Orientador: Patrícia de Oliveira Prada / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-23T21:18:55Z (GMT). No. of bitstreams: 1 Castro_Giselede_M.pdf: 1355339 bytes, checksum: 9e121a8d79b7d1be25b7e55dcea89b10 (MD5) Previous issue date: 2013 / Resumo: A obesidade é uma doença caracterizada pelo acúmulo excessivo de gordura corporal, acarretando diversas alterações metabólicas. Estas alterações influenciam o controle do balanço energético. O hipotálamo é considerado o órgão chave para o controle do balanço energético. Recebe informações da periferia por meio de nutrientes, sinais neurais e hormônios. Dentre estes hormônios, a insulina participa do controle da ingestão alimentar, tendo efeitos anorexigênicos. Esta regulação ocorre pela ativação de sua via de sinalização IR/PI3q/Akt. Em animais obesos ocorre uma menor ativação desta via como consequência ao estresse do retículo endoplasmático e ativação de proteínas da via inflamatória. No presente estudo destacamos a amígdala como uma importante região do sistema nervoso central envolvida na regulação da ingestão alimentar em resposta à insulina. Essa regulação foi dependente da ativação da via PI3q, pois a injeção de LY aboliu os efeitos da insulina no controle da ingestão. A insulina diminuiu os níveis de RNAm do neuropeptídeo orexigênico NPY e aumentou os níveis do RNAm do neuropeptídeo anorexigênico ocitocina na amígdala de animais controles via PI3q, o que pode ter contribuído para a hipofagia. Além disso, em animais obesos não houve redução na ingestão alimentar em resposta a insulina e a fosforilação da Akt foi diminuída na amígdala, sugerindo resistência à insulina nessa região do sistema nervoso central. A resistência à insulina foi associada com o estresse do retículo endoplasmático e inflamação na região da amígdala. A inibição do estresse do retículo endoplasmático com PBA (ácido fenil butírico), inibidor do estresse de retículo, reverteu a ação e sinalização da insulina, diminuindo a expressão do RNAm do NPY e aumentando a expressão do RNAm da ocitocina na amígdala de animais obesos. Em conjunto, os resultados sugerem que a amígdala é sensível a ação anorexigênica da insulina, sendo este efeito dependente da via PI3q. A ação da insulina na amígdala provavelmente modula a ingestão por alterar os níveis de neuropeptídeos como NPY e ocitocina. Animais obesos apresentam resistência à ação e sinalização da insulina em amígdala. O mecanismo molecular pelo qual ocorre resistência à insulina parece estar relacionado à ativação da via inflamatória e ao estresse de retículo endoplasmático / Abstract: Insulin acts in hypothalamus decreasing food intake (FI) by IR/PI3K/Akt pathway. This pathway is impaired in obese animals and endoplasmic reticulum (ER) stress and low grade inflammation are possible mechanisms involved in this impairment. Here, we highlighted amygdala as an important brain site of FI regulation in response to insulin. This regulation was dependent on PI3K/AKT pathway similar to the hypothalamus. Insulin was able to decrease NPY and increase oxytocin mRNA levels in the amygdala via PI3K, which may contribute to hypophagia. Additionally, obese rats did not reduce FI in response to insulin and AKT phosphorylation was decreased in the amygdala, suggesting insulin resistance. Insulin resistance was associated with ER stress and low grade inflammation in this brain region. The inhibition of ER stress with PBA reverses insulin action/signaling, decreases NPY and increases oxytocin mRNA levels in the amygdala from obese rats, suggesting that ER stress is probably one of the mechanisms that induce insulin resistance in the amygdala / Mestrado / Metabolismo e Biologia Molecular / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

Les signalisations oncogéniques induisent une consommation accrue de glucose qui n'est que partiellement satisfaite par le microenvironnement. Pour s'adapter et survivre à ce stress métabolique, les cellules malignes mettent en jeu des mécanismes qui restent mal compris. Nos travaux montrent que cette limitation en glucose a pour principale conséquence de déclencher une apoptose via la voie de signalisation PERK-CHOP de la réponse à un stress du réticulum endoplasmique (SRE), nommée Unfolded Protein Response (UPR). Nous avons découvert que le RE est capable de sentir la carence en glucose via la diminution de la disponibilité en UDP Nacétylglucosamine produit par la voie des hexosamines. La délétion du facteur pro-Apoptotique CHOP dans un modèle de cancer spontané du poumon induit par KrasG12V chez la souris augmente l'incidence tumorale, confirmant que le SRE constitue un mécanisme cellulaire de sauvegarde anti-Tumoral. Nous montrons également que le franchissement de cette barrière implique l'atténuation sélective de la voie PERK-CHOP par la protéine chaperon p58IPK, qui permet aux cellules de bénéficier en retour des effets protecteurs des autres voies d'un UPR devenu chronique. Ces résultats révèlent une dualité fonctionnelle pour le stress du RE dans la tumorigenèse contrôlée, au moins pour partie, par la protéine p58IPK / During carcinogenesis, oncogene activation induces high glucose avidity that outstrips the microenvironment supply until angiogenesis occurs. How malignant cells cope with this potentially lethal metabolic stress remains poorly understood. We found that oncogene-Driven glucose shortage triggers apoptosis through the PERK-CHOP pathway of the endoplasmic reticulum (ER) unfolded protein response (UPR). Deletion of the pro-Apoptotic UPR effector CHOP in a mouse model of KrasG12V induced lung cancer increases tumour incidence, strongly supporting the notion that ER stress serves as a barrier to malignancy. Overcoming this barrier requires the selective attenuation of the PERK-CHOP arm of the UPR by the molecular chaperone p58IPK. Furthermore, p58IPK-Mediated adaptive response enables cells to benefit from the protective features of chronic UPR. Altogether, these results show that ER stress activation and p58IPK expression control the fate of malignant cells facing glucose shortage

Orientador: Marcelo Fábio Gouveia Nogueira / Resumo: Embora estágios iniciais de embriões de mamíferos tenham aparentemente uma grande plasticidade, eles, assim como o oócito em maturação, são bastante sensíveis a estresses exógenos. Responder a esses estresses é uma parte vital da fisiologia celular. Cada vez mais é aparente que um dos principais mecanismos, para iniciar a resposta celular a uma variedade de estressores exógenos, reside no retículo endoplasmático (RE). O RE é uma importante organela responsável pela síntese, processamento e transporte de proteínas e lipídeos. No entanto e in vitro, a perturbação do microambiente do RE pode causar alterações na estrutura das proteínas levando ao acúmulo de proteínas com o incorreto dobramento, uma condição denominada estresse do RE. Dependendo da intensidade do estresse, o RE pode desencadear a apoptose pela Unfolded Protein Response. Em várias espécies, estudos observaram uma melhoria da competência do oócito e no desenvolvimento do embrião quando foram adicionados inibidores do estresse do RE no meio de maturação e/ou meio de cultivo in vitro (CIV). Dentre os inibidores do estresse do RE, o ácido tauroursodesoxicólico (TUDCA) se mostrou benéfico na produção in vitro de embriões. Entretanto, pouco é conhecido sobre o seu efeito na maturação oocitária e no desenvolvimento do embrião bovino in vitro. Portanto, os objetivos gerais desta Tese foram: Capítulo 1 - investigar o efeito da adição de TUDCA durante a maturação in vitro (MIV) sobre a competência do oócito e do embrião [ma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although early stages of mammalian embryos appear to retain great plasticity, they and maturing oocytes are quite sensitive to exogenous stresses. Responding to those stresses is a vital part of cellular physiology, and it is increasingly apparent that one of the main mechanisms for initiating that response is based on the endoplasmic reticulum (ER). ER is an important organelle responsible for the synthesis, processing, folding and transport of proteins and lipids. However, in vitro, the disturbance of the ER microenvironment can cause changes in the structure and folding of proteins leading to the accumulation of misfolding proteins, a condition called ER stress. Depending on the intensity of the stress, the ER can trigger apoptosis by Unfolded Protein Response. In several species, studies have reported an improvement in oocyte and embryo competence when ER stress inhibitors have been added to the in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. Among the ER stress inhibitors, tauroursodeoxycholic acid (TUDCA) has been shown to be beneficial in in vitro embryo production. However, little is known about TUDCA’s effect on oocyte maturation and the development of bovine embryo in vitro. Therefore, the main objectives of this Thesis were: Chapter 1 - to investigate the effect of adding different TUDCA concentrations during IVM on the oocyte and embryo competence [nuclear maturation, mitochondrial activity, reactive oxygen species (ROS) production, pronucle... (Complete abstract click electronic access below) / Doutor

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23080号 / 医博第4707号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 伊藤 貴浩, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Enterohemorrhagic E. coli (EHEC) infections are a leading cause of foodborne illness in the United States. Shiga-like toxins are produced that can cause hemorrhagic colitis and can lead to dangerous complications, such as acute kidney injury and hemolytic uremic syndrome (HUS). There are currently no specific treatments for HUS, and therefore more research into EHEC and HUS needs to be done. Our study focuses on Shiga-like toxin induction of endoplasmic reticulum (ER) stress in in vitro and in vivo systems, using human monocyte-like THP-1 cells and a non-human primate model of HUS. We used qPCR to determine the levels of ER stress marker expression induced by both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) challenges. We also looked at ER stress marker expression in non-human primates that survived a lethal Stx2 challenge after being given a Stx2 binding tetravalent peptide. We expected to see increased ER stress marker expression in THP-1 cells challenged with both Shiga-like toxins and in animals that received lethal doses of the toxins. Although results were inconclusive for THP-1 cell experiments, our preliminary non-human primate data suggest that the timing of ER stress marker production is important, and Shiga-like toxins may suppress the unfolded protein response (UPR) in some baboon tissues. We also show that the therapeutic peptide TVP may reverse this UPR suppression and relieve ER stress leading to animal survival. Our study, along with the current literature, shows that Shiga-like toxin induced ER stress is a promising area for future study.

CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36-/- mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36+/+ mice. CD36-/- mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36-/- mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.

Excessive endoplasmic reticulum (ER) stress leads to cell loss in many diseases, e.g., contributing to endothelial cell loss after spinal cord injury. Here, we determined whether ER stress-induced mitochondrial dysfunction could be explained by interruption of the focal adhesion kinase (FAK)-mitochondrial STAT3 pathway we recently discovered. ER stress was induced in brain-derived mouse bEnd5 endothelial cells by thapsigargin or tunicamycin and caused apoptotic cell death over a 72 h period. In concert, ER stress caused mitochondrial dysfunction as shown by reduced bioenergetic function, loss of mitochondrial membrane potential and increased mitophagy. ER stress caused a reduction in mitochondrial phosphorylated S727-STAT3, known to be important for maintaining mitochondrial function. Normal activation or phosphorylation of the upstream cytoplasmic FAK was also reduced, through mechanisms that involve tyrosine phosphatases and calcium signaling, as shown by pharmacological inhibitors, bisperoxovanadium (bpV) and 2-aminoethoxydiphenylborane (APB), respectively. APB mitigated the reduction in FAK and STAT3 phosphorylation, and improved endothelial cell survival caused by ER stress. Transfection of cells rendered null for STAT3 using CRISPR technology with STAT3 mutants confirmed the specific involvement of S727-STAT3 inhibition in ER stress-mediated cell loss. These data suggest that loss of FAK signaling during ER stress causes mitochondrial dysfunction by reducing the protective effects of mitochondrial STAT3, leading to endothelial cell death. We propose that stimulation of the FAK-STAT3 pathway is a novel therapeutic approach against pathological ER stress.

Persistent viral infections are a major health concern, with persistently infected (PI) cells being a source of continued shedding of virus and generation of viral mutants. Here, we hypothesized that cells persistently infected with the enveloped virus parainfluenza virus 5 (PIV5) would show altered expression of endoplasmic reticulum (ER) stress proteins and increased resistance to death caused by drug-induced ER stress. To test this, lysates of mock-infected, PIV5 acute-infected, and PIV5 PI human lung A549 cells were collected and levels of ER stress proteins were compared. Western blotting revealed that immunoglobulin heavy chain binding protein (BiP/GRP78) was present in higher levels in acute-infected and PI cells compared to naïve cells, indicating increased ER stress in both acutely infected and PI cells. Interestingly, basal levels of the ER stress-sensing protein IRE1-alpha were upregulated in PI compared to naïve and acutely infected cells, but PI cells showed decreased activation of IRE1-alpha compared to acutely infected cells. Naïve, acute-infected, and PI A549-NLR cells were treated with ER stress-inducing drugs tunicamycin, thapsigargin, and epigallocatechin gallate and monitored in real-time viability assays for drug-induced cell death. PI cells showed lower levels of stress-induced cell death compared to naive cells, whereas acute-infected cells experienced the greatest extent of cell death when challenged with ER stress-inducing drugs. Together, these results support the hypothesis that PIV5 persistently infected cells display altered ER stress response pathways and that PI cells are more resistant to death caused by ER stress-inducing drugs. Additionally, these results suggest that IRE1-alpha plays a key role in the shift from acute to persistent infection. These results have implications for the treatment of persistent viral infections, as well as the potential for these viruses to be used for oncolytic virotherapy in the future.

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Pancreatic beta-cells are responsible for secreting insulin into the circulation to maintain whole body glucose homeostasis. While pancreatic beta-cells have a large capacity to secrete insulin, their function progressively deteriorates during the pathogenesis of type 2 diabetes as a result of both genetic predisposition and environmental factors. Obesity is the largest risk factor for developing type 2 diabetes and is associated with various conditions that can impair normal beta-cell function, including excess free fatty acids, inflammation and insulin resistance. Accumulating evidence in the literature suggests that endoplasmic reticulum (ER) stress contributes to the molecular mechanism of pancreatic beta-cell failure during the progression of type 2 diabetes. In this thesis, I have examined the role of the ER stress sensor ATF6-alpha and also the ER-resident chaperone GRP78 in pancreatic beta-cell homeostasis and function. Work presented in Chapter 2 examined the function of naturally occurring ATF6-alpha protein variants associated with type 2 diabetes. I also examined the role of endogenous ATF6-alpha in pancreatic beta-cells, which is described in Chapter 3. Results from these analyses suggest that the ATF6-alpha gene is not a type 2 diabetes susceptibility gene; however, ATF6-alpha protein expression is important to beta-cell function and survival. Finally, ER stress markers have been detected in pancreatic beta-cells and insulin sensitive tissues (such as adipose and liver), which promote beta-cell dysfunction and insulin resistance, respectively. In Chapter 4, I examined the contribution of ER stress in beta-cell dysfunction specifically by generating transgenic mice over-expressing GRP78. The mice were subsequently challenged by high fat diet to determine their susceptibility to developing symptoms of type 2 diabetes. Indeed increased chaperone capacity in pancreatic beta-cells protected against obesity-induced glucose intolerance and insulin resistance. Overall, these data support the hypothesis that ER stress contributes to beta-cell dysfunction in type 2 diabetes progression.