Necroptosis is a pathway of inflammatory cell death that is associated with several pathologies and is induced by ligation of surface TLR or cytokine receptors in macrophages. Many signaling pathways depend on endocytosis, a process mediated by GTPases such as dynamin. We evaluated the role of dynamin-dependent endocytosis in the necroptosis of macrophages using various dynamin inhibitors. Using flow cytometry, we confirmed that during necrosome signaling, various dynamin inhibitors (e.g. Dyngo 4a and Dynasore) blocked the internalization of TLR4, which also resulted in the inhibition of cytokine production. Despite the similar impact of Dynasore and Dyngo 4a on TLR4 endocytosis and cytokine production, only Dyngo 4a prevented TLR4-induced necroptosis of macrophages. Further studies indicated that Dyngo 4a was a potent stimulator of the p38 MAPK pathway, and activation of this pathway by Dyngo 4a was responsible for the inhibition of necroptosis of macrophages following TLR4 signaling. Thus, these studies reveal the previously unknown role of the p38 MAPK pathway in regulating the activation of necrosome signaling.

El síndrome antifosfolípido (SAF) es una enfermedad autoinmune sistémica, que se caracteriza por la presencia conjunta de trombosis vascular o complicaciones obstétricas (pérdida fetal o abortos espontáneos a repetición) y la presencia de anticuerpos antifosfolípidos (aFLs) en el plasma de los pacientes. El mecanismo por el cual esos anticuerpos producen la enfermedad no se comprende totalmente, y esto se debe a la poli especificidad de estos anticuerpos, la multiplicidad de los sitios de acción y por la variabilidad del contexto clínico en donde se presenta la enfermedad. Mediante estudios in vitro e in vivo se demostró que la actividad trombogénica de los aFLs esta determinada entre otras por la interferencia que producen estos anticuerpos con el sistema hemostático: inhiben el camino de la proteína C activada, impiden la normal fibrinolisis y producen la activación celular, principalmente células endoteliales, monocitos y plaquetas. En las células endoteliales (CE) y en los monocitos, los aFLs incrementan la expresión de FT y moléculas de adhesión, mientras que en las plaquetas se observó un aumento en la producción plaquetaria de TXA2, agregación y liberación de los gránulos plaquetarios en presencia de dosis subagregantes de diferentes agonistas. Con el fin de dilucidar los mecanismos intracelulares involucrados en la activación celular mediada por los aFLs, se estudió el rol de la p38 MAPK, una proteína serina/treonina quinasa, que constituye la mayor cascada de transducción de señales inflamatorias desde la superficie celular hacia el núcleo. En este trabajo de tesis se comprobó que los aFLs producen la activación de la p38 MAPk en las plaquetas y en las CE, y que esta vía de activación intracelular es en gran parte responsable del incremento de la activación plaquetaria y de las CE, debido a que los efectos protrombóticos y proinflamatorios de los aFLs son disminuyen significativamente cuando se inhibe la p38MAPk con el inhibidor especifico SB20380. / Antiphospholipid Syndrome (APS) is a systemic autoimmune disease, characterized by the joint presence of vascular thrombosis or obstetric complications (fetal loss or recurrent spontaneous abortions) and the presence of antiphospholipid antibodies (aPL) in the plasma of patient. The mechanism by which these antibodies cause disease is not fully understood, and this is due to the poly specificity of these antibodies, multiple sites of action and variability of clinical context where the disease occurs. Using in vitro and in vivo showed that the thrombogenic activity of aPL is determined among others by the interference caused by these antibodies with the hemostatic system: the way to inhibit activated protein C, prevent normal fibrinolysis and cell activation occur mainly endothelial cells, monocytes and platelets. In endothelial cells (EC) and monocytes, AFLS increase TF expression and adhesion molecules, whereas in platelets was observed an increase in platelet production of TXA2, platelet aggregation and release in the presence of doses of subagregantes different agonists. In order to elucidate the intracellular mechanisms involved in mediated cell activation by the aPL, we studied the rol of p38 MAPK, a protein serine / threonine kinase, which constitutes the major transduction cascade of inflammatory signals from the cell surface to the nucleus. In this thesis aPLs were found to produce the activation of p38 MAPK in platelets and EC, and that the intracellular activation pathway is largely responsible for the increased platelet activation and EC, because that prothrombotic and proinflammatory effects of aPLs are significantly diminished when p38MAPK was inhibited with specific inhibitor SB20380.

Le glioblastome multiforme (GBM) est la tumeur cérébrale la plus fréquente et la plus agressive. Il s’agit d’une tumeur capable de survivre même dans des conditions d’oxygénation faible ou hypoxie. En effet, les cellules cancéreuses du GBM activent des voies de survie en réponse à cette privation de dioxygène, dont l’autophagie. Il s’agit d’un mécanisme catabolique conduisant à la dégradation des constituants cellulaires, générant ainsi des précurseurs pour l’anabolisme cellulaire ainsi que de l’ATP. Nous avons étudié l’activation de l’autophagie en réponse à l’hypoxie, dans trois lignées cellulaires de GBM humain, les U87MG, les M059K et les M059J. Une autophagie de survie est activée dans les trois lignées cellulaires, en réponse à l’hypoxie. L’inhibition du flux autophagique par la chloroquine (CQ), induit une accumulation des autophagosomes, soulignant ainsi l’efficacité du processus. L’inhibition de l’autophagie par la CQ ou par des siRNA spécifiques dirigés contre les transcrits de Beclin1 ou d’Atg5, entraîne une diminution significative de l’activité métabolique cellulaire, ainsi qu’un retard de prolifération. Toutefois, nous n’avons pas détecté de mort apoptotique dépendante des caspases. Nous avons donc étudié une deuxième voie de signalisation de survie cellulaire, la signalisation neurotrophique. Une augmentation significative des transcrits de TrkC FL et T1 (TrkC tronqué) ainsi que de leur ligand, la NT-3 a été observée dans les cellules U87MG cultivées en hypoxie. De même, le taux de production des protéines TrkC FL et T1 a significativement augmenté en hypoxie. L’augmentation de l’expression du TrkC FL était accompagnée par une augmentation de sa phosphorylation et de celle de la p38 MAPK. L’inhibition de cette dernière par siRNA induit un clivage de la PARP, qui est d’autant plus important suite à l’ajout de la CQ. Ces effets étaient plus marqués au niveau des cellules cultivées en hypoxie. L’inhibition de l’autophagie par la CQ, augmente l’expression de TrkC FL et la phosphorylation de la p38, ce qui suggère qu’en absence de l’autophagie, les cellules s’adapteraient en augmentant la signalisation du TrkC.La recherche des zones hypoxiques et autophagiques sur des coupes de tumeurs issues de patients atteints de GBM, confirme le caractère hypoxique de cette tumeur, et montre une induction du processus autophagique. En comparaison avec le cavernome (tumeur cérébrale bénigne), les patients atteints de GBM montrent une augmentation significative de l’expression de TrkC et de NT-3, ce qui renforce l’importance de la signalisation neurotrophique dans la survie des cellules de GBM. / Glioblastoma multiform (GBM), a primary brain tumor that is the most common and the most aggressive. It’s characterized by a high degree of hypoxia and a resistance to therapy because of its adaptation capacities including autophagy. This degradation process allows recycling of cellular components to produce precursors for anabolism and ATP. We have studied the hypoxia-induced autophagy in three human GBM cell lines, the U87MG, M059K and M059J. We have found a survival hypoxia-induced autophagy that was efficient in all cell lines. Indeed, we observed an accumulation of autophagosomes when we inhibited the autophagic flux with chloroquine (CQ). Treatment with CQ or interference of Beclin1 or Atg5 expression by specific siRNA in GBM cells significantly decreased their metabolic activity and growth. However, we did not detect PARP cleavage by western blotting. Thus, we verified the neurotrophic signaling as another survival pathway by which GBM cells resist to hypoxia. After hypoxia, the transcription level of TrkC FL (full length), TrkC-T1 (truncated TrkC) and the NT-3 (the TrkC ligand) significantly increases in the U87MG cell lines, as far as the translation level of TrkC FL and TrkC-T1. When we explored the TrkC FL signaling pathway, there was an increase in the phosphorylation level of p38. After inhibition of this MAPK, we observed PARP cleavage, which was particularly important in hypoxia conditions. This cleavage was further enhanced upon CQ treatment. The autophagy inhibition using either CQ, siBeclin1 or siAtg5, increases TrkC FL and T1 expression, suggesting that in the absence of autophagy, cells would adapt by increasing TrkC signaling.Finally, we have verified for hypoxic (BNIP3) and autophagic (LC3) markers on tumor sections from patients with GBM. We confirmed the hypoxic character of GBM, and showed important autophagy activation, after autophagosomes quantification. In comparison with cavernoma (benign brain tumor), patients with GBM showed a significant increase in TrkC and NT-3 expression, highlighting the importance of neurotrophic signaling in GBM tumor cell survival.

Submitted by Francine Silva (francine.silva@unipampa.edu.br) on 2016-09-28T21:17:01Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Potencial protetor do Extrato de Psidium guajava frente à toxicidade induzida pelo organofosforato Clorpirifós em Drosophila melanogaster.pdf: 1278673 bytes, checksum: fb86ba9285bb81b1327ca36aa998f903 (MD5) / Made available in DSpace on 2016-09-28T21:17:01Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Potencial protetor do Extrato de Psidium guajava frente à toxicidade induzida pelo organofosforato Clorpirifós em Drosophila melanogaster.pdf: 1278673 bytes, checksum: fb86ba9285bb81b1327ca36aa998f903 (MD5) Previous issue date: 2015-05-20 / Clorpirifós (CP) é um inseticida organofosforado amplamente utilizado no controle de pragas agrícolas e domésticas. O principal dano causado pelo CP é a neurotoxicidade induzida pela inibição da enzima acetilcolinesterase, o que ocasiona um aumento no neurotransmissor acetilcolina e promove uma hiperexitação no sistema nervoso central e junções musculares, levando a perturbações do funcionamento fisiológico. A exposição ocupacional é uma das principais formas de intoxicação humana por organofosforados e as terapias atuais para estes compostos não são totalmente eficientes. Nesse sentido a procura por compostos capazes de reverter esses danos tem se intensificado e alguns estudos têm focado seus esforços sobre os efeitos de proteção de plantas ou compostos naturais em várias condições neuropatológicas. Psidium guajava é uma planta amplamente utilizada na medicina popular e a sua atividade antioxidante foi descrita, no Brasil as folhas e os frutos são utilizados para a anorexia, cólera, diarréia, problemas digestivos, disenteria, insuficiência gástrica, inflamação das membranas mucosas, laringite, problemas de pele, dor de garganta, úlceras, entre outros. Neste estudo foi avaliado o potencial antioxidante e protetor do extrato hidroalcoólico de P. guajava (HEPG) contra a toxicidade induzida por CP na mosca da fruta Drosophila melanogaster. A atividade antioxidante de HEPG in vitro foi confirmada pelos ensaios de ABTS, DPPH, fenóis totais e FRAP. A exposição das moscas ao CP causou aumento da mortalidade, deficiências locomotoras e inibição da acetilcolinesterase. Moscas expostas ao CP apresentaram aumento de ROS e peroxidação lipídica, acompanhado por uma diminuição significativa na viabilidade mitocondrial. Como resposta ao aumento do estresse oxidativo, moscas expostas ao CP mostraram aumento da atividade da GST e nos níveis de GSH. A expressão de mRNA de NRF2 e MPK2 (que codifica p38MAPK em D. melanogaster) também foram significativamente super regulados. HEPG foi capaz de restaurar todos os danos e alterações bioquímicas/moleculares causados pelo CP. Os nossos resultados mostram pela primeira vez o potencial efeito protetor de P. guajava contra a toxicidade causada por clorpirifós, sugerindo a Psidium guajava como um tratamento alternativo adjunto para o envenenamento por compostos organofosforados. / Chlorpyrifos (CP) is an organophosphate insecticide widely used for control agricultural and household pests. The main damage caused by the CP is the neurotoxicity induced by inhibition of the enzyme acetylcholinesterase, which causes an increase in the neurotransmitter acetylcholine and promotes hiperexitação the central nervous system and muscle junctions, leading to disruption of physiologic function. Occupational exposure is a major form of human poisoning by organophosphates and current therapies for these compounds are not fully efficient. In this sense the search for compounds that can reverse this damage has intensified and some studies have focused their efforts on plants protection purposes or natural compounds in various neuropathological conditions. Psidium guajava is a plant widely used in popular medicine and its antioxidant activity was described in Brazil leaves and fruit are used for anorexia, cholera, diarrhea, digestive problems, dysentery, gastric insufficiency, inflammation of mucous membranes, laryngitis, skin problems, neck pain, ulcers, among others. In this study we evaluated the antioxidant and protective potential of the hydroalcoholic extract of P. guajava (HEPG) against CP induced toxicity in the fruit fly Drosophila melanogaster. HEPG in vitro antioxidant activity was confirmed by ABTS, DPPH, Total Phenolics and FRAP assays. The exposure of flies to CP caused increased mortality, locomotor deficits and inhibition of acetylcholinesterase. Flies exposed to CP presented elevated ROS and lipid peroxidation which was accompanied by a significant decrease in mitochondrial viability. As a response to increased oxidative stress, CP exposed flies showed increased in GST activity and GSH levels. The mRNA expression of NRF2 and MPK2 (which encodes D. melanogaster p38MAPK) were also significantly up-regulated. HEPG was able to restore all the damage and biochemical/molecular alterations caused by CP. Our results show for the first time the potential of P. guajava protective effect against the toxicity caused by Chlorpyrifos, suggesting Psidium guajava as an adjunct alternative treatment for poisoning by organophosphorus compounds.

No description available.

Le cetuximab est une thérapie ciblée dirigée contre le récepteur du facteur de croissance épidermique (EGFR) utilisée dans le cancer colorectal (CCR) en combinaison avec des chimiothérapies (5-FU, irinotécan et oxaliplatine). Sa fixation inhibe les voies de signalisation en aval du récepteur conduisant à une diminution de la prolifération et de la survie des cellules ciblées. Cependant, chez les patients atteint de CCR présentant une protéine KRAS mutée, le cetuximab est inefficace, de plus, la moitié des patients présentant une protéine KRAS wild-type ne répond pas non plus au cetuximab. Afin de découvrir des nouveaux biomarqueurs de sélection des patients qui pourraient bénéficier de ce traitement ou d'améliorer la réponse des patients, une meilleure connaissance des mécanismes d'action du cetuximab est nécessaire. Par exemple récemment, des études ont montré que la protéine p53 participait à la réponse au cetuximab. Il serait intéressant de savoir si le statut de cette protéine peut constituer un bon marqueur de réponse. Précédemment, nous avons montré que l'activation de la « mitogen activated protein kinase » p38 (MAPK p38) induit une résistance à l'irinotécan in vitro et in vivo et est un marqueur de non réponse à cette drogue. De plus, d'autres équipes ont montré que la MAPK p38 bloque également la réponse au 5-FU et participe à la cytotoxicité de l'oxaliplatine. Il semble alors que la MAPK p38 soit impliquée dans les mécanismes d'action des anti-tumoraux. Il est donc intéressant de savoir si la MAPK p38 participe également à la cytotoxicité des thérapies ciblées comme le cetuximab. Pour répondre à ces questions nous avons choisi de comparer deux lignées cellulaires de CCR KRAS wild-type mais au statut TP53 différent et qui répondent différemment au cetuximab: la lignée Caco2 (30% d'inhibition de la survie, TP53 muté) et la lignée DiFi (80% d'inhibition de la survie, TP53 sauvage). Nous avons effectué des tests de cytotoxicité combinant le cetuximab et l'inhibition transcriptionnelle ou pharmacologique de p38 ou l'inhibition transcriptionnelle de p53. Nous avons également testé l'apoptose et la prolifération induite par le cetuximab en l'absence de la MAPK p38 ou de p53. Enfin nous avons testé l'expression des gènes BIM, p27 et PUMA impliqués dans l'apoptose et la prolifération. Dans cette étude, nous avons démontré que la MAPK p38 et p53 participent à l'effet cytotoxique du cetuximab dans les cellules DiFi. En revanche, dans les cellules Caco2, la MAPK p38 bloque partiellement l'effet du cetuximab. Nous avons également montré l'implication de p38 et p53 dans l'apoptose induite par le cetuximab et l'implication de p38 dans l'inhibition de la prolifération. La protéine p38 est impliquée dans la régulation de ERK et dans la localisation nucléaire de FOXO3a responsable de l'expression des gènes BIM et p27. Enfin, nous avons montré que p38 a également un rôle dans le mécanisme d'action d'inhibiteurs de tyrosine kinase ciblant l'EGFR (lapatinib et erlotinib). Pour conclure, nous avons déterminé que p38 et p53 sont impliquées dans le mécanisme d'action du cetuximab. Nous avons décris l'implication de la voie p38-FOXO3a dans le mécanisme d'action du cetuximab. Enfin, p38 et p53 semblent être de bons biomarqueurs de réponse au cetuximab. / Cetuximab is used in colorectal cancer (CRC), as targeted therapy against the Epithelial Growth Factor receptor (EGFR), in association with chemotherapy (5-FU, oxaliplatin and irinotecan). Its binding inhibits signaling pathways downstream to the receptor leading to a decrease in proliferation and surviving. In KRAS mutated CRC patients, Cetuximab is ineffective, and half of KRAS Wild Type (WT) patients does not respond to Cetuximab either. Thus, a better knowledge of Cetuximab mechanism of action will help to improve response rate and to find new biomarkers of response. Recently, studies have shown that p53 protein is involved in the response to cetuximab. It would be interesting to know if the status of this protein could be a biomarker of response. Previously, we have shown that activation of the mitogen activated protein kinase p38 (p38MAPK) induces irinotecan resistance in vitro and in vivo and is a predictive factor of response to irinotecan. Moreover, others teams found that p38 MAPK also partially block 5-FU response and participate to oxaliplatin cytotoxicity. It seems that p38 is involved in mechanism of action of anti-tumor agent. The aim of our project is to determine, in KRAS WT colorectal cells, if p38 MAPK is involved as well in the cetuximab effect. In this aim, experiments were done on two KRAS WT CRC cell lines but with different p53 status which respond differently to Cetuximab: Caco2 cells (30% of survival inhibition, TP53 mutated) and DiFi cells (80% of survival inhibition, TP53 wild-type). Cytotoxic experiments combining cetuximab treatment and inhibition of p38MAPK or p53 by transcriptional inhibition or using a pharmacological inhibitor of p38 (SB202190) were performed. We assessed apoptosis and inhibition of proliferation by FACS analysis of cell cycle and DNA synthesis. In addition, BIM, PUMA and p27 expression were analyzed by QPCR and Western Blot. Our results showed that inhibition of p38MAPK enhances Cetuximab cytotoxic effect in Caco2 cells but impairs it in DiFi cells as inhibition of p53. We also observed that inhibition of p38 MAPK and p53 decreases cetuximab induced apoptosis and inhibition of p38 decrease anti-proliferative effect in DiFi cells. The prevention of cell death by SB202190 in cetuximab treated DiFi cells could be explained by ERK pathway activation and the decrease of FOXO3a nuclear localization leading to p27 and BIM expression decrease, respectively involved in cellular proliferation and mitochondrial apoptosis. Our results have shown the same inhibiting effect of SB202190 on the cytotoxic effect of two tyrosine-kinase inhibitors targeting EGFR (lapatinib and erlotinib) in DiFi cells indicating that p38MAPK implication is linked to inhibition of EGFR kinase activity not to Cetuximab only. We have shown that p38MAPK is involved in response to the inhibition of EGFR activity via nuclear localization of FOXO3a. p53 protein has also a role in cetuximab response. Both seem to be predictive factors of response to cetuximab therapy.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

This project was carried out in the Cell Signaling Research Group headed by Dr. Carme Caelles at IRB Barcelona. As a part of the research-line that deals with physiological and pharmacological (anti-inflammatory and/or anti-diabetic) actions conducted by some nuclear receptor (NR) ligands through negative interference with the c-Jun N-terminal kinase (JNK) signaling pathway, this project was focused on studying the mechanism of cross-talk between those pathways. The results of the study show the ligand-dependent LXR inhibition of the LPS-activated SAPK (JNK and p38MAPK) pathways. Moreover, PP5, a serine/threonine phosphatase previously shown to regulate MAPK pathways, is suggested as a novel target of LXR that negatively regulates LPS-induced activation of SAPK pathways. Furthermore, it is proposed that through the inhibition of SAPK activity, and thereby cJun/AP-1 activity, PP5 is mediating negative regulation of LPS-induced Mmp13 gene expression by LXR in murine primary macrophages. / Este proyecto se llevó a cabo en el Grupo de Investigación en Señalización Celular del IRB Barcelona y fue dirigido por la Dra. Carme Caelles. El trabajo se centra en el estudio del mecanismo de interferencia entre las vías de los receptores nucleares (NR) y la señalización de la quinasa c-Jun N-terminal Kinase (JNK). Esta inhibición forma parte de la línea investigación sobre las acciones fisiológicas y farmacológicas (anti-inflamatorias y / o anti-diabéticas) realizadas por los ligandos de algunos NR. El estudio demuestra la inhibición de las vías SAPK (JNK y p38MAPK) en respuesta a LPS a través de la activación dependiente de ligando de LXR. Además, PP5, una fosfatasa serina/treonina que previamente se demostró que regula las vías de las MAPKs, se sugiere como el mediador de esta inhibición. Esta interacción estaría inhibiendo la expresión en respuesta a LPS del gen Mmp13 en macrófagos de ratón.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.