Abstract Background The majority of patients requiring mechanical ventilation are over the age of 65 and advanced age is known to increase the severity of ventilator-induced lung injury (VILI) and mortality. However, the mechanisms which predispose aging ventilator patients to increased mortality rates are not fully understood. Pulmonary edema is a hallmark of VILI and the severity of edema increases with age. Ventilation with conservative fluid management decreases mortality rates in acute respiratory distress (ARDS) patients, but has not been investigated in VILI. We hypothesized that age-associated increases in pulmonary edema promote age-related increases in ventilator-associated mortality. Endoplasmic reticulum (ER) stress can disrupt cellular functions and plays a key role in many disease states. The severity of ER stress also increases with age. We hypothesized that age-associated increases in ER stress also increase in the severity of VILI. Finally, serum Vitamin C (VitC) levels also decrease with age. VitC treatments have been shown to decrease mortality rates in murine models of ARDS by and attenuate pulmonary edema. We hypothesize that VitC treatments will attenuate ventilator induced pulmonary edema in our aged murine subjects. Methods Mechanical Ventilation: Young and old mice were mechanically ventilated with either high tidal volume (HVT) or low tidal volume (LVT) for with either liberal or conservative fluid support. One group received VitC treatment prior to ventilation. Cell Stretch: Alveolar epithelial cells (ATIIs) from young and old mice were harvested, cultured, and mechanically stretched. Treatment groups received ER stress inhibitor 4-PBA. Results Both advanced age and HVT ventilation significantly increased inflammation, injury, and decreased survival rates. Conservative fluid support significantly diminished pulmonary edema decreased mortality rates. VitC treatments significantly decreased pulmonary edema and improved pulmonary mechanics. Mechanical stretch promoted ER Stress and upregulated proinflammatory gene expression and secretion in aged ATIIs. ER stress inhibition attenuated all of these effects. Conclusion Conservative fluid management alone attenuated age-associated increases in ventilator-associated mortality. VitC treatments decreased pulmonary edema and partially restore pulmonary mechanics in old mice ventilated with HVT. ER stress inhibition decreased stretch induced proinflammatory gene expression and protein secretion in aged mechanically stretched ATII cells.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

On solid surfaces wild strains of Saccharomyces cerevisiae form biofilm-like, structured colonies enabling them to survive long-term in hostile environments in the wild. However, the molecular mechanisms underlying the spatio-temporal development of colonies and their resistance to hostile conditions are still largely unknown. In this study, we analyzed the effect of the HAC1 gene on the colony morphology of wild strains of S. cerevisiae. The transcription factor Hac1p activates the unfolded protein response (UPR), which leads to activation of the expression of genes encoding components of the protein secretory pathway, and genes involved in stress responses in the endoplasmic reticulum (ER). The impact of HAC1 deletion is significant even under non-stress conditions and causes a radical reduction of structured colony architecture in hac1∆ strains derived from two wild S. cerevisiae strains (PORT and BR-F-Flo11p-GFP) and one laboratory ΣSh strain forming semi-fluffy or fluffy colonies. The hac1∆ strains exhibit a decreased vegetative growth rate, reduced cell attachment to the agar and an ineffective cell-cell adhesion resulting in decreased flocculation. The hac1∆ strains derived from BR-F-Flo11p-GFP contain a low level of Flo11p surface adhesin which is considered very important for the proper...

Ehlers-Danlos Syndrome (EDS) comprises a spectrum of heritable connective tissue disorders with varying genetic origins and clinical manifestations such as soft tissue fragility and skin hyperextensibility. There are multiple EDS subtypes, the first few of which were defined by collagen mutations. Many new EDS variants have been discovered involving mutations that do not necessarily implicate collagen biosynthesis but do involve extracellular matrix (ECM) proteins. One of these proteins, Aortic Carboxypeptidase-Like Protein (ACLP), is a large secreted protein encoded by the AEBP1 (adipocyte enhancer binding protein 1) gene. Previous research has shown that ACLP plays a vital role in binding collagen via its discoidin domain and therefore regulates connective tissue assembly. Thus far, individuals from 7 different families have been identified with different EDS-causing ACLP mutations. Some mutations are ACLP null whereas other mutations lead to expressed mutant ACLP. One of these mutations is characterized by a single-nucleotide deletion that causes the insertion of 40 amino acids in the discoidin domain of ACLP. It is therefore denoted “ACLP-Ins40”. The goal of this research was to characterize the ACLP-Ins40 protein and investigate how mutations in ACLP disrupt ECM homeostasis and cause EDS. We initially sought to determine if the ACLP-Ins40 mutation would alter ACLP’s ability to bind collagen. To achieve this goal we generated expression vectors of full length human ACLP carrying the Ins40 mutation. By western blot, it was determined that ACLP-Ins40 was not secreted from fibroblasts and was retained intracellularly. We then hypothesized that the retention of ACLP-Ins40 in the secretory pathway would induce ER stress due to misfolding. 3T3 fibroblasts were co-transfected with the ACLP-Ins40 expression vector and an XBP1u-EGFP sensor of ER stress. Immunofluorescence imaging revealed that in comparison to WT, fibroblasts expressing ACLP-Ins40 experienced ER stress with significantly increased spliced XBP1. This may then cause cell death, the improper secretion of other important ECM proteins, or defective collagen scaffolding, all which could contribute to symptoms of EDS. These studies contribute to our current understanding of how mutations in the AEBP1 gene and alterations in the ACLP protein cause EDS. This connection provides a framework for future research and for targeted interventions to treat EDS. / 2021-06-17T00:00:00Z

Des altérations de fonctions physiologiques du réticulum endoplasmique (RE) induisent un processus appelé stress RE. Dans le domaine cardiovasculaire, plusieurs travaux ont montré que le stress RE contribue au développement de la majorité des pathologies cardiaques. En réponse au stress RE, la réponse UPR (Unfolded Protein Response) est activée afin de restaurer l’homéostasie du RE et de permettre la survie de la cellule. Néanmoins, dans le cas d’un stress RE excessif ou prolongé, les altérations ne pouvant plus être compensées, la cellule est éliminée par apoptose contribuant au développement de la pathologie cardiaque. Une thérapie prometteuse pour lutter contre ce type de pathologie consisterait donc à moduler la réponse au stress RE afin d’inhiber l’apoptose des cardiomyocytes. Au cours de ma thèse, je me suis intéressé aux modifications induites en réponse au stress RE dans le cœur et au rôle de la sirtuine 1 (SIRT1) dans la modulation de cette réponse. SIRT1 est une déacétylase activée par différents stress cardiaques et connue pour favoriser la survie cellulaire. D’une part, j’ai mis en évidence que le stress RE induit une modification importante de l’architecture des cardiomyocytes et en particulier une augmentation des contacts RE/mitochondries associée à une altération de la fonction mitochondriale. D’autre part, en utilisant une lignée cellulaire (H9c2), des cardiomyocytes de rat adulte et des souris invalidées pour SIRT1, j’ai démontré in vitro et in vivo (i) que SIRT1 est activée et joue un rôle cardioprotecteur en réponse au stress RE, (ii) que SIRT1 limite la réponse UPR en régulant spécifiquement la voie PERK, et (iii) que SIRT1 régule la voie PERK en déacétylant le facteur d’initiation de la traduction, eIF2 sur deux résidus lysine. Ces résultats montrent donc pour la première fois que SIRT1 est impliquée dans la régulation de la réponse apoptotique au stress RE des cardiomyocytes et suggèrent que cette déacétylase serait une cible thérapeutique intéressante pour prévenir l’apoptose dans les pathologies cardiaques liées au stress RE. / Impairment of physiological functions of the endoplasmic reticulum (ER) induces the so-called ER stress. ER stress has been implicated in many cardiovascular diseases including ischemic heart, hypertrophy and heart failure. To overcome the deleterious effect of ER stress, an evolutionarily conserved adaptive response known as Unfolded Protein Response (UPR) is activated in order to restore ER homeostasis and promote cell survival. Nevertheless, in the case of prolonged or severe ER stress, apoptotic cell death is ultimately activated to eliminate stressed cells, thus contributing to the development of the pathology. The modulation of ER stress response, in order to reduce cardiomyocyte apoptosis, thus appears as a promising therapeutic strategy for such pathologies. During my Ph.D thesis, I studied the modification that occur during ER stress response in the heart and the role of the sirtuine 1 (SIRT1) in the modulation of this response. SIRT1 is a deacetylase activated in response to many cardiac stresses to promote cell survival. First, we showed that ER stress induces important structural modifications of cardiomyocytes and in particular an increase in contact sites between ER and mitochondria associated with an alteration of the mitochondrial function. Secondly, using a cell line (H9c2), freshly isolated adult rat ventricular cardiomyocytes and SIRT1-KO mice, we demonstrated in vitro and in vivo (i) that SIRT1 is activated and plays a cardioprotective role in ER stress response, (ii) that SIRT1 attenuates the UPR by specifically regulating the PERK pathway, and (iii) that SIRT1 modulates PERK axis by deacetylating the translation initiation factor, eIF2on two lysine residues. Collectively, our results provide the first evidence that SIRT1 modulates ER stress-induced apoptosis in the heart and suggest that this deacetylase may represent a therapeutic target to prevent apoptosis in cardiac pathologies associated to ER stress.

En réponse aux inducteurs de la mort cellulaire immunogénique, la calréticuline (CRT) est transportée depuis sa localisation orthotopique dans la lumière du réticulum endoplasmique (RE) vers la surface cellulaire où elle va jouer un rôle de signal puissant d’endocytose à l’endroit des cellules présentatrices d’antigène. Ici nous rapportons qu’une autre protéine, la lysyl-ARNt-synthétase (KRS), est exposée à la surface des cellules stressées où elle colocalise avec la CRT au niveau des radeaux lipidiques. La déplétionde KRS à l’aide d’ARNs interférents annihile l’exposition de la CRT induit par les anthracyclines ou les rayonnements UVC. A l’inverse de la CRT, KRS est aussi retrouvée dans le surnageant des cellules stressées. De plus, la protéine KRS recombinante (KRSr) est ici incapable d’influencer la liaison de laCRT recombinante à la surface cellulaire. Et KRSr ne possède pas la capacité de stimuler des macrophages in vitro. Enfin nous révélons que le statut de phosphorylation d’eIF2α constitue un critère différentiel entre l’oxaliplatine et la cisplatine, capable de rendre compte de l’incapacité de cette dernière à entrainer l’exposition de la CRT, et ce malgré une très grande ressemblance chimique entre ces deux composés. Ces résultats mettent donc en exergue la contribution respective de KRS et du stress du RE dans l’émission du signal prototypique de la mort immunogénique. / In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopiclocalization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as a potent engulfment signal for antigen-presenting cells. Here, we report that another protein, the lysyl-tRNA-synthetase (KRS), was exposed on the surface of stressed cells, on which KRS co-localized with CRT in lipid rafts. Depletion of KRS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KRS was also found in the supernatant of stressed cells. Recombinant KRS (rKRS) protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, rKRS protein was unable to stimulate macrophages invitro. Finally, we reveal that the phosphorylation status of eIF2α constitute a differential criteria between oxaliplatin and cisplatin (CDDP), accounting for the incapacity of CDDP to trigger the exposure ofCRT, despite their structural and chemical similarities. These results underscore the respective contribution of KRS and ER stress to the emission of the prototypic signal of immunogenic cell death.

A adaptação dos cimentos endodônticos, Ketac-Endo, AH Plus e Endomethásone, à dentina radicular submetida à ação prévia dos agentes de limpeza final laser de Er:YAG, EDTA a 17% e Solução Salina Fisiológica a 0,9% foi estudada pelas microscopias óptica e eletrônica de varredura. Foram empregados 90 caninos humanos extraídos, que após instrumentação pela técnica escalonada de memória e irrigação com hipoclorito de sódio a 1%, foram divididos em três grupos de 30, de acordo com o agente de limpeza final empregado. No grupo A, empregou-se o laser de Er:YAG intracanal, com parâmetros de 100mJ (energia real de 44mJ), 10pps, durante dez segundos, sob refrigeração a ar, com movimentos circulares no sentido apicocoronário, seguido de irrigação com 20ml de solução salina fisiológica a 0,9%. No grupo B, empregaram-se 5ml de EDTA a 17% durante cinco minutos, seguido de irrigação com solução salina fisiolólgica a 0,9%. No grupo C, empregaram-se 20ml de Solução Salina Fisiológica a 0,9%. Após secagem, os canais radiculares foram armazenados em solução salina fisiológica a 0,9% durante 48 horas. Cada grupo foi então dividido em três subgrupos de dez,de acordo com o cimento endodôntico. Os 90 canais radiculares foram obturados pela técnica clássica, seguida da condensação lateral ativa da guta-percha. Após comprovação radiográfica da obturação os canais foram vedados com Cimpat no terço cervical e forame apical, e as raízes armazenadas em solução salina fisiológica a 0,9% em umidade de 100% a 37oC, durante 30 dias. Em seguida, foram seccionadas transversalmente a 5mm do ápice radicular, fotografadas com aumento de quatro vezes e submetidas à análise em microscopia óptica. Para a análise em microscopia eletrônica de varredura, as secções apicais foram moldadas em silicona Aquasil e os moldes obtidos foram metalizados e fotografados em aumentos que variaram de 50 a 200 vezes. As fotos obtidas pelas microscopias foram escaneadas e transferidas para um programa de medida de área (SigmaScan) para mensuração das áreas preenchidas pelos materiais obturadores e das possíveis fendas entre material obturador e parede do canal radicular. Diante dos resultados obtidos com a metodologia empregada e da discussão apresentada pudemos concluir que: 1- Os agentes de limpeza final empregados exerceram efeito diferenciado na adaptação dos cimentos testados. O laser de Er:YAG interferiu na adaptação de todos os cimentos, o EDTA melhorou a adaptação do Endomethásone e do AH Plus em relação ao laser de Er:YAG e à solução salina fisiológica a 0,9% e a solução salina fisiológica melhorou a adaptação do Ketac-Endo, em ambas microscopias empregadas. 2- O Endomethásone apresentou a pior adaptação entre os cimentos e o AH Plus apresentou a melhor adaptação com EDTA na microscopia eletrônica de varredura. 3- A associação das microscopias óptica e eletrônica de varredura mostrou ser um método confiável na detecção das fendas, sendo que a microscopia eletrônica de varredura propiciou melhor visualização. 4- Todos os cimentos testados apresentaram alteração dimensional, verificando-se contração para o Ketac-Endo, expansão para o Endomethásone e AH-Plus, sendo que o AH Plus mostrou-se mais próximo da estabilidade dimensional. / The adaptation of endodontic sealers, Ketac-Endo, AH Plus and Endomethásone to dentinal root submitted to the previous action of final Er:YAG laser cleaning agents, EDTA at 17% and a saline solution at 0.9% was studied by Optical and Scanning Electron Microscopy. Ninety extracted human canines were employed, and following the step back technique preparation and irrigation with sodium hypochlorite solution they were divided into three groups with thirty teeth each, according to the final cleaning agent used. In group A, Er:YAG laser was used intracanal, with the following parameters: 100mJ (real energy 44mJ), 10pps, for 10 seconds under air cooling with circular movements in apicocervical direction and the canals were irrigated with 20ml saline solution at 0.9%. In group B, 5ml of EDTA at 17% irrigated the root canals for 5 minutes, followed by a saline solution at 0.9%. In group C, a saline solution at 0.9% was employed to irrigate the root canals. After drying, the roots were stored in a saline solution at 0.9% for 48 hours. Each group was divided into three subgroups of ten, according to the selected endodontic sealer. The ninety root canals were filled through standard technique, followed by gutta-percha lateral condensation. Following radiographic filling proof, the root canals were sealed with Cimpat in the cervical third and at the apical foramen, and the roots stored in a 0.9% saline solution at 37oC and 100% humidity for 30 days. Afterwards, they were transversally secctioned at 5mm to the root apex, and photographed with a 4x magnification, and submitted to optical microscopic analysis. Before the Scanning Electron Microscopic analysis, the apical root sections were molded with Aquasil and the obtained replicas metallized and photographed with 50x and 200x magnifications. The resulting images, in both microscopies, were scanned and transferred to the area measurement software program (SigmaScan) to measure the areas filled by endodontic materials and the failed areas between the endodontic material and the root canal walls. Through the results with the selected methodology and by means of the presented discussion, we could conclude that: 1- The final cleaning agents employed in the root canals presented different effects in the adaptation of the endodontic sealers. The Er:YAG laser was harmful to the adaptation of all the endodontic sealers; the EDTA improved the adaptation of Endomethásone and AH Plus and the 0.9% saline solution improved the adaptation of Ketac-Endo in both microscopies. 2- Endomethásone presented the worst adaptation compared to the other sealers and AH Plus presented the best adaptation with EDTA by Scanning Electron Microscopic analysis. 3- The Optical Microscopy associated with the Scanning Electron Microscopy proved to be a safe method in detecting filling failures, being, the Scanning Electron Microscopy the best. 4- All endodontic sealers presented dimensional alterations, Ketac-Endo presented contraction, Endomethásone and AH Plus presented expansion, being, AH Plus closer to dimensional stability.

Au cours de cette thèse, nous nous sommes intéressés à la différenciation normale et pathologique des cellules germinales (CGs). Dans une première partie, la différenciation normale a été étudiée chez l'amphibien urodèle Pleurodeles waltl. L'acide rétinoïque (AcR) est impliqué dans l'induction de l'entrée en méiose des cellules germinales (CGs) chez les amniotes (Bowles et al., 2006 ; Smith et al., 2008). Nous avons voulu savoir si l'AcR était aussi impliqué dans ce processus chez le pleurodèle. Nous avons montré que l'expression de PwDmc1 (marqueur de méiose), survient plus précocement dans les gonades femelles (avant la métamorphose) que dans les gonades mâles (après la métamorphose). In vitro, l'application d'AcR sur des gonades mâles et femelles en culture organotypique ainsi que l'inhibition de la dégradation de l'AcR endogène induisent l'entrée en méiose des CGs. Nous avons également montré que la balance synthèse/dégradation l'AcR endogène est modulée par les hormones stéroïdes. Les mécanismes de différenciation des CGs décrits chez les mammifères semblent donc conservés chez les urodèles. La seconde partie de la thèse s'inscrit dans le contexte de la différenciation pathologique des CGs.De nombreuses études ont montré qu'une exposition in utero à des xénobiotiques pouvait provoquer des altérations de la différenciation des CGs. Ces cellules altérées restent en dormance pendant l'enfance. A la puberté, elles prolifèrent et donnent naissance aux tumeurs testiculaires, suggérant que le développement de ces tumeurs est hormono-sensible (McIntyre et al., 2008). Nous avons étudié les effets des hormones stéroïdes sur la prolifération des cellules séminomateuses humaines TCam2. L'oestradiol et la testostérone stimulent leur prolifération ainsi que l'expression d'une isoforme tronquée du récepteur alpha des oestrogènes ER[alpha]36. Ce récepteur est induit par la voie GPER-AMPc/PKA et semble nécessaire à l'expression du récepteur à l'EGF. Il pourrait donc constituer une cible thérapeutique potentielle. / Retinoic acid (RA) is involved in germ cells meiosis onset (CGs) in amniotes (Bowles et al., 2006 ; Smith et al., 2008). The aim of the first part of our study was to determine if RA is able to induce meiosis entry in the urodele amphibian Pleurodeles waltl. Our data on PwDmc1 (meiosis marker) expression indicate an earlier meiosis onset in females compared with males depending on RA biosynthesis. In vitro, the treatment of male and female gonads with RA induces an earlier meiosis entry in both sexes. Moreover, we have shown that RA biosynthesis is regulated by steroid hormones. Therefore, the mechanisms which trigger GCs differentiation in mammals might be conserved in urodeles. Many studies indicated that in utero xenobiotic exposure is able to induce alterations in GCs differentiation. These neoplasic cells enter in a period of dormancy until after puberty where they proliferate and the testicular tumours emerge. This prepubertal dormancy suggests that the testicular cancer development is hormone sensitive (McIntyre et al., 2008). In the second part we studied the effects of steroid hormones on the proliferation of the seminoma cell line TCam2. Our results indicate that estradiol and testosterone can induce the proliferation of this cell line and the expression of ER[alpha]36, a truncated form of estrogen receptor alpha. This expression is stimulated by GPER-cAMP/PKA signalisation pathway and activates EGFR expression. ER[alpha]36 could be a potential therapeutic target.

Submitted by PPG Comunica??o Social (famecos-pg@pucrs.br) on 2018-05-14T11:08:38Z No. of bitstreams: 1 LUIZ_GUILHERME_DOS_SANTOS_JUNIOR_TES.pdf: 3115882 bytes, checksum: 07a8cceae2d838a2c5081b04988d9878 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-05-17T11:20:05Z (GMT) No. of bitstreams: 1 LUIZ_GUILHERME_DOS_SANTOS_JUNIOR_TES.pdf: 3115882 bytes, checksum: 07a8cceae2d838a2c5081b04988d9878 (MD5) / Made available in DSpace on 2018-05-17T11:32:46Z (GMT). No. of bitstreams: 1 LUIZ_GUILHERME_DOS_SANTOS_JUNIOR_TES.pdf: 3115882 bytes, checksum: 07a8cceae2d838a2c5081b04988d9878 (MD5) Previous issue date: 2018-03-26 / Cette th?se pr?sente une analyse des mises en sc?ne de l??rotique-grotesque dans le cin?ma de Cla?dio Assis ? partir des films Amarelo Manga (2002), Baixio das Bestas (2006) et Febre do Rato (2011). Nous avons comme objectif d?analyser les repr?sentations de l??rotique-grotesque par certains personnages de la trilogie filmique de Cla?dio Assis, et ? travers de cat?gories li?es ? la mise en sc?ne cin?matographique, notamment les cadrages, les plans, les angles, la lumi?re et les mouvements de cam?ra. En ce qui concerne la mise en sc?ne, nous proposons un dialogue entre le th??tre et le cin?ma dans le but de mieux comprendre les diff?rences de focalisation, d?apr?s les ?tudes de Pavis (2010), Aumont (2007), Comolli (2008), Oliveira Jr. (2013), Bordwell Thompson (2013) et Bazin (2014). Pour l?analyse du grotesque, nous partons des concepts des auteurs qui se penchent sur ce th?me dans les ?tudes litt?raires, le th??tre et les arts visuels. Pour la compr?hension du grotesque, nous utilisons les apports de Hugo (2007), Kaiser (1986), Stam (1992), Russo (2000) et Sodr? & Paiva (2002), soulignant les propositions conceptuelles de Bakhtin (2013) sur les analogies entre corps et ? image grotesques ?, mais aussi Didi- Huberman (2015) qui analyse le corps ? difforme ? d?apr?s la pens?e esth?tique de Georges Bataille. Pour la m?thodologie, nous suivons le concept de ? d?coupage ? sugg?r? par Gardies (2006) sur a place des corps dans les dimensions visuelles du cadrage. Nous concluons que les films de la trilogie de Cl?udio Assis, malgr? les diff?rences d?ordre th?matique, mettent en sc?ne l??rotique-grotesque ? partir de la r?currence d?images qui comprennent le bas du corps, les comportements abjects, la violence, la cruaut?, le difforme et les transgressions ?rotiques. / A tese analisa as encena??es do er?tico-grotesco no cinema de Cl?udio Assis, a partir dos filmes Amarelo Manga (2002), Baixio das Bestas (2006) e Febre do Rato (2011). Objetivamos analisar as representa??es do er?tico-grotesco a partir de determinados personagens da trilogia f?lmica de Cl?udio Assis, e por meio de categorias referentes ? encena??o cinematogr?fica, dentre elas, enquadramentos, planos, ?ngulos, ilumina??o e movimentos de c?mera. No que diz respeito ? encena??o, realizamos um tr?nsito entre o teatro e cinema, com o intuito de compreender as diferen?as de enfoque, segundo os estudos de Pavis (2010); Aumont (2006); Comolli (2008); Oliveira Jr. (2013), Bordwell; Thompson (2013) e Bazin (2014). Para a an?lise do grotesco, partimos de conceitos oriundos de autores que versam sobre o tema nos estudos liter?rios, no teatro e nas artes visuais. Para o entendimento do grotesco, utilizamos os aportes de Hugo (2007); Kaiser (1986); Stam (1992); Russo (2000) e Sodr? & Paiva (2002), com ?nfase nas proposi??es conceituais de Bakhtin (2013) acerca das analogias entre corpo e ?imagem grotesca?; e Didi-Huberman (2015), que analisa o corpo ?disforme? com base no pensamento est?tico de Georges Bataille. Para a metodologia e o m?todo de an?lise, seguimos o conceito de ?decupagem? sugeridos por Aumont e Marie (2004); para o m?todo anal?tico dos frames, partimos das sugest?es de Gardies (2006) sobre o lugar dos corpos nas dimens?es visuais do enquadramento. Centralizamos a an?lise em dois personagens de cada filme, com a inten??o de interpretar os modos de representa??o do grotesco no espa?o dos quadros (enquadramentos). Conclu?mos que os filmes da trilogia de Cl?udio Assis, mesmo com diferen?as de cunho tem?tico, encenam o er?tico-grotesco a partir da recorr?ncia de imagens que abrangem o baixo-corporal, comportamentos abjetos, viol?ncia, crueldade, o disforme e transgress?es er?ticas.

Fibroblastos são células essenciais na cicatrização da derme, sintetizando e degradando matriz extracelular (MEC) e sua função é alterada pela hiperglicemia. Demonstramos previamente uma deficiência na migração de fibroblastos de ratos diabéticos, acompanhada de dilatação das cisternas do retículo endoplasmático (RE) e aumento no número de mitocôndrias. Neste estudo avaliamos se a glicose elevada (HG) induz estresse do RE, resultando na ativação da via UPR (Unfolded Protein Response). Conforme observado por microscopia eletrônica de transmissão, fibroblastos NIH-3T3 expostos por 3 dias a HG apresentaram dilatação do RE e alteração na morfologia das mitocôndrias. A expressão dos marcadores de estresse do RE: BiP, XBP1 spliced (XBP1s) e CHOP não foi alterada pela HG. Resultados semelhantes foram observados em fibroblastos de ratos diabéticos. A marcação com um indicador fluorescente do potencial de membrana mitocondrial não foi modificada pela glicose elevada. Esses resultados sugerem que a disfunção de fibroblastos frente à HG não está relacionada a estresse de RE. / Fibroblasts are essential cells during dermis wound healing, synthesizing and degrading extracellular matrix (ECM); their function is altered by hyperglycemia. We have previously demonstrated an impairment of migration of fibroblasts derived from diabetic rats, accompanied by the enlargement of endoplasmic reticulum (ER) cisternae and an increase in the number of mitochondria. This study assessed whether high glucose (HG) induces stress of ER, resulting in the activation of UPR (Unfolded Protein Response). As observed by transmission electron microscopy, NIH-3T3 fibroblasts exposed for 3 days to HG showed enlargement of the ER cisternae and changes in the morphology of mitochondria. The expression of the ER stress markers: BiP, spliced XBP1 (XBP1s) and CHOP was not altered by HG. Similar results were observed in fibroblasts derived from diabetic rats. Labeling with a fluorescent indicator of mitochondrial membrane potential was not altered by high glucose. These results suggest that dysfunction of fibroblasts exposed to HG is not related to ER stress.