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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

Régulation de l'expression des immunoglobulines au cours du développement lymphocytaire B tardif / Regulation of immunoglobulins expression during late B lymphocytes development

Ashi, Mohamad Omar 30 March 2018 (has links)
Le processus aléatoire des recombinaisons V(D)J permet d’obtenir un répertoire d’anticorps (Ac) ou immunoglobulines (Ig) hautement diversifié. En revanche, le caractère imprécis des jonctions V(D)J conduit à l’apparition de décalages du cadre de lecture dans deux tiers des cas. Ainsi, la plupart des cellules B hébergent des allèles d’Ig avec des réarrangements V(D)J non-productifs au sein de leur génome. Plusieurs études incluant celles menées au laboratoire ont montré que ces allèles non-productifs sont transcrits mais subissent une régulation post-transcriptionnelle impliquant le mécanisme de dégradation des ARNm appelé NMD « Nonsense-Mediated mRNA Decay ». Cette surveillance ARN diminue ainsi le taux d’ARNm codant pour des chaînes d’Ig tronquées. En revanche, l’impact de l’épissage alternatif des transcrits d’Ig non-productifs sur la production d’Ig aberrantes reste jusqu’ici peu exploré. L’étude de ce processus appelé NAS (« Nonsense-associated Altered Splicing »), et en particulier du phénomène de saut d’exon, présente un grand intérêt car cet épissage alternatif peut permettre la synthèse d’Ig tronquées présentant des délétions internes. Les projets développés lors de cette thèse ont révélé la toxicité des chaînes d’Ig dépourvues de domaine variable (V) dans les plasmocytes, et mis en évidence l’existence d’un nouveau point de contrôle au cours de la différenciation plasmocytaire. Ce phénomène nommé TIE-checkpoint (Truncated-Ig Exclusion) conduisant à l’élimination des plasmocytes exprimant des Ig tronquées, est la conséquence d’un saut d’exon lors de l’épissage des transcrits Ig non-productifs. Pour étudier les évènements de NAS lors de l’épissage des transcrits d’Ig dans les plasmocytes, il faut par conséquent limiter l’activation du TIE-checkpoint. A l’aide d’un modèle murin présentant un exon non-sens additionnel au locus IgH, nous avons pu analyser in vivo l’épissage alternatif par « saut d’exon » des transcrits d’Ig non-productifs. En effet, l’élimination de cet exon addtionnel aboutit à la synthèse d’une chaîne d’Ig normale et non à la production de chaînes tronquées. Cette étude a été menée dans des cellules B primaires et des plasmocytes. Les résultats obtenus ont révélé que l’hypertranscription des gènes d’Ig, qui accompagne la différenciation plasmocytaire, favorise l’épissage alternatif des transcrits d’Ig non-productifs, par un phénomène de saut d’exon. Nous avons également étudié les éventuelles connexions entre le mécanisme de NMD, impliqué dans la surveillance des ARNm, et l’UPR (« Unfolded Protein Response ») permettant de réguler l’homéostasie protéique dans les plasmocytes. De façon originale, nous avons identifié une boucle de régulation positive entre les processus de surveillance ARN (NMD) et protéique (UPR, autophagie, protéasome). La mise en évidence de cette coopération dans les plasmocytes constitue un exemple unique au vue de la littérature et, aurait pour effet de limiter la synthèse d’Ig tronquées tout en autorisant la synthèse massive d’Ig. Enfin, nous avons étudié le rôle de l’épissage des transcrits d’Ig non-codants (appelés transcrits I « germinaux ») au cours du processus de CSR « Class Switch Recombination ». Cette étude a apporté des précisions sur le rôle des sites donneurs d’épissage des exons I et révélé que la reconnaissance de ces sites d’épissage module l’intensité de la transcription de la région « switch » S adjacente, et par conséquent, son accessibilité à AID « Activation-Inducedcytidine Deaminase » lors de la CSR. / The random V(D)J recombination process contributes to the generation of a vast immunoglobulin (Ig) repertoire. However, imprecise V(D)J junctions lead to the appearance of frameshift mutations in two-third of the cases. Hence, numerous B-lineage cells retain non-productively V(D)J rearranged Ig alleles in their genome. Several studies including ours have shown that these non-productive alleles are transcribed but rapidly degraded by NMD « Nonsense-Mediated mRNA Decay », thus decreasing the level of mRNA encoding truncated Ig. However, less is known about the impact of alternative splicing on non-productive Ig transcripts, and especially « exon skipping », with regard to the production of truncated Ig with internal deletions. During my thesis, we have shown that truncated Ig chains lacking variable (V) domain exhibted toxic effects in plasma cells revealing a new « Truncated-Ig Exclusion » (TIE-) checkpoint during plasma cell differentiation. The TIE-checkpoint eliminates plasma cell-expressing truncated Ig, as a consequence of exon skipping during splicing of non-productive Igκ transcripts. However, the TIE checkpoint activation limits the analysis of NAS (« Nonsense associated Altered Splicing ») of Ig transcripts in plasma cells. Using a mouse model harboring an additional frameshift-inducing V exon at the IgH chain locus, we could analyze NAS of non-productive Ig transcripts in primary B cells and plasma cells. This study revealed that hypertranscription of Ig genes accompanying plasma cell differentiation favors alternative splicing of non-productive Ig transcripts. We also investigated potential connections between the NMD mechanism, involved in mRNA surveillance, and the UPR (« Unfolded Protein Response ») pathway that regulates protein homeostasis in plasma cells. Interestingly, we identified a positive regulatory loop between RNA (NMD) and protein (UPR, autophagy, proteasome) surveillance processes. In view of the literature, the occurrence of such cooperation is unique to plasma cells, and this should help to limit the expression of truncated Ig while allowing massive Ig synthesis. Finally, we studied other aspects of Ig RNA splicing, and investigated the role of splice donor site on non-coding « germline » I transcripts during CSR (« Class Switch Recombination »). Using dedicated mouse models, we found that the deletion of Iƴ1 splice donor site drastically decreased CSR to IgG1. Overall, this study demonstrated that the recognition of I exon donor splice site enhances transcription of « switch » regions S, facilitating their opening and the subsequent recruitment of AID « Activation-Induced cytidine Deaminase » during CSR.
42

O papel da UPR (Unfolded Protein Response) na resistência a drogas de céluas endoteliais em resposta ao estresse causado pelo pH ácido tumoral

Visioli, Fernanda January 2011 (has links)
A terapia antiangiogênica surgiu como uma alternativa promissora para o tratamento do câncer. No entanto, evidências recentes mostram que as células endoteliais isoladas diretamente de um tumor maligno são mais resistentes a diferentes drogas do que as células endoteliais presentes no mesmo tecido normal. Essas diferenças podem ser atribuídas em parte à adaptação das células endoteliais ao microambiente tumoral. Uma característica singular do microambiente tumoral é a consistente acidificação do meio extracelular, cujos efeitos nas células endoteliais não são conhecidos. Acidez extracelular pode alterar múltiplas funções biológicas, causar estresse do retículo endoplasmático (RE) e ativação da Unfolded Protein Response (UPR). Células endoteliais humanas primárias de derme (HDMEC) cultivadas em pH 6.4, ajustado tanto com ácido lático tanto com ácido clorídrico, apresentaram aumento da expressão de proteínas relacionadas à UPR, como GRP78, ATF4, elf2α fosforilada e aumento na clivagem do mRNA de XBP1. Nessas condições massiva morte celular ocorreu após 48 horas. Em contrapartida, quando as células endoteliais eram expostas à acidez crônica não-letal com pH 7.0 durante sete dias, essas foram capazes de se adaptar coincidentemente com um aumento da expressão da proteína GRP78 Após sete dias sob pH 7.0, as células HDMEC apresentaram maior resistência à morte celular quando tratadas com as drogas Etoposide, Adriamicina e Sunitinib em doses que variavam entre 0.0025μM a 100μM. O silenciamento do gene GRP78 com ShRNA reverteu esse fenótipo resistente. Para determinar os níveis de UPR in vivo utilizou-se captura por microdissecção à laser de células endoteliais em lâminas histológicas de 14 carcinomas espinocelulares bucais. Observou-se um aumento significativo dos níveis de mRNA de GRP78, ATF4 e CHOP em células endoteliais dos tumores quando comparadas a células endoteliais primárias (HDMEC). Além do mais, células endoteliais tumorais apresentaram intensa imunomarcação para GRP78 comparativamente a células endoteliais de mucosa bucal normal. A acidez, uma importante fonte de estresse no microambiente tumoral, pode ativar uma UPR adaptativa em células endoteliais. Aumento da expressão de GRP78 em células endoteliais é associado com maior resistência a drogas quimioterápicas. Os resultados sugerem que a resistência mediada pela UPR pode contribuir com o insucesso terapêutico na resposta a drogas antitumorais. / Antiangiogenic therapy has emerged as a promising alternative for cancer treatment. However, growing evidence has shown that endothelial cells isolated directly from malignant tumors are more resistant to different drugs than endothelial cells from normal tissues. These differences may due to the adaptation of endothelial cells to the tumor microenvironment. A unique feature of tumor microenvironment is the consistent acidification of the extracellular environment, whose effects on endothelial cells are not known. Extracellular acidity can alter multiple biological functions, including endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR). Primary human dermal microvascular endothelial cells (HDMEC) cultured at medium pH 6.4, adjusted with either lactic acid or either hydrochloric acid, showed strong up-regulation of the UPR-related proteins: GRP78, ATF4, phospho-elf2α and increased XBP1 mRNA splicing. However massive cell death occurred after 48 hours. In contrast, when endothelial cells were exposed to chronic nonlethal acidic stress at pH 7.0 for up to seven days, cells were able to adapt, coincidental with a marked increase in GRP78 protein expression. After 7 days at pH 7.0, HDMEC cells showed increased resistance to cell death when exposed to Etoposide, Adriamycin and Sunitinib at doses ranging from 0.0025μM to 100μM. Knockdown of GRP78 by shRNA reversed the resistance phenotype. To determine the levels of UPR in vivo, laser capture microdissection of endothelial cells from oral squamous cell carcinomas biopsies was done. There is a significant increase in mRNA levels of GRP78, ATF4 and CHOP on endothelial cells of tumors compared to untreated primary endothelial cells (HDMEC). Moreover, tumor 16 endothelial cells showed strong GRP78 immunostaining compared to endothelial cells from normal oral mucosa. Low pH, an important source of cellular stress in the tumor microenvironment, can activate an adaptive UPR response in endothelial cells. Increased expression of GRP78 in endothelial cells is associated with chemoresistance. The results suggest that UPR-mediated resistance may contribute to therapeutic failures in response to anticancer drugs.
43

Role of SLMAP in Endoplasmic Reticulum Stress and Unfolded Protein Response

Mahmood, Ahsan 13 August 2013 (has links)
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.
44

Examining the Role of Endoplasmic Reticulum Stress in Pancreatic Beta-cell Biology

Teodoro, Tracy 31 August 2012 (has links)
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.
45

Cellular mechanisms affecting redox homeostasis in response to stress in Saccharomyces cerevisiae

Tan, Shixiong , Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2009 (has links)
Maintainence of appropriate redox homeostasis is crucial for processes such as protein folding in the endoplasmic reticulum (ER) and to minimise genesis of oxidative stress. Previous studies have indicated a possible link between ER stress and production of reactive oxygen species (ROS) although the cellular mechanisms involved were not fully elucidated. To investigate the cellular mechanisms involved in tolerance to oxidative stress and ER stress, genome-wide screens were performed to identify mutants sensitive to chronic ER stress induced by dithiothreitol and tunicamycin. These screens identified the Cu,Zn superoxide dismutase (SOD1) and genes involved in NADPH generation (RPE1, TKL1) as important for chronic ER stress tolerance. Superoxide anion has been identified as one of the ROS generated during ER stress. The ER oxidoreductase Ero1p, previously implicated in ROS production in vitro, did not appear to be a source of superoxide when the protein was over-expressed. It was also found that cellular NADP(H) levels affected induction of the unfolded protein response (UPR), since cells lacking TKL1 or RPE1 exhibited decreased UPR induction. These data indicate an important role for superoxide dismutase and cellular NADP(H) in survival of cells during ER stress. Subsequent analysis determined that NADPH generation was also required for adaptation to H2O2. Mutants affected in NADPH production were chronically sensitive to H2O2 but resistant to an acute dose. These mutants over-accumulated reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This over- production of GSH was not regulated at the transcriptional level of GSH1 encoding ??- glutamyl cysteine synthetase. These data raise the important question as to how cells maintain cellular glutathione redox balance. To better understand how cells respond to perturbations in glutathione redox homeostasis, cells deleted for GLR1, encoding GSSG reductase, were exposed to extracellular oxidised glutathione (GSSG) and intracellular GSH and GSSG were monitored over time. Intriguingly cells lacking GLR1 showed increased levels of GSH accumulation upon GSSG treatment in a manner independent of GSH synthesis. It was subsequently found that the cytosolic thioredoxin-thioredoxin reductase system contributes to the reduction of GSSG in vivo.
46

O papel da UPR (Unfolded Protein Response) na resistência a drogas de céluas endoteliais em resposta ao estresse causado pelo pH ácido tumoral

Visioli, Fernanda January 2011 (has links)
A terapia antiangiogênica surgiu como uma alternativa promissora para o tratamento do câncer. No entanto, evidências recentes mostram que as células endoteliais isoladas diretamente de um tumor maligno são mais resistentes a diferentes drogas do que as células endoteliais presentes no mesmo tecido normal. Essas diferenças podem ser atribuídas em parte à adaptação das células endoteliais ao microambiente tumoral. Uma característica singular do microambiente tumoral é a consistente acidificação do meio extracelular, cujos efeitos nas células endoteliais não são conhecidos. Acidez extracelular pode alterar múltiplas funções biológicas, causar estresse do retículo endoplasmático (RE) e ativação da Unfolded Protein Response (UPR). Células endoteliais humanas primárias de derme (HDMEC) cultivadas em pH 6.4, ajustado tanto com ácido lático tanto com ácido clorídrico, apresentaram aumento da expressão de proteínas relacionadas à UPR, como GRP78, ATF4, elf2α fosforilada e aumento na clivagem do mRNA de XBP1. Nessas condições massiva morte celular ocorreu após 48 horas. Em contrapartida, quando as células endoteliais eram expostas à acidez crônica não-letal com pH 7.0 durante sete dias, essas foram capazes de se adaptar coincidentemente com um aumento da expressão da proteína GRP78 Após sete dias sob pH 7.0, as células HDMEC apresentaram maior resistência à morte celular quando tratadas com as drogas Etoposide, Adriamicina e Sunitinib em doses que variavam entre 0.0025μM a 100μM. O silenciamento do gene GRP78 com ShRNA reverteu esse fenótipo resistente. Para determinar os níveis de UPR in vivo utilizou-se captura por microdissecção à laser de células endoteliais em lâminas histológicas de 14 carcinomas espinocelulares bucais. Observou-se um aumento significativo dos níveis de mRNA de GRP78, ATF4 e CHOP em células endoteliais dos tumores quando comparadas a células endoteliais primárias (HDMEC). Além do mais, células endoteliais tumorais apresentaram intensa imunomarcação para GRP78 comparativamente a células endoteliais de mucosa bucal normal. A acidez, uma importante fonte de estresse no microambiente tumoral, pode ativar uma UPR adaptativa em células endoteliais. Aumento da expressão de GRP78 em células endoteliais é associado com maior resistência a drogas quimioterápicas. Os resultados sugerem que a resistência mediada pela UPR pode contribuir com o insucesso terapêutico na resposta a drogas antitumorais. / Antiangiogenic therapy has emerged as a promising alternative for cancer treatment. However, growing evidence has shown that endothelial cells isolated directly from malignant tumors are more resistant to different drugs than endothelial cells from normal tissues. These differences may due to the adaptation of endothelial cells to the tumor microenvironment. A unique feature of tumor microenvironment is the consistent acidification of the extracellular environment, whose effects on endothelial cells are not known. Extracellular acidity can alter multiple biological functions, including endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR). Primary human dermal microvascular endothelial cells (HDMEC) cultured at medium pH 6.4, adjusted with either lactic acid or either hydrochloric acid, showed strong up-regulation of the UPR-related proteins: GRP78, ATF4, phospho-elf2α and increased XBP1 mRNA splicing. However massive cell death occurred after 48 hours. In contrast, when endothelial cells were exposed to chronic nonlethal acidic stress at pH 7.0 for up to seven days, cells were able to adapt, coincidental with a marked increase in GRP78 protein expression. After 7 days at pH 7.0, HDMEC cells showed increased resistance to cell death when exposed to Etoposide, Adriamycin and Sunitinib at doses ranging from 0.0025μM to 100μM. Knockdown of GRP78 by shRNA reversed the resistance phenotype. To determine the levels of UPR in vivo, laser capture microdissection of endothelial cells from oral squamous cell carcinomas biopsies was done. There is a significant increase in mRNA levels of GRP78, ATF4 and CHOP on endothelial cells of tumors compared to untreated primary endothelial cells (HDMEC). Moreover, tumor 16 endothelial cells showed strong GRP78 immunostaining compared to endothelial cells from normal oral mucosa. Low pH, an important source of cellular stress in the tumor microenvironment, can activate an adaptive UPR response in endothelial cells. Increased expression of GRP78 in endothelial cells is associated with chemoresistance. The results suggest that UPR-mediated resistance may contribute to therapeutic failures in response to anticancer drugs.
47

O papel da UPR (Unfolded Protein Response) na resistência a drogas de céluas endoteliais em resposta ao estresse causado pelo pH ácido tumoral

Visioli, Fernanda January 2011 (has links)
A terapia antiangiogênica surgiu como uma alternativa promissora para o tratamento do câncer. No entanto, evidências recentes mostram que as células endoteliais isoladas diretamente de um tumor maligno são mais resistentes a diferentes drogas do que as células endoteliais presentes no mesmo tecido normal. Essas diferenças podem ser atribuídas em parte à adaptação das células endoteliais ao microambiente tumoral. Uma característica singular do microambiente tumoral é a consistente acidificação do meio extracelular, cujos efeitos nas células endoteliais não são conhecidos. Acidez extracelular pode alterar múltiplas funções biológicas, causar estresse do retículo endoplasmático (RE) e ativação da Unfolded Protein Response (UPR). Células endoteliais humanas primárias de derme (HDMEC) cultivadas em pH 6.4, ajustado tanto com ácido lático tanto com ácido clorídrico, apresentaram aumento da expressão de proteínas relacionadas à UPR, como GRP78, ATF4, elf2α fosforilada e aumento na clivagem do mRNA de XBP1. Nessas condições massiva morte celular ocorreu após 48 horas. Em contrapartida, quando as células endoteliais eram expostas à acidez crônica não-letal com pH 7.0 durante sete dias, essas foram capazes de se adaptar coincidentemente com um aumento da expressão da proteína GRP78 Após sete dias sob pH 7.0, as células HDMEC apresentaram maior resistência à morte celular quando tratadas com as drogas Etoposide, Adriamicina e Sunitinib em doses que variavam entre 0.0025μM a 100μM. O silenciamento do gene GRP78 com ShRNA reverteu esse fenótipo resistente. Para determinar os níveis de UPR in vivo utilizou-se captura por microdissecção à laser de células endoteliais em lâminas histológicas de 14 carcinomas espinocelulares bucais. Observou-se um aumento significativo dos níveis de mRNA de GRP78, ATF4 e CHOP em células endoteliais dos tumores quando comparadas a células endoteliais primárias (HDMEC). Além do mais, células endoteliais tumorais apresentaram intensa imunomarcação para GRP78 comparativamente a células endoteliais de mucosa bucal normal. A acidez, uma importante fonte de estresse no microambiente tumoral, pode ativar uma UPR adaptativa em células endoteliais. Aumento da expressão de GRP78 em células endoteliais é associado com maior resistência a drogas quimioterápicas. Os resultados sugerem que a resistência mediada pela UPR pode contribuir com o insucesso terapêutico na resposta a drogas antitumorais. / Antiangiogenic therapy has emerged as a promising alternative for cancer treatment. However, growing evidence has shown that endothelial cells isolated directly from malignant tumors are more resistant to different drugs than endothelial cells from normal tissues. These differences may due to the adaptation of endothelial cells to the tumor microenvironment. A unique feature of tumor microenvironment is the consistent acidification of the extracellular environment, whose effects on endothelial cells are not known. Extracellular acidity can alter multiple biological functions, including endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR). Primary human dermal microvascular endothelial cells (HDMEC) cultured at medium pH 6.4, adjusted with either lactic acid or either hydrochloric acid, showed strong up-regulation of the UPR-related proteins: GRP78, ATF4, phospho-elf2α and increased XBP1 mRNA splicing. However massive cell death occurred after 48 hours. In contrast, when endothelial cells were exposed to chronic nonlethal acidic stress at pH 7.0 for up to seven days, cells were able to adapt, coincidental with a marked increase in GRP78 protein expression. After 7 days at pH 7.0, HDMEC cells showed increased resistance to cell death when exposed to Etoposide, Adriamycin and Sunitinib at doses ranging from 0.0025μM to 100μM. Knockdown of GRP78 by shRNA reversed the resistance phenotype. To determine the levels of UPR in vivo, laser capture microdissection of endothelial cells from oral squamous cell carcinomas biopsies was done. There is a significant increase in mRNA levels of GRP78, ATF4 and CHOP on endothelial cells of tumors compared to untreated primary endothelial cells (HDMEC). Moreover, tumor 16 endothelial cells showed strong GRP78 immunostaining compared to endothelial cells from normal oral mucosa. Low pH, an important source of cellular stress in the tumor microenvironment, can activate an adaptive UPR response in endothelial cells. Increased expression of GRP78 in endothelial cells is associated with chemoresistance. The results suggest that UPR-mediated resistance may contribute to therapeutic failures in response to anticancer drugs.
48

Role of SLMAP in Endoplasmic Reticulum Stress and Unfolded Protein Response

Mahmood, Ahsan January 2013 (has links)
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.
49

Defining cellular and molecular mechanisms of hereditary transthyretin amyloidosis

Giadone, Richard Michael 29 May 2020 (has links)
Hereditary transthyretin amyloidosis (ATTR amyloidosis) is a multi-system protein folding disorder that results from >100 described mutations in the transthyretin (TTR) gene. In the disease, non-natively folded TTR, originally produced by the liver, travels throughout circulation and deposits extracellularly at downstream target organs. The multi-tissue etiology of the disease makes it difficult to study in vitro, while no mouse model accurately recapitulates disease pathology. Therefore, we utilized patient-specific induced pluripotent stem cells (iPSCs) to test the hypothesis that production of and exposure to destabilized TTRs results in distinct cellular and molecular changes. The liver’s contribution to the deposition of TTR at distal tissues is understudied. As a result, in Aim 1 we sought to assess the effects of destabilized TTR production on effector hepatic cells. To this end, we utilized gene editing to generate isogenic, patient iPSCs expressing either mutant or wild-type TTR. Combining this tool with single cell RNAseq, we identified hepatic proteostasis factors, including unfolded protein response (UPR) pathways, whose expression coincided with the production of destabilized TTR. Enhancing endoplasmic reticulum (ER) proteostasis within patient hepatic cells via exogenous activation of adaptive UPR signaling, we demonstrated preferential reduction in the secretion of pathogenic TTR. In turn, we demonstrated that production of disease-associated TTR correlates with expression of proteostasis factors capable of regulating TTR secretion and in turn downstream pathogenesis. ATTR amyloidosis patients exhibit extreme phenotypic variation (e.g. TTR fibril deposits at cardiac tissue and/or peripheral nerves). In Aim 2, we sought to define responses of target cell types to pathologically-diverse TTRs. To accomplish this, we profiled transcriptomic changes resulting from exposure to a variety of destabilized TTRs to determine 1) target cell response to TTR exposure and 2) how this response changes across diverse variants and cell types. In doing so, we found that TTR exposure elicits distinct variant- and cell type-specific transcriptional responses. Herein, we addressed our central hypothesis by profiling destabilized TTR production within hepatic cells and TTR exposure at target cell types. Collectively, these data may result in the discovery of unidentified and potentially druggable pathologically-associated pathways for ATTR amyloidosis and other systemic amyloid diseases.
50

Mort cellulaire immunogène : du stress du reticulum endoplasmique à l'exposition de la calréticuline / Immunogenic Cell Death : Endoplasmic Reticulum Stress to Calreticulin Exposure

Bezu, Lucillia 18 September 2017 (has links)
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 cliniques. / Conventional 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.

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