory defects and loss of beta-cell mass, the latter most likely through increases in the rate of apoptosis. Although the mechanisms underlying these beta-cell defects are unclear, evidence suggests that chronic exposure of beta-cells to elevated fatty acid (FA) plays a role in disease development in genetically susceptible individuals. Furthermore, it has been postulated that endoplasmic reticulum (ER) stress signalling pathways (the unfolded protein response; UPR) play a role in FA-induced beta-cell dysfunction. The broad aim of this thesis was to explore the nature of these relationships. Experiments detailed in this thesis demonstrate that MIN6 beta-cells mount a comprehensive ER stress response with exposure to elevated saturated fatty acid palmitate, but not the unsaturated fatty acid, oleate, within the low elevated physiological range. This response was time-dependent and involved both transcriptional and translational changes in UPR transducers and targets. The differential activation of ER stress in MIN6 beta-cells by saturated, but not unsaturated FA species may represent a mechanism of differential beta-cell death described in many studies with these FA. Furthermore, these experiments describe defects in ER to Golgi trafficking with chronic palmitate treatment, but not oleate or thapsigagin treatment, identifying this as a potential mechanism by which palmitate treatment induces ER stress. Moreover, these studies have shown the relevance to ER stress to a whole body model of T2D by demonstrating UPR activation in the islets of the db/db mouse. In conclusion, studies detailed in this thesis have demonstrated that ER stress occurs in in vitro and in vivo models of beta-cell lipotoxicity and apoptosis. In addition, these studies have identified defects in ER to Golgi trafficking as a mechanism by which palmitate treatment induces ER stress. These studies highlight the importance of ER stress in the development of T2D.
9
Katsoulieris, Elias. "Oxidatives and Endoplasmic Reticulum Stress in Kidney Priximal Tubule Cells." Thesis, University of Brighton, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506517.
10
Niederreiter, Lukas. "Endoplasmic reticulum (ER) stress transcription factor Xbp1 in intestinal tumourigenesis." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708846.
11
Gaifem, Joana Filipa Madureira. "" Role Of In Endoplasmic Reticulum Stress Response In Sacccharoromyces cerevisiae "." Master's thesis, Instituto de Ciências Biomédicas Abel Salazar, 2011. http://hdl.handle.net/10216/56926.
12
Bonilla, Myriam. "Endoplasmic reticulum stress linked to calcium signaling in saccharomyces cerevisiae." Available to US Hopkins community, 2003. http://wwwlib.umi.com/dissertations/dlnow/3080627.
13
Das, Indrajit. "Therapeutic Targeting of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease." Thesis, Griffith University, 2012. http://hdl.handle.net/10072/365999.
Abstract:
Endoplasmic reticulum (ER) stress occurs when proteins misfold during biosynthesis in the ER. ER stress in intestinal secretory cells has been implicated in the aetiology of inflammatory bowel diseases (IBD) and intestinal inflammation in mice. Intestinal secretory cells are susceptible to ER stress due to high rates of protein synthesis, and ER stress in these cells results in reduced production of cell surface and secreted proteins leading to thinner mucus with a lower anti-microbial content, allowing penetration by luminal microbes, leading to inflammation. Cells experiencing ER stress attempt to restore homeostasis via the unfolded protein response (UPR), which enables the cells to increase the protein folding capacity of the ER. The primary focus of this thesis is to examine whether the drugs that are efficacious in IBD treatment modulate goblet cell function and ER homeostasis, and whether drugs that modulate ER stress can suppress intestinal inflammation and restore intestinal homeostasis. In order to explore the ER stress-inflammation nexus I utilized well established IBD anti-inflammatory agents, 5-Aminosalicylate (5-ASA), 6-thioguanine (6-TG), the anti-TNF antibody, infliximab, and the glucocorticoid dexamethasone (DEX). Chemical chaperones (TUDCA and sodium 4-PBA) and UPR modulators (guanabenz, salubrinal and 4μ8C) were used to investigate how modulation of ER stress affects goblet cell function and intestinal inflammation. In vivo studies were carried out in Winnie mice, which have ER stress in goblet cells due to a Muc2 misfolding mutation resulting in colitis involving both innate and adaptive immunity. To understand the direct effect of these drugs on ER stress in goblet cells in the absence of confounding inflammatory factors, in vitro experiments were performed using the human colonic adenocarcinoma LS174T cell line, a cell line containing cells with goblet cell differentiation, where ER stress was induced either by inhibition of N-glycosylation by tunicamycin or by depletion of Ca2+ by thapsigargin.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text
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Gendrisch, Fabian [Verfasser], and Stefan F. [Akademischer Betreuer] Martin. "The role of endoplasmic reticulum stress responses in contact dermatitis." Freiburg : Universität, 2018. http://d-nb.info/1211956318/34.
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Martin, Rachel E. "Targeted sensors to monitor oxidative stress in the endoplasmic reticulum." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5884/.
Abstract:
Hydrogen peroxide has a diverse array of functions in cells. Not only as a mediator of oxidative stress, it is also involved in signalling in many pathways including tyrosine phosphorylation, sumoylation, proliferation and differentiation and cysteine oxidation [1]. There are a number of different producers of hydrogen peroxide in the cell including the NADPH oxidase (Nox) family of enzymes [2], the electron transport chain in the mitochondria [3] and disulfide bond formation in the endoplasmic reticulum (ER) [4]. In order to prevent a toxic build-up of hydrogen peroxide the cell also deploys a range of antioxidant enzymes including catalase, the glutathione peroxidases (GPxs) and the peroxiredoxins (Prxs) [5]. Therefore at all times a balance must be maintained to allow enough hydrogen peroxide production to allow for signalling to occur but at the same time ensuring the concentration does not increase enough to cause any oxidative damage. Oxidative damage is associated with aging in general and diseases including cancer, cardiovascular disorders and neurodegenerative diseases. For such diseases to occur the balance of hydrogen peroxide production and removal must be tipped and it is the elucidation of these processes with regards to the ER that we hope to achieve. We present the development and use of a novel sensor, BGB, which specifically detects hydrogen peroxide in the ER. BGB works in conjunction with the SNAPtag system to allow specific targeting of a small molecule probe to the ER. BGB is selective for hydrogen peroxide over other reactive oxygen species due to the use of a boronic acid group as the reactive moiety. We have developed MALDITOF mass spectrometry methods for quantitative analysis of this probe when used both and . Also included is the development of Pep-B, a probe which was designed to measure hydrogen peroxide in the ER, but using a peptide targeting system rather than SNAP-tag, an investigation into the synthesis of a fluorophore based probe and the development of an alternative method of analysis of BGB using an antibody based system which negates the requirement of MALDI-TOF.
16
Gaifem, Joana Filipa Madureira. "" Role Of In Endoplasmic Reticulum Stress Response In Sacccharoromyces cerevisiae "." Dissertação, Instituto de Ciências Biomédicas Abel Salazar, 2011. http://hdl.handle.net/10216/56926.
17
Wang, Yongchao. "THE ROLE OF ENDOPLASMIC RETICULUM STRESS IN ETHANOL-INDUCED NEURODEGENERATION." UKnowledge, 2019. https://uknowledge.uky.edu/pharmacol_etds/33.
Abstract:
Heavy ethanol use causes neurodegeneration manifested by neuronal loss and dysfunction. It is becoming imperative to delineate the underlying mechanism to promote the treatment of ethanol-induced neurodegeneration. Endoplasmic reticulum (ER) stress is a hallmark and an underlying mechanism of many neurodegenerative diseases. This study aims to investigate the role of ER stress in ethanol-induced neurodegeneration. In experimental design, adult mice were exposed to binge ethanol drinking by daily gavage for 1, 5, or 10 days and the response of ER stress was examined. We found the induction of ER stress appeared at 5 days and remained at 10 days. Moreover, the induction of ER stress was accompanied by an increase in neurodegeneration. With cell culture, we demonstrated that ethanol exposure resulted in neuronal apoptosis and that blocking ER stress by sodium phenylbutyrate (4-PBA) abolished ethanol-induced neuronal apoptosis, suggesting that ER stress contributes to ethanol-induced neurodegeneration.
Mesencephalic astrocyte-derived neurotrophic factor (MANF) responds to ER stress and has been identified as a protein upregulated in ethanol-exposed developmental mouse brains. To investigate its implication in ethanol-induced neurodegeneration, we established a central nervous system (CNS)-specific Manf knockout mouse model and examined the effects of MANF deficiency on ethanol-induced neuronal apoptosis and ER stress using a third-trimester equivalent mouse model. We found MANF deficiency worsened ethanol-induced neuronal apoptosis and ER stress and that blocking ER stress abrogated the harmful effects of MANF deficiency on ethanol-induced neuronal apoptosis. Moreover, a whole transcriptome RNA sequencing supported the involvement of MANF in ER stress modulation and revealed candidates that may mediate the ER stress-buffering capacity of MANF. Collectively, these data suggest that MANF is neuroprotective against ethanol-induced neurodegeneration via ameliorating ER stress.
Because MANF is a neurotrophic factor, we also examined the effects of MANF deficiency on neurogenesis. We observed that MANF deficiency increased neurogenesis in the subgranular zone of the hippocampal dentate gyrus and subventricular zone of the lateral ventricles in the mouse brain. Mechanistically, this finding was supported by a decrease of cell cycle inhibitors (p15 and p27), an increase of G2/M marker (phospho-histone H3), and an increase of neural progenitor markers (Sox2 and NeuroD1) in the brain of conditional Manf knockout mice. Our in vitro studies demonstrated that the gain-of-function of MANF inhibited cell cycle progression, whereas the loss-of-function of MANF promoted cell cycle progression. Taken together, these data suggest that MANF may affect the process of neurogenesis through altering cell cycle progression.
18
Kerbiriou, Mathieu. "Étude du stress du reticulum endoplasmique (RE) dans la mucoviscidose." Brest, 2008. http://www.theses.fr/2008BRES3204.
Abstract:
La mucoviscidose est une maladie génétique causée par des mutations dans le gène codant pour le canal chlorure CFTR (cystic fibrosis transmenbrane conductance regulator). La plus fréquente de ces mutations est la delF508 qui entraîne l’expression d’une protéine mal formée qui est retenue dans le RE. Or, l’accumulation de protéines mal formées dans le RE provoque un stress et le déclenchement d’une réponse appelée UFR (unfolded protein response) qui induit entre autres la baisse de la synthèse protéique et l’augmentation des capacités de dégradation des protéines. De plus, l’UPR est activée par des facteurs exogènes présents dans la mucoviscidose comme l’inflammation et les infections. Ainsi, nous avons voulu savoir si le CFTR delF508 pouvait induire un stress du KB et déclencher I’UPR. Dans la première partie de cette thèse, nous montrons pour la première fois que l’UPR est déclenchée dans des cellules exprimant le CFTR delF508. De plus, nous avons observé que l’inhibition sélective d’un composant de I’UPR (ATF6) permet une restauration partielle de l’activité du CFTR delF5O8. L’UPR peut également mener à l’apoptose lors d’un stress du RE prolongé ou trop intense. Or, le déclenchement de l’apoptose est altéré dans la mucoviscidose. Ainsi, nous avons comparé l’apoptose induite par un stress du RE entre des cellules exprimant le CFTR sauvage et muté delF5OS (CF, cystic fibrosis). Dans la seconde partie de cette thèse, nous montrons que la voie apoptotique impliquant le calcium, la m-calpaïne, la caspase 12 et la caspase 3 est altérée dans les cellules CF. L’UPR est impliquée dans la physiopathologie de la mucoviscidose et sa régulation est une cible thérapeutique potentielleCystic fibrosis is a genetic disease caused by mutations in the CFTR (cystic fibrosis transmenbrane conductance regulator) chloride channel gene. The most common of these mutations is the deletion of the phenylalanine at position 508 (delF508) of the protein which leads to the expression of a misfiled protein which is retained in the endoplasmic reticulum (ER). Now, the accumulation of misfiled proteins in the ER induces a stress and the triggering of an adaptative response called UPR (unfolded protein response) which induces among other things the decrease of protein synthesis and the augmentation of proteins degradation capacities. Moreover, UPR can be induced by exogenous factors which are present in cystic fibrosis (CF) such as inflammation and infections. Thus, we wanted to know whether delF508-CFTR could induce a stress and trigger 13FR. Hi the first part of this thesis, we show for the first time that UPR is triggered in delF508-CFTR expressing cells. Moreover, we have observed that the selective inhibition of a component of UFR (ATF6, activating transcription factor 6) allows a partial restauration of the delF508-CFTR channel activity. UFR can also land to apoptosis during a prolonged or too intense ER stress. Now, apoptosis is altered in CF. Thus, we compared ER stress-induced apoptosis between Wt (Wild type) and CF cells. In the second part of this thesis, we show that the apoptotic cascade involving calcium, m-calpain, caspase 12 and caspase 3 is altered in delF508-CFTR expressing cells, suggesting its implication in CF physiopathology. These results show that UPR is involved in CF physiopathology and that its regulation is a potential therapeutic target
19
Mihai, Adina Daniela. "Obesity-related factors involved in endoplasmic reticulum stress induction in adipocytes." Thesis, Durham University, 2015. http://etheses.dur.ac.uk/11637/.
Abstract:
Obesity is the most common nutritional disorder in the developed world and represents a major risk factor for associated diseases like type 2 diabetes mellitus, cardiovascular diseases and hypertension. The condition affects the whole body homeostasis but mainly the adipose tissue and is characterised by low grade inflammation, insulin resistance and hyperlipidemia. In adipocytes, it has been associated with endoplasmic reticulum stress (ER stress) induction and activation of the unfolded protein response (UPR). ER stress has been shown to play a central role in the molecular events leading to inflammation and insulin resistance in obese adipocytes, but the physiological triggers of ER stress are still unknown. The aim of my thesis was to investigate the role of obesity-related factors such as high concentrations of saturated fatty acids, cholesterol, proinflammatory cytokines or tissue remodelling-induced hypoxia and glucose starvation in ER stress induction My results indicate for the first time that glucose starvation and hypoxia, two markers of adipose tissue remodelling in obesity, represent physiological triggers of ER stress in in vitro differentiated 3T3-F442A and 3T3-L1 adipocytes. High concentrations of saturated fatty acid palmitic acid, cholesterol or proinflammatory cytokines (TNFα, IL-6 and IL-1β), although shown to be potent inducers in other cell lines, do not induce ER stress in my model of in vitro differentiated adipocytes. In conclusion, my results suggest that the adipose tissue remodelling process in obesity could play a central role in ER stress induction in adipocytes.
20
Park, Soon Hyang. "The role of endoplasmic reticulum stress signaling in isolated islet apoptosis." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32257.
Abstract:
A major obstacle to islet transplantation is β-cell death following isolation. Isolation exposes islets to various stresses including endoplasmic reticulum (ER) stress inducers; therefore, the role of ER stress signaling in isolated islet apoptosis was investigated. Activation of eIF2α and JNK1 and XBP1 splicing followed by an increase in caspase-3 activity were observed in isolated human islets. Since the absence of protein-tyrosine phosphatase 1B (PTP1B) was previously shown to reduce ER stress-induced signaling and apoptosis in fibroblasts, the role of PTP1B in ER stress signaling was investigated in β-cells. While encouraging data emerged, using an inhibitor and miRNA targeting PTP1B, a conclusive link between PTP1B inhibition and improved β-cell survival has not yet been seen. This study provides the first evidence that ER stress signaling may influence isolated islet apoptosis and could point to novel therapeutic approaches in islet transplantation.Un obstacle majeur lors de la transplantation des îlots pancréatiques est la perte des cellules β lors de la procédure d'isolation. En effet, lors de ce processus, les îlots sont exposés à divers stress cellulaires incluant ceux qui induisent un stress au niveau du réticulum endoplasmique (RE). Cette étude porte donc sur la signalisation menant à l'apoptose en réponse au stress du RE sur les îlots isolés. L'activation d'eIF2α, de JNK1 et de l'épissage de XBP1 qui est suivi par une augmentation de l'activité de la caspase-3 fut observées sur des îlots isolés chez l'humain. L'absence de la protéine tyrosine phosphatase 1B (PTP1B) avait précédemment été démontrée comme pouvant contribuer à la diminution de la signalisation déclenchée par le stress du RE et l'apoptose chez les fibroblastes. Malgré des résultats encourageants concernant l'utilisation d'un inhibiteur et d'un miRNA qui ciblent PTP1B, un lien concluant entre l'inhibition de cette enzyme et l'amélioration de la survie des cellules β n'a pas été observé Cette étude fournit la première évidence qui clarifie le rôle de la signalisation induite par le stress du RE lors de l'apoptose des îlots pancréatiques. De plus, elle pourrait résulter en une nouvelle approche thérapeutique pour augmenter la survie des cellules β lors de la transplantation d'îlots.
21
Hühn, Martin [Verfasser]. "Endoplasmic Reticulum (ER)-stress signalling in the alveolar epithelium / Martin Hühn." Gießen : Universitätsbibliothek, 2013. http://d-nb.info/1065395310/34.
22
Mahmood, Ahsan. "Role of SLMAP in Endoplasmic Reticulum Stress and Unfolded Protein Response." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/24399.
Abstract:
Cardiac function is regulated by the molecular components of the sarco/endoplasmic reticulum (ER/SR). Disruptions in homeostatic balance of these proteins and calcium regulation results in activation of ER stress response. Sarcolemmal membrane-associated proteins (SLMAPs) are found in cell membrane, SR/ER, and mitochondria. Overexpression of SLMAP in the myocardium has shown to impair excitation-contraction (E-C) coupling in the transgenic (Tg) mice. ER stress response was examined in Tg mice overexpressing SLMAP in the myocardium. In Tg hearts, changes observed in the expression of proteins involved in ER stress were dependent on the age and sex. SLMAP overexpression results in maladaptive ER stress response, as the mice age. Neonatal cardiomyocytes isolated from the Tg hearts showed decreased viability, upregulation of ER stress response proteins, which were sensitized to thapsigargin-induced stress, and desensitized to palmitate-induced oxidative stress. These findings suggest that normal SLMAP levels are important for proper cardiac function, and cell viability.
23
Nakano, Kenzo. "Chloroquine induces apoptosis in pancreatic neuroendocrine neoplasms via endoplasmic reticulum stress." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263541.
24
Brown, Max Adam. "Investigation of how endoplasmic reticulum stress causes insulin resistance and neuroinflammation." Thesis, Durham University, 2015. http://etheses.dur.ac.uk/11438/.
Abstract:
Endoplasmic reticulum (ER) stress is caused by the accumulation of mis/unfolded proteins in the ER. ER stress signalling pathways termed the unfolded protein response are employed to alleviate ER stress through increasing the folding capacity and decreasing the folding demand of the ER as well as removing mis/unfolded proteins. However, ER stress signalling pathways induce diverse cellular changes beyond changes to the ER. This study aims to further investigate some of these ER stress-mediated events. ER stress can cause activation of JNK. Prolonged ER stress-mediated JNK activation is reported to promote apoptosis whilst both acute and long-lasting JNK activation is proposed to cause insulin resistance. To begin with it is reported in this thesis that acute ER stress-induced JNK activation, which is dependent on IRE1α and TRAF2, promotes survival. In contrast to other studies, this thesis provides evidence that acute ER stress-mediated JNK activation does not inhibit insulin signalling during ER stress in several cell lines. However, prolonged ER stress, in four different cell lines, does inhibit insulin signalling in a JNK independent manner. This study argues that ER-stress-induced insulin resistance during prolonged ER stress involves inhibition of trafficking of newly synthesised insulin receptors through the secretory pathway to the plasma membrane. Finally ER stress can activate inflammatory signalling pathways other than JNK and thus ER stress may promote inflammation. Neuroinflammation and ER stress are reported in Parkinson’s disease (PD) yet a link between them has so far not been investigated. Using a cellular model of PD, it is reported in this thesis that ER stress has the potential to activate neuroinflammation in PD.
25
Fonseca, Sonya G. "Role of WFS1 in Regulating Endoplasmic Reticulum Stress Signaling: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/414.
Abstract:
The endoplasmic reticulum (ER) is a multi-functional cellular compartment that functions in protein folding, lipid biosynthesis, and calcium homeostasis. Perturbations to ER function lead to the dysregulation of ER homeostasis, causing the accumulation of unfolded and misfolded proteins in the cell. This is a state of ER stress. ER stress elicits a cytoprotective, adaptive signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can moderate stress, cells can produce the proper amount of proteins and maintain a state of homeostasis. If the UPR, however, is dysfunctional and fails to achieve this, cells will undergo apoptosis.
Diabetes mellitus is a group of metabolic disorders characterized by persistent high blood glucose levels. The pathogenesis of this disease involves pancreatic β-cell dysfunction: an abnormality in the primary function of the β-cell, insulin production and secretion. Activation of the UPR is critical to pancreatic β-cell survival, where a disruption in ER stress signaling can lead to cell death and consequently diabetes. There are several models of ER stress leading to diabetes. Wolcott-Rallison syndrome, for example, occurs when there is a mutation in the gene encoding one of the master regulators of the UPR, PKR-like ER kinase (PERK).
In this dissertation, we show that Wolfram Syndrome 1 (WFS1), an ER transmembrane protein, is a component of the UPR and is a downstream target of two of the master regulators of the UPR, Inositol Requiring 1 (IRE1) and PERK. WFS1 mutations lead to Wolfram syndrome, a non-autoimmune form of type 1 diabetes accompanied by optical atrophy and other neurological disorders. It has been shown that patients develop diabetes due to the selective loss of their pancreatic β-cells. Here we define the underlying molecular mechanism of β-cell loss in Wolfram syndrome, and link this cell loss to ER stress and a dysfunction in a component of the UPR, WFS1. We show that WFS1 expression is localized to the β-cell of the pancreas, it is upregulated during insulin secretion and ER stress, and its inactivation leads to chronic ER stress and apoptosis.
This dissertation also reveals the previously unknown function of WFS1 in the UPR. Positive regulation of the UPR has been extensively studied, however, the precise mechanisms of negative regulation of this signaling pathway have not. Here we report that WFS1 regulates a key transcription factor of the UPR, activating transcription factor 6 (ATF6), through the ubiquitin-proteasome pathway. WFS1 expression decreases expression levels of ATF6 target genes and represses ATF6-mediated activation of the ER stress response (ERSE) promoter. WFS1 recruits and stabilizes an E3 ubiquitin ligase, HMG-CoA reductase degradation protein 1 (HRD1), on the ER membrane. The WFS1-HRD1 complex recruits ATF6 to the proteasome and enhances its ubiquitination and proteasome-mediated degradation, leading to suppression of the UPR under non-stress conditions. In response to ER stress, ATF6 is released from WFS1 and activates the UPR to mitigate ER stress.
This body of work reveals a novel role for WFS1 in the UPR, and a novel mechanism for regulating ER stress signaling. These findings also indicate that hyperactivation of the UPR can lead to cellular dysfunction and death. This supports the notion that tight regulation of ER stress signaling is crucial to cell survival. This unanticipated role of WFS1 for a feedback loop of the UPR is relevant to diseases caused by chronic hyperactivation of ER stress signaling network such as pancreatic β-cell death in diabetes and neurodegeneration.
26
Tan, Zhijia, and 谭志佳. "Molecular analyses of chondrocyte differentiation and adaptation to ER stress." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/209435.
Abstract:
Endochondral bone development depends on the progression of chondrocyte proliferation, hypertrophy and terminal differentiation, which requires precise transcriptional regulation and signaling coordination. Disturbance of this process would disrupt chondrocyte differentiation and lead to chondrodysplasias. In cells, a highly conserved mechanism, ER stress signaling, has been developed to sense the protein load and maintain the cellular homeostasis. In humans, mutations in COL10A1 induce ER stress and result in metaphyseal chondrodysplasia type Schmid (MCDS). Previous analysis of a MCDS mouse model (13deltg mouse) had revealed a novel mechanism of chondrocyte adaptation to ER stress. The hypertrophic chondrocytes survive ER stress by reverting to a pre-hypertrophic like state (Tsang et al., 2007). To dissect the underlying mechanisms that coordinate chondrocyte survival, reverted differentiation and adaptation to ER stress, different chondrocyte populations in the wild type and 13del growth plates were fractionated for global gene expression analyses.
The genome-wide expression profiles of proliferating chondrocytes, prehypertrophic chondrocytes, hypertrophic chondrocytes and terminally differentiated chondrocytes in the wild type growth plate provide molecular bases to understand the processes underlying both physiological and pathological bone growth. Systematic analyses of these transcriptomic data revealed the gene expression patterns and correlation in the dynamics of endochondral ossification. Genes associated with sterol metabolism and cholesterol biosynthesis are enriched in the prehypertrophic chondrocytes. Selected genes (Wwp2, Zbtb20, Ppa1 and Ptgis) that may potentially contribute to endochondral ossification were identified differentially expressed in the growth plate. Bioinformatics approaches were applied to predict regulatory networks in chondrocytes at different differentiation stages, implying the essential and dominant roles of Sox9 in coordination of stage specific gene expression. We further confirmed that Sox9 directly regulates the transcription of Cyr61, Lmo4, Ppa1, Ptch1 and Trps1, suggesting that Sox9 integrates different steps of chondrocyte differentiation via regulation of its target genes and partially crosstalk with IHH signaling pathway.
The information on gene expression and regulation from physiological growth plate provides important basis to understand the molecular defects of chondrodysplasia. The hypertrophic zone in 13del growth plate was fractionated into upper, middle and lower parts for microarray profiling, corresponding for the onset of ER stress, onset of reverted differentiation and adaptation phase. Comparative transcriptomics of wild type and 13del growth plates revealed genes related to glucose, amino acid and lipid metabolisms are up regulated in response to ER stress. Fgf21 was identified as a novel ER stress inducible factor regulated by ATF4. Removal of Fgf21 results in increasing cell apoptosis in 13del hypertrophic zone without affecting the reverted differentiation process. Up regulation of genes expression related to hypoxic stress (Slc2a1, Hyou1, Stc2 and Galectin3) in 13del hypertrophic chondrocytes suggested that survival and adaptation of chondrocytes to ER stress involve cross-regulation by other stress pathways. Our findings have provided a new insight into the mechanisms that facilitate chondrocyte survival under ER stress in vivo, and propose the integrative effects of hypoxic stress pathway during the stress adaptation process, which broaden the molecular horizons underlying chondrodysplasias caused by protein folding mutations.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
27
Sarkar, Deboleena Dipak. "Potential Role Of Endoplasmic Reticulum Redox Changes In Endoplasmic Reticulum Stress And Impaired Protein Folding In Obesity-Associated Insulin Resistance." Diss., The University of Arizona, 2013. http://hdl.handle.net/10150/306999.
Abstract:
Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule’s reduction potential compared with a dithiol redox buffer like lipoic acid. From this study, we concluded that the glutathione redox couple in ER significantly raised the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in ER with high reliability in real-time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggest a range of ER redox potential consistent with those determined by earlier approaches that did not involve fluorescent sensors. Our primary aim in developing eroGFP1-iL as a redox-sensing tool was to be able to assess whether redox changes represent an early initiator of ER stress in obesity-induced reduction in high molecular weight (HMW) adiponectin in circulation. Hypoxia is a known mediator of redox changes. We found that oligomerization of HMW adiponectin was impaired in the hypoxic conditions observed in differentiated fat cells. The redox-active antioxidant ascorbate was found capable of reversing hypoxia-induced ER stress. Lastly, we demonstrated that changes in ER redox condition is associated with ER stress response and is implicated in the mechanism of action of the insulin-sensitizing agent troglitazone and desensitizing agent palmitate. Using the redox sensing property of eroGFP1-iL, palmitate was found to be an effective modulator of redox changes in the ER and troglitazone was found to cause oxidizing changes in the ER. The action of palmitate in causing aberrant ER redox conditions was associated with aberrant HMW adiponectin multimerization. Palmitate-induced ER stress was ameliorated by troglitazone. Taken together, the data suggest a potential role of ER redox changes in ER stress and impaired protein folding in adipocytes.
28
Shank, Karin Janel. "Coordination of the endoplasmic reticulum stress response and lipid metabolism in plants." NCSU, 2000. http://www.lib.ncsu.edu/theses/available/etd-20000729-161702.
Abstract:
The endoplasmic reticulum (ER) stress response is an important signal transduction pathway that senses ER stress caused by misfolded proteins or increased secretory protein traffic and induces molecular chaperone expression to counter such stress. The response has been well characterized in yeast and mammals where it has been associated with a variety of metabolic pathways, such as phospholipid biosynthesis, translational inhibition, and ER associated degradation. In plants, however, the connections of the ER stress response with metabolic pathways other than those involved in chaperone biosynthesis have not been characterized. This study defines a connection between phospholipid synthesis and the ER stress response in plants. Two model systems were used to characterize this association, the maize mutant floury-2 (fl2), which displays a unique endosperm specific ER stress response mediated by a mutant seed storage protein, and soybean cell cultures treated with the pharmacological agents tunicamycin (Tm) or azetidine-2-carboxylic acid (AZC). These chemicals interfere with normal protein synthesis and processing events, and are well characterized inducers of ER stress responses in animals, plants, and yeast. Investigation of both of these systems revealed a common theme; induction of the ER stress response in plants leads to increased activity and/or expression of various phospholipid biosynthetic enzymes. These increases were correlated with previously described amplifications in expression of the molecular chaperones binding protein (BiP), protein disulfide isomerase (PDI) and calreticulin. Certain aspects of the ER stress response may be unique to plants. A seed-specific result of the ER stress response was the accumulation of triacylglycerols (TG), which specifically increased in the endosperm of the fl2 mutant to more than 3-fold higher than normal endosperm levels by 36 days after pollination (DAP). The maize and soybean systems used in this study provide a starting point for the investigation of other details of the ER stress response in plants and represent important tools for future efforts to define the components of the signaling pathway.
29
Misiewicz, Michael. "Identification of a novel endoplasmic reticulum stress response element regulated by XBP1." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116963.
Abstract:
The Prion protein (PrP), which is the causative agent of scrapie diseases, has a still unclear physiological function, despite 30 years of research on its nature. However, a preponderance of evidence is beginning to support the idea that cellular prion protein (PrPC) has a pro-survival function. Here, we study the regulation of the prion protein gene (PRNP) in this context, as the regulation of the PRNP gene is not well understood. By homology, we identified in the PRNP a novel promoter element which bears similarity to the Endoplasmic Reticulum Stress Response Element (ERSE). This novel ERSE ("ERSE-26") is able to regulate PRNP endogenously in response to endoplasmic reticulum (ER) stress. In order to determine whether or not the ERSE-26 exists elsewhere in the genome and what is co-regulated with PRNP, we conducted a bioinformatic search and identified 38 other genes with an ERSE-26. Their expression was confirmed by treating cultured primary human neurons and MCF-7 cells with the ER stressors Brefeldin A, Tunicamycin and Thapsigargin and conducting Reverse Transcriptase PCR or Quantitative PCR. We found that the genes SESN2, GADD45B and PRNP were significantly upregulated, and others showed an upward trend. Finally, a luciferase reporter construct containing the ERSE-26 only was used to identify that the ER stress transcription factor XBP1 is a transcription factor that induces ERSE-26 activity. Finally, we conducted a literature search to determine what functions of the cell are co-regulated with PRNP with the ERSE-26. Oxidative stress response and pro-survival genes were found in the ERSE-26 genes and found to be the most upregulated by the ERSE-26, strengthening the case for PrPC as a pro-survival gene.La protéine Prion (PrP), qui est l'agent infectieux causant les encéphalopathies transmissibles, n'a pas toujours un rôle bien identifié dans la cellule, malgré 30 ans de recherche sur sa fonction physiologique. Cependant, de plus en plus de preuves commencent à impliquer PrP dans des fonctions de protection dans la cellule. Dans cette étude, nous avons étudié la régulation peu connue du promoteur du gène qui encode PrP (PRNP). Par homologie de séquence, nous avons identifié un nouvel élément dans le promoteur de PRNP qui ressemble à l'Endoplasmic Reticulum Stress Response Element (ERSE). Ce nouvel ERSE (appelé ERSE-26) est capable de réguler l'expression du PRNP de manière endogène en réponse au stress dans le réticulum endoplasmique (RE). Pour savoir si l'ERSE-26 existe ailleurs dans le génome et afin de trouver d'autres gènes régulé avec PRNP, nous avons fait une recherche bioinformatique dans le génome entier. Nous avons identifié 38 gènes contenant aussi un ERSE-26 dans leur promoteur. Afin de confirmer l'expression de ces gènes en réponse au stress ER, nous avons traité des cultures de neurones primaires humains et des cellules MCF-7 avec les activateurs du stress RE Brefeldin A, Tunicamycin et Thapsigargin, puis vérifié l'expression par Transcriptase Inverse PCR (RT-PCR) ou RT-PCR quantitative. Nous avons montré l'induction des gènes GADD45B, SESN2 et PRNP, et d'autres ont montré une tendance positive. Ensuite, un plasmide rapporteur luciferase contenant l'ERSE-26 seulement a été utilisé pour montrer que le facteur de transcription du stress ER XBP1 est un facteur de transcription responsable pour l'activité de l'ERSE-26. Finalement, nous avons fait une recherche dans la littérature afin de déterminer la fonction des gènes contenant ERSE-26. Les gènes répondant au stress oxydant et les gènes pro-survie étaient parmi les gènes ERSE-26, et aussi ont été le plus induits, soutenant le rôle protecteur du PrP dans la cellule.
30
Lari, Federica. "Resolution of proteotoxic stress in the endoplasmic reticulum by ubiquitin ligase complexes." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:871e0484-3de4-4d0d-8206-4af16a8b743e.
Abstract:
The eukaryotic endoplasmic reticulum (ER) is a multifunctional organelle, primarily responsible for the folding and maturation of secretory proteins, as well as lipid metabolism, calcium homeostasis, ubiquitin-dependent signalling and cell fate decisions. ER-associated degradation (ERAD) oversees protein folding and delivers misfolded proteins for degradation by the proteasome via ubiquitin conjugation mediated by RING-type E3 ubiquitin ligases. An intact ERAD is crucial to cellular homeostasis, as unresolved protein imbalances cause ER stress that ultimately lead to apoptosis. The human ER accommodates at least 25 E3s, however our understanding is mostly limited to Hrd1 and AMFR/gp78, both of which have a defined function in ERAD. To understand the contribution of ER E3s to cellular and organelle homeostasis, this study used mass spectrometry of purified E3 complexes to identify cofactors and build interaction networks of ER-resident E3s. These findings will form the foundation for investigating the biological roles of these ubiquitin ligases. Transcriptional analysis highlighted the centrality of Hrd1 among all ER-resident E3s in response to protein misfolding in the ER. Additionally, the contribution of individual Hrd1 complex components to resolving proteotoxic stress was assessed using a misfolded antibody subunit (IgM heavy chain), rather than conventional pharmacological treatments. The ERAD components essential for substrate degradation and survival under proteotoxic stress were identified, highlighting the pivotal role of Hrd1, its cofactor SEL1L and the Derlin family members. Finally, it was demonstrated that autophagy induction in response to proteasome inhibition is key to relieve the burden of protein misfolding in the ER, as it sustained the survival of cells defective for ERAD. Importantly, this study proposes a potential involvement of Hrd1 in signalling from the ER to autophagy, suggesting potential crosstalk between the ERAD and autophagic pathways.
31
Pallet, Nicolas. "Rôle du stress du reticulum endoplasmique dans la néphrotoxicité de la ciclosporine." Paris 5, 2009. http://www.theses.fr/2009PA05S007.
Abstract:
L'introduction des inhibiteurs de la calcineurine (IC), la ciclosporine (CsA) et le tacrolimus (TRL), a révolutionné le pronostic à court terme de la greffe rénale en réduisant de manière significative l'incidence des rejets aigus. Cependant avec le temps, un greffon subit des détériorations structurelles et fonctionnelles irréversibles aboutissant à une néphropathie chronique d'allogreffe. L'origine de la néphropathie d'allogreffe est multifactorielle et la néphrotoxicité des IC contribue significativement à son développement. Il n'existe pas à l'heure actuelle de traitement prévenant la néphrotoxicité des IC et une meilleure compréhension des mécanismes responsables est nécessaire afin d'améliorer la prise en charge des patients transplantés rénaux. Les techniques d'analyse globale sont un moyen d'émettre de nouvelles hypothèses physiopathologiques et l'étude du transcriptome pourrait contribuer à mettre en évidences des mécanismes nouveaux de néphrotoxicité des ICs. L'objectif de ce travail est de caractériser le profil d'expression génique des cellules tubulaires rénales en réponse à la CsA afin de mettre en évidence des mécanismes originaux de néphrotoxicité. En effet, les cellules tubulaires ont un rôle important dans le développement et l'aggravation de nombreuses néphropathies chroniques. Dans la thèse présentée ici, des puces à ADN ont été utilisées afin de comparer le transcriptome de cellules tubulaires rénales par CsA et sirolimus, un immunosuppresseur non néphrotoxique. L'analyse des profils d'expression génique a mis en évidence que la CsA induisait un stress du réticulum endoplasmique (RE), ce qui constitue un mécanisme original de néphrotoxicité. Le stress du RE constitue une cible thérapeutique car le salubrinal, une molécule qui limite les effets délétères du stress du RE, protège contre la néphrotoxicité de la CsA in vitro et in vivo. Nous avons également mis en évidence que ce stress contribuait au développement de modifications phénotypiques épithéliales évocatrices de transition épithélio-mésenchymateuse. Enfin, l'étude des conséquences du stress du RE sur les cellules tubulaires nous a également permis de caractériser un processus adaptatif original dans ce contexte de toxicité de la CsA qui est l'autophagie. Ce travail soulève donc la problématique du rôle stress du RE dans le développement de la néphrotoxicité de la CsA et par extension, au cours de la néphropathie d'allogreffe. Appliqué à des problématiques spécifiques comme l'ischémie-reperfusion ou l'ischémie chronique, ce champ d'investigation nouveau pourrait déboucher sur le développement de biomarqueurs de souffrance tissulaire ainsi que de nouvelles thérapeutiques néphroprotectricesThe molecular mechanisms by which cyclosporine (CsA) induces chronic nephrotoxicity remain poorly understood. Previous transcriptomic study suggested that CsA might induce endoplasmic reticulum (ER) stress in human tubular cells. The aim of this study was to characterize the features of the tubular ER stress induced by CsA and to investigate its consequences on cell differentiation and viability. We confirmed that CsA is responsible for ER stress in vitro in primary cultures of human tubular cells and in vivo in the rat. CsA and other ER stress inducers were responsible for epithelial phenotypic changes (EPCs) leading to the generation of protomyofibroblasts, independently of TGF-0 signaling. RNA interference directed against cyclophilin A gene supported the role of its inhibition in the ER stress and EPCs induced by CsA. Salubrinal, a selective inhibitor of eIF2oc dephosphorylation known to protect cells from ER stress, significantly reduced EPCs and cytotoxicity induced by CsA. Finally, ER stress detection in tubular cells on 56 kidney transplant protocol biopsies was significantly associated with tubular atrophy and interstitial fibrosis. In conclusion, we describe here a new mechanism that initiates dedifferentiation and tubular cell death during CsA treatment, and that may be implicated in the occurrence of the tubulo-interstitial lesions observed in CsA chronic nephrotoxicity. These results provide an interesting framework for further nephroprotective therapies by targetting ER stress
32
Levet, Clémence. "Mild Endoplasmic Reticulum Stress Protects From Cell Death : The Role Of Autophagy." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10209.
Abstract:
Ces dernières années ont été très fructueuses pour l'identification des mécanismes fondamentaux de l'apoptose chez les métazoaires. Cependant, il reste beaucoup à apprendre sur la manière dont sont régulés les programmes de mort en fonction du type cellulaire. Un excès d'apoptose des neurones peut conduire à différentes pathologies neurodégénératives comme les maladies de Huntington, Parkinson ou Alzheimer. La neurodégénérescence est souvent associée à un stress du réticulum endoplasmique (RE), à l'autophagie et à un stress oxydatif. Cependant le rôle de ces mécanismes dans la régulation de la neurodégénérescence n'a pas été clairement établi. Afin de tester l'implication du stress du RE dans la régulation de la mort neuronale, nous avons utilisé différents modèles de neurodégénérescence chez la Drosophila melanogaster et les mammifères. Nous avons d'abord montré que l'induction d'un stress modéré du réticulum protégeait les photorécepteurs de l'oeil de drosophile contre l'apoptose. Nous avons ensuite montré que l'effet protecteur du stress du RE était conservé dans les modèles de maladie de Parkinson chez la drosophile et la souris. Afin de caractériser l'effet protecteur du stress du réticulum, nous avons étudié l'activation de mécanismes protecteurs lors d'un stress du réticulum. Nous avons montré que dans la rétine de drosophile l'activation du stress du RE dans un contexte de mort neuronale induisait une réponse antioxydante et l'autophagie. Nous nous sommes plus particulièrement intéressés au rôle de l'autophagie dans le rôle protecteur du stress du RE. Nous avons montré que l'activation de l'autophagie était nécessaire à l'effet protecteur du stress du RE contre l'apoptose. Nous avons donc pu mettre en évidence que la réponse au stress du RE ne participait pas uniquement à réduire l'accumulation de protéines mal-conformées mais permettait aussi de protéger les neurones contre la mort cellulaire en activant l'autophagieThe last years have been very successful in identifying mechanisms, which control apoptosis in metazoan. However, the regulation of cell death in specific cell type remains to be determined. An excess of neuron apoptosis can lead to neurodegenerative diseases such as Huntington, Parkinson or Alzheimer diseases. Neurodegeneration is usually associated to Endoplasmic Reticulum stress (ER stress), autophagy or oxidative stress. However, the role of these mechanisms in the regulation of neurodegeneration is not clearly established. To test the role of ER stress in the regulation of neuronal death, we used several models of neurodegeneration in Drosophila and mammals. First, we have shown that the genetic induction of ER stress protected photoreceptors of the Drosophila eye from apoptosis. Then, we have shown that the protective effect of ER stress is conserved in both Drosophila and mouse models of Parkinson disease. In order to characterize the protective effect of ER stress, we have studied the activation of protective mechanisms upon ER stress. We have shown that in the Drosophila retina, ER stress can induce an anti-oxidative response and autophagy. Interestingly, autophagy is only activated in presence of both ER stress and cell death signal. We have focused on the role of autophagy in the protective effect of ER stress. We have shown that the activation of autophagy was required for the protective effect of ER stress. Thus, we have shown that ER stress response is not only involved in the reduction of misfolded protein accumulation, but can also protect neurons form cell death by activating autophagy
33
Page, Lindsay N. "Endoplasmic Reticulum Stress Contributes to Cyclosporine A-Induced Lens Epithelial Cell Loss." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1585948655611948.
34
Bicknell, Alicia Anne. "Two MAP kinases regulate novel aspects of the endoplasmic reticulum stress response." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3369545.
Abstract:
Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed September 14, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 160-185).
35
Pino, Steven C. "Role of Endoplasmic Reticulum Stress Response Signaling in T Cells: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/381.
Abstract:
T cells play a central role in cellular-mediated immunity and must become activated to participate as effector cells in the immune response. The activation process is highly intricate and involves stimulation of a number of downstream signaling pathways enabling T cells to proliferate and produce cytokines that are vital for proper effector function. This increase in protein production and protein folding activity adds to the normal physiological strain on cellular machinery. One cellular compartment that has generated a mechanism to mitigate the stress induced by increased protein production is the endoplasmic reticulum (ER).
In general, an increase in cellular production of proteins that overwhelms a cell’s protein folding capability can alter ER homeostasis and lead to ER stress. To counteract this stress, an adaptive cellular mechanism known as the ER stress response (ERSR) is initiated. The ERSR allows a cell to cope with normal physiological stress within the ER caused by increased protein translation. In this dissertation, we show that in vitro and in vivoT cell activation involving T cell receptor (TCR) ligation in the presence of costimulation initiates the physiological ERSR. Interestingly, the ERSR was also activated in T cells exposed only to TCR ligation, a treatment known to induce the ‘non-responsive’ states of anergy and tolerance. We further identified a key component of the downstream TCR signaling pathway, protein kinase C (PKC), as an initiator of physiological ERSR signaling, thus revealing a previously unknown role for this serine/threonine protein kinase in T cells. Therefore, induction of the physiological ERSR through PKC signaling may be an important ‘preparatory’ mechanism initiated during the early activation phase of T cells.
If ER stress is persistent and ER homeostasis is not reestablished, physiological ER stress becomes pathological and initiates cellular death pathways through ER stress-induced apoptotic signaling. We further present data demonstrating that absence of functional Gimap5, a putative GTPase implicated to play a role in TCR signaling and maintenance of overall T cell homeostasis, leads to pathological ER stress and apoptosis. Using the BioBreeding diabetes-prone (BBDP) rat, a model for type 1 diabetes (T1D), we link pathological ER stress and ER stress-induced apoptotic signaling to the observed T cell lymphopenic phenotype of the animal. By depleting the ER stress apoptotic factor CHOP with siRNA, we were able to protect Gimap5-/-BBDP rat T cells from ER stress-induced death. These findings indicate a direct relationship between Gimap5 and maintenance of ER homeostasis for T cell survival.
Overall, our findings suggest that the ERSR is activated by physiological and pathological conditions that disrupt T cell homeostasis. TCR signaling that leads to PKC activation initiates a physiological ERSR, perhaps in preparation for a T cell response to antigen. In addition, we also describe an example of pathological ERSR induction in T cells. Namely, we report that the absence of functional Gimap5 protein in T cells causes CHOP-dependent ER stress-induced apoptosis, perhaps initiated by deregulation of TCR signaling. This indicates a dual role for TCR signaling and regulation in the initiation of both the physiological and pathological ERSR. Future research that provides insights into the molecular mechanisms that govern ERSR induction in TCR signaling and regulation may lead to development of therapeutic modalities for treatment of immune-mediated diseases such as T1D.
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Thomas, Sally Edwina. "The role of cell cycle checkpoints in the survival of endoplasmic reticulum stress." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608043.
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Chan, Cheuk-wing Wilson, and 陳卓榮. "ER stress in the pathogenesis of osteochondrodysplasia." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085192.
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Adolph, Timon Erik. "ER stress converts autophagy defects into intestinal inflammation." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708683.
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Gwiazda, Kamila Sabina. "Role of endoplasmic reticulum calcium stores in beta-cell ER stress and lipotoxicity." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/12553.
Abstract:
There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. The present study sought to determine the mechanism by which palmitate affects intracellular Ca²⁺ in pancreatic β-cells, and in particular the role of the endoplasmic reticulum (ER). In the MIN6 β-cell line, palmitate rapidly increased cytosolic Ca²⁺ through a combination of Ca²⁺ store release and extracellular Ca²⁺ influx. Palmitate caused a reversible lowering of ER Ca²⁺, measured directly with the fluorescent protein-based ER Ca²⁺ sensor, D1ER. Using another genetically encoded indicator, long-lasting oscillations of cytosolic Ca²⁺ in palmitate-treated cells were observed. The kinetics of pharmacological SERCA inhibition on the β-cell ER stress response were characterized, and the ER calcium sensor PERK was found to be rapidly activated in response to irreversible ER calcium depletion. ER calcium depletion in palmitate-treated cells also induced rapid phosphorylation of PERK, as well as other subsequent downstream ER stress signals. In summary, the effects of the free fatty acid palmitate on pancreatic β-cell Ca²⁺ homeostasis were characterized in this thesis. This study provides the first direct evidence that free fatty acids reduce ER Ca²⁺ and sheds light on pathways involved in β-cell ER stress, lipotoxicity and the pathogenesis of type 2 diabetes.
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Tillman, Erik J. (Erik James). "Genetic analysis of endoplasmic reticulum homeostasis during stress and infection of Caenorhabditis elegans." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/119917.
Abstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Animals experience intrinsic and extrinsic stressors throughout development and adulthood. To maintain cellular and organismal homeostasis, eukaryota and metazoa rely on conserved, integrated stress response pathways. Throughout its life cycle, the free-living nematode Caenorhabditis elegans encounters diverse microbial taxa, including both nutritional and pathogenic species. Intestinal infection with the pathogenic bacteria Pseudomonas aeruginosa induces a transcriptional innate immune response leading to the secretion of immune effector molecules into the intestinal lumen. Previous work has demonstrated a critical role for the endoplasmic reticulum unfolded protein response in surviving immune activation during larval development. Specifically, the most ancient IRE-1/XBP-1 branch of the UPR is required for larval development during immune activation, whether or not pathogen is present. To understand additional mechanisms regulating ER homeostasis in C. elegans, we conducted a forward genetic screen and identified suppressors of xbp-1 mutant larval lethality on P. aeruginosa. In this work, I outline the characterization of several identified mutations that each affect a gene encoding a broadly conserved transcriptional regulator. A mutation in the gene encoding the forkhead DNA binding domain-containing transcription factor FKH-9 enhances ER homeostasis outside the context of infection and immune activation, but paradoxically sensitizes animals to perturbations in cytosolic proteostasis. My results suggest that loss of fkh-9 enhances translocation of misfolded proteins out of the ER, thereby disrupting cytosolic proteostasis and decreasing proteasomal function. These findings implicate a critical need for balancing proteostasis across cellular compartments during organismal stress, and further investigation of the additional characterized mutants will elucidate the breadth of this phenomenon.
by Erik J. Tillman.
Ph. D.
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Nugent, Ashleigh Elizabeth. "The Presence of Extracellular Matrix Alters the Chondrocyte Response to Endoplasmic Reticulum Stress." Kent State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=kent1271375344.
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Mlynarczyk, Coraline. "Regulation of p21CDKN1A in the endoplasmic reticulum stress response : mechanism, function and implications." Paris 7, 2013. http://www.theses.fr/2013PA077128.
Abstract:
Dans les tumeurs solides, un apport réduit en nutriments et oxygène génère un stress au niveau du réticulum endoplasmique (RE). Nous avons précédemment montré que la réponse physiologique déclenchée (UPR) induit p53/47, une protéine isoforme de p53. P53/47 provoque un arrêt du cycle cellulaire en G2, via 14-3-3 sigma, pour rétablir l'homéostasie du RE. Ceci contraste avec le blocage en Gl par p21 qui est activé par p53 suite à un dommage de F ADN. La majorité des traitements contre le cancer étant basée sur l'utilisation d'agents génotoxiques, nous avons cherché à comprendre comment un stress du RE pouvait influencer la réponse à un dommage de l'ADN. Il apparaît que l'expression de p21 est réduite en conditions de stress du RE. Cet effet est dominant par rapport à l'induction de p21 lorsque l'ADN est endommagé, aboutissant à une augmentation de la mort cellulaire par apoptose. La suppression de p21 implique l'action coordonnée de p53 et p53/47 à un niveau transcriptionnel et traductionnel. S'il est présent, p21 empêche l'arrêt en G2, en induisant la dégradation protéasomique de 14-3-3 sigma, via l'enzyme ubiquitine ligase COP1. En conséquence, ce travail i) décrit la façon dont p53 contrôle l'équilibre entre les phases Gl et G2 du cycle cellulaire en fonction du stress ; ii) dévoile un nouveau mécanisme permettant à p53 de réguler l'expression de ses gènes cibles ; iii) révèle une fonction pro-proliférative de p21 dont la suppression est essentielle dans la réponse au stress du RE and iv) détermine des conditions présentes dans les cellules tumorales qui peuvent être utilisées pout renforcer l'effet cytotoxique de molécules anticancéreuses utilisées en cliniqueStress to the endoplasmic reticulum (ER) occurs during nutrient or oxygen limitation as can be found in solid tumors and is characterized by an accumulation of misfolded proteins in the ER lumen. We have previously shown that the resulting unfolded protein response (UPR) induces the p53 isoform p53/47 and a subsequent 14-3-3 sigma-mediated G2 arrest that helps to restore ER homeostasis. This contrasts with the p21coKNiA-dependent Gl arrest caused by p53 following DNA damage. A large proportion of anticancer therapies are based on genotoxic agents and we sought to determine how ER stress affects the p53-mediated response to DNA damage. It appeared that the UPR down regulates expression of p21 in a p53-dependent manner and prevents p21 induction by DNA damage, resulting in increased apoptosis. P53/47 impedes p21 promoter transactivation by p53 and the coordinated activity of both isoforms represses de novo p21 protein synthesis, via codons 76 to 164 of the p21 mRNA. It is further demonstrated that p21, if not suppressed, prevents ER stress-induced G2 arrest via directing 14-3-3 sigma degradation by the E3 ubiquitin ligase COP1. Therefore, this work i) illustrates how p53 controls an intrinsic balance between the Gl and G2 checkpoints in response to different stress signals; ii) uncovers a novel level at which p53 can regulate expression of its target genes; iii) reveals a pro-proliferative role for p21, whose suppression is essential during ER stress and iv) identifies conditions displayed by solid tumor cells that may be used to potentiate the cytotoxicity of existing anticancer drugs
43
Zha, Beth Shoshana. "HIV Protease Inhibitors Trigger Lipid Metabolism Dysregulation Through Endoplasmic Reticulum Stress and Autophagy." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/273.
Abstract:
HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART). HIV PIs are extremely effective at suppressing viral load, but have been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease. Recent studies indicate that activation of endoplasmic reticulum (ER) stress is an important cellular mechanism underlying HIV PI-induced dysregulation of lipid metabolism. However, the exact role of ER stress in HIV PI-associated lipodystrophy and dyslipidemia remains to be identified. Hepatocytes and adipocytes are important players in regulating lipid metabolism and the inflammatory state. Dysfunction of these two cell types is closely linked to various metabolic diseases. In this dissertation research, we aimed to define the role of activation of ER stress in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes and further identifty the potential molecular mechanisms. Both cultured and primary mouse adipocytes and hepatocytes were used to examine the effect of individual HIV PIs on ER stress activation and lipid metabolism. The results indicated that HIV PIs differentially activate ER stress through depletion of ER calcium stores, activating the unfolded protein response (UPR). UPR activation further lead to an alteration of cellular differentiation through downstream transcription factor CHOP. At the same time, HIV PIs also altered adipogenesis via differential regulation of the adipogenic transcription factor PPARγ. HIV PI-induced ER stress was closely linked to dysregulation of autophagy activation through CHOP, and upstream ATF-4, signaling pathways. In hepatocytes, the integrase inhibitor raltegravir abrogated HIV PI-induced lipid accumulation by inhibiting ER stress activation and dysregulation of autophagy pathway. Our studies suggest that both ER stress and autophagy are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV HAART-treated patients.
44
Egawa, Naohiro. "The endoplasmic reticulum stress sensor, ATF6α, protects against neurotoxin-induced dopaminergic neuronal death." Kyoto University, 2011. http://hdl.handle.net/2433/142092.
45
Lipson, Kathryn L. "The Role of Endoplasmic Reticulum Stress Signaling in Pancreatic Beta Cells: a Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/363.
Abstract:
Protein folding in the endoplasmic reticulum (ER) is essential for proper cellular function. However, the sensitive environment in the ER can be perturbed by both pathological processes as well as by physiological processes such as a large biosynthetic load placed on the ER. ER stress is a specific type of intracellular stress caused by the accumulation of immature or abnormal misfolded or unfolded proteins in the ER. Simply defined, ER stress is a disequilibrium between ER load and folding capacity. Cells have an adaptive response that counteracts ER stress called the "Unfolded Protein Response” (UPR). The ability to adapt to physiological levels of ER stress is especially important for maintaining ER homeostasis in secretory cells. This also holds true for pancreatic β-cells, which must fold and process large amounts of the hormone insulin.
Pancreatic β-cells minimize abnormal levels of glycemia through adaptive changes in the production and regulated secretion of insulin. This process is highly sensitive, so that small degrees of hypo- or hyperglycemia result in altered insulin release. The frequent fluctuation of blood glucose levels in humans requires that β-cells control proinsulin folding in the ER with exquisite sensitivity. Any imbalance between the load of insulin translation into the ER and the actual capacity of the ER to properly fold and process the insulin negatively affects the homeostasis of β-cells and causes ER stress.
In this dissertation, we show that Inositol Requiring 1 (IRE1), an ER-resident kinase/endoribonuclease and a central regulator of ER stress signaling, is essential for maintaining ER homeostasis in pancreatic β-cells. Importantly, IRE1 has a crucial function in the body’s normal production of insulin in response to high glucose. Phosphorylation and subsequent activation of IRE1 by transient exposure to high glucose is coupled to insulin biosynthesis, while inactivation of IRE1 by siRNA or inhibition of IRE1 phosphorylation abolishes insulin biosynthesis. IRE1 signaling under these physiological ER stress conditions utilizes a unique subset of downstream components of IRE1 and has a beneficial effect on pancreatic β-cell homeostasis.
In contrast, we show that chronic exposure of β-cells to high glucose causes pathological levels of ER stress and hyperactivation of IRE1, leading to the degradation of insulin mRNA. The term “glucose toxicity” refers to impaired insulin secretion by β-cells in response to chronic stimulation by glucose and is characterized by a sharp decline in insulin gene expression. However, the molecular mechanisms of glucose toxicity are not well understood. We show that hyperactivation of IRE1 caused by chronic high glucose treatment or IRE1 overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1 signaling using a dominant negative form of the protein prevents insulin mRNA degradation in β-cells. Additionally, islets from mice heterozygous for IRE1 retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.
This work suggests that the rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism may represent an essential element in the adaptation of β-cells to chronic hyperglycemia. This adaptation is crucial for the maintenance of β-cell homeostasis and may explain in part why the β-cells of diabetic patients with chronic hyperglycemia stop producing insulin without simply undergoing apoptosis. This work implies that prolonged activation of IRE1 signaling is involved in the molecular mechanisms underlying glucose toxicity.
This work therefore reveals two distinct activities elicited by IRE1 in pancreatic β-cells. IRE1 signaling activated by transient exposure to high glucose enhances proinsulin biosynthesis, while chronic exposure of β-cells to high glucose causes hyperactivation of IRE1, leading to the degradation of insulin mRNA. Physiological IRE1 activation by transient high glucose levels in pancreatic β cells has a beneficial effect on insulin biosynthesis. However, pathological IRE1 activation by chronic high glucose or experimental drugs negatively affects insulin gene expression. In the future, a system to induce a physiological level of IRE1 activation, and/or reduce the pathological level of IRE1 activation could be used to enhance insulin biosynthesis and secretion in people with diabetes, and may lead to the development of new and more effective clinical approaches to the treatment of this disorder.
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Herrenbruck, Adrienne Rose. "EFFECTS OF HIGH FAT EXPOSURE ON SKELETAL MUSCLE AUTOPHAGY AND ENDOPLASMIC RETICULUM STRESS." UKnowledge, 2018. https://uknowledge.uky.edu/khp_etds/53.
Abstract:
Autophagy is a major degradation mechanism, responsible for clearing damaged and dysfunctional organelles, including the endoplasmic reticulum, a structure essential for protein synthesis and myocellular hypertrophy. Alterations in autophagy throughout various tissues of the body have been linked to various negative side effects such as decreased myocellular hypertrophy and insulin resistance. High fat diets lead to changes (both increases and decreases) in autophagy in various tissues throughout the body in a tissue-specific manner.
Skeletal muscle autophagy is decreased in myotubes cultured from obese women, however the mechanism by which this occurs is unknown. As the largest organ system in the human body, skeletal muscle serves an important role in overall metabolic health. Therefore, sufficient skeletal muscle autophagy is important for proper metabolic function. Moreover, a decrease in liver and pancreas autophagy has been found to lead to endoplasmic reticulum (ER) stress and the development of insulin resistance. Understanding the relationship between autophagy and ER stress in the skeletal muscle following a high fat diet may help elucidate a novel target for decreasing negative side effects.
Interestingly, both acute and chronic exercise have been shown to increase skeletal muscle autophagy. This points to a potential therapeutic treatment for those suffering with decreased skeletal muscle autophagy and may help improve ER stress.
The purpose of this study was to compare the in vivo and in vitro effects of high fat exposure on skeletal muscle autophagy. Additionally, the relationship of autophagy and ER stress in skeletal muscle was explored. Lastly, this project identified changes in skeletal muscle autophagy and ER stress following cyclic stretch, an in vitro model of exercise in C2C12 myotubes.
Eight-week-old C57BL/6J were fed a high fat diet for 16 weeks and tibialis anterior muscle examined for changes in autophagy markers. Gene expression (mRNA content) of autophagy markers Atg3 (p=0.011, fold change 1.37), Atg12 (p=0.026, 1.38), and Atg16L (p=0.004, 1.49) were increased in skeletal muscle of obese mice. Protein content was also measured, where increases in Atg3 (p = 0.04, 1.22), Atg12 (p = 0.027, 1.21), and Atg16L1(p = 0.021, 1.59) were found. However, there was no difference in LC3 II:I ration. No changes were seen in Atg5 or LC3.
Additionally, C2C12 myotubes were treated with equimolar palmitate and oleate for 24h then assessed for mRNA content of genes involved in autophagy and ER stress. Autophagy genes Atg5 (p = 0.007, fold change 1.78), Atg12 (p = 0.001, fold change 1.99), and LC3 (p = 0.01, fold change 2.02) were decreased with high fat treatment. Paradoxically, there was an increase in Atg16L (p = 0.005, fold change 1.90). There were no changes in protein content. ER stress was increased indicated by an increase of sXBP1 (p = 0.005, fold change 1.33). Furthermore, inhibition of autophagy lead to changes in ER morphology and ER stress.
To identify the impact of cyclic stretch on skeletal muscle autophagy and ER stress, C2C12 myotubes were subjected to 30 minutes of equibaxial stretch and examined for changes in autophagy and ER stress. Autophagy flux, measured by tyrosine release, increased by 34% (p = 0.04) following exercise and ER stress was decreased.
In conclusion, this study provides the novel finding that decreased skeletal muscle autophagy is sufficient for inducing ER stress. Additionally, cyclic stretch increases autophagy and improves ER homeostasis.
47
Bezu, Lucillia. "Mort cellulaire immunogène : du stress du reticulum endoplasmique à l'exposition de la calréticuline." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS246.
Abstract:
Les traitements actuels anticancéreux ont une action cytotoxique directe sur les cellules tumorales mais également sur les cellules saines des systèmes immunitaires et hématopoïétiques. De plus, ces traitements ont l’incapacité de stimuler le système immunitaire et de prévenir les récidives. Cependant, certains agents tels que les anthracyclines, la radiothérapie ou encore la thérapie photodynamique ont la capacité d’induire une mort cellulaire dite immunogène. Durant cette modalité, l’exposition à la surface des cellules tumorales mourantes d’une protéine chaperonne du reticulum endoplasmique (RE), la calréticuline (CALR), est une étape essentielle et caractéristique de l’immunogénicité ainsi que la libération d’HMGB1, l’autophagie et la sécrétion d’ATP. Des données issues de la littérature prouvent que les anthracyclines sont également capables d’activer des marqueurs du stress du RE. Notre travail a consisté à étudier les liens moléculaires entre le stress du RE et les marqueurs de la mort cellulaire immunogène. Grâce à un criblage à haut débit, nous avons déterminé que les drogues dites immunogènes activaient la phosphorylation d’eIF2α (eukaryotic initiation factor 2 alpha) et qu’il existait une corrélation forte avec l’exposition de la CALR (R score 0,73 ; p<0,01). De manière surprenante, ces agents échouent à activer la voie de signalisation sous-jacente ainsi que les deux autres voies du stress du RE médiées par ATF6 (activating transcription factor 6) et XBP1s (spliced X-box binding protein 1). Par ailleurs, une anthracycline appelée mitoxantrone inhibe activement les trois voies du stress du RE en co-traitement avec un inducteur du stress du RE et inhibiteur de la N-glycosylation: la tunicamycine. Ces données in vitro ont également été validées dans un modèle in vivo de souris immunodéficientes xénogreffées. De plus, grâce à un algorithme reliant les propriétés physico-chimiques des drogues anti-cancéreuses avec leurs capacités à induire les marqueurs de mort cellulaire immunogène suivants: P-eIF2α, CALR, HMGB1, granules de stress et autophagie, un score prédictif d’immunogénicité a pu être déterminé. Nous devrons déterminer dans le futur comment utiliser ce score comme marqueur prédictif d’une réponse immunogène au cours des traitements cliniquesConventional anticancer chemotherapies display a high degree of toxicity with certain specificity for tumor cells. However most of these approaches fail to activate immune system-related bystander effects and thus do often fail to prevent from recurrence. Despite these premises, certain anticancer treatments (including anthracycline-based chemotherapy, radiotherapy and photodynamic therapy) have the ability to induce an immunogenic cell death (ICD) modality. The exposure of calreticulin (CALR) during the course of ICD is quintessential for the transfer of tumor antigen from dying tumors to dendritic cells of the immune system as well as translocation of high mobility group box 1 (HMGB1), autophagy and ATP secretion. Previous studies have shown that certain anticancer agents including anthracylins are able to activate markers of endoplasmic reticulum stress (ER stress). Here we investigated the molecular mechanisms that link ER stress responses with hallmarks of ICD. In a drug screening approach, we showed that ICD-inducing drugs triggered the phosphorylation of the eukaryotic initiation factor 2 alpha (P-eIF2α) and that this correlated with CALR exposure (R score 0.73, p<0.01). Surprisingly though the agents failed to induce downstream ER stress pathways including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6). In addition, we found that mitoxantrone actively inhibited all three arms of the unfolded protein response, when co-administered with the inhibitor of N-linked glycosylation tunicamycin, whereas tunicamycin alone triggered all arms of ER-stress. These findings were validated in vivo in immunodeficient animals xenografted with biosensors for ER-stress responses. Moreover, using a machine learning approach that integrates physicochemical properties of oncologic drugs with their ability to elicit immunogenic hallmarks including the phosphorylation of eIF2α, the exposure of CALR, the translocation of HMGB1, the formation of stress granules and the induction of autophagy we established an in silico approach for ICD prediction. For the future, we further aim to investigate the possibility to use these score comprising P-eIF2α and its downstream consequences as biomarker for immunogenic responses during anticancer treatment in patients
48
Lo, Ling-kit Rebecca. "ER-stress signaling and chondrocyte differentiation in mice." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B3861926X.
49
Lo, Ling-kit Rebecca, and 羅令潔. "ER-stress signaling and chondrocyte differentiation in mice." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B3861926X.
50
Clavier, Thomas. "Etude de l 'expression du stress du reticulum endoplasmique au cours de l'inflammation systémique aigue chez l'homme Gene expression of protein tyrosine phosphatase 1B and endoplasmic reticulum stress during septic shock." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMR015.
Abstract:
Le syndrome de réponse inflammatoire systémique est une entité fréquente en réanimation. Ses causes sont nombreuses et parfois intriquées : sepsis, traumatisme, chirurgie lourde, circulation extra-corporelle (CEC), ischémie-reperfusion, … Sa physiopathologie repose sur la synthèse de cytokines, l’activation des cellules immunitaires et de la coagulation et l’altération de la fonction endothéliale. Ces anomalies induisent une dysfonction vasculaire pouvant être responsable d’un état de choc et d’un syndrome de défaillance d’organes (Syndrome de Détresse Respiratoire Aigüe, insuffisance rénale, syndromes confusionnels, …). Des données récentes ont montré qu’un mécanisme cellulaire adaptatif, le stress du réticulum endoplasmique (SRE), est impliqué dans la dysfonction endothéliale et les défaillances d’organes induites par l’inflammation systémique. Le SRE correspond à l’accumulation de protéines non repliées au sein du réticulum endoplasmique, accumulation pouvant altérer le fonctionnement cellulaire et aller jusqu’à induire l’apoptose. L’Unfolded Protein Response (UPR) est un mécanisme de réponse au SRE qui permet de replier ces protéines, en particulier via la synthèse de protéines chaperonnes. L’UPR permet donc la résolution du SRE. Des travaux pré-cliniques ont montré que l’inhibition du SRE au cours du sepsis ou de l’inflammation stérile via des chaperonnes chimiques diminuait la défaillance d’organe et améliorait la survie. Il n’existe cependant aucune description de cette réponse lors de l’inflammation systémique aigüe chez l’Homme. Nous montrons dans ce travail que les trois voies de l’UPR sont activées au cours du sepsis et de l’inflammation induite par la CEC. Au cours du sepsis l’expression d’une voie de l’UPR était associée au score de défaillances d’organes SOFA. Chez les patients bénéficiant d’une chirurgie cardiaque sous CEC, une réponse UPR relativement faible et une moindre synthèse de chaperonnes étaient associées à une défaillance d’organe post-opératoire. Nos données confirment donc que l’UPR est activée chez l’Homme lors d’une inflammation systémique et que l’intensité de son expression semble associée à la défaillance d’organeSystemic inflammatory response syndrome is a common phenomenon in intensive care unit. Its origins are multiple and sometimes intricate: sepsis, trauma, major surgery, cardiopulmonary bypass (CPB), ischemia-reperfusion, acute pancreatitis, ... Its physiopathology is based on cytokine synthesis, activation of immune cells and coagulation, and alteration of endothelial function (in particular nitrogen monoxide synthesis). These abnormalities induce vascular dysfunction which may be responsible for a shock and multiple organ failure syndrome (Acute Respiratory Distress Syndrome, renal failure, confusional syndromes, ...). Recent data have shown that endoplasmic reticulum stress (ERS), is involved in endothelial dysfunction and organ failure induced by systemic inflammation. ERS corresponds to the accumulation of unfolded proteins in the endoplasmic reticulum, accumulation that can alter cell function and even induce apoptosis. The Unfolded Protein Response (UPR) is a response to ERS that allows the proteins to be folded, in particular through the synthesis of chaperone proteins and therefore allows the ERS resolution. Pre-clinical studies have shown that ERS inhibition during sepsis or sterile inflammation via chemical chaperones reduces organ failure and improves survival. However, there is no description of UPR during systemic inflammation in humans. We show in this work that all the three UPR pathways are activated during sepsis and CPB-induced inflammation. During sepsis the expression of one UPR pathway was associated with the SOFA organ failure score. In patients undergoing cardiac surgery with CPB, a relatively low UPR response and a decreased chaperone synthesis were associated with post-operative organ failure. Our data therefore confirm that UPR is activated in humans during systemic inflammation and that the intensity of its expression appears to be associated with organ failure