<p>Introduction: Mitochondrial dysfunction and oxidative stress increase with aging and may contribute to age-associated muscle atrophy (sarcopenia). Resistance exercise (RE) can promote the accretion of muscle mass, increase strength, and ultimately improve function in the elderly. Such beneficial effects are thought to be mitigated solely by increased muscle mass and strength; however, the contribution of the mitochondria to the beneficial effects of RE in aging have not been thoroughly characterized. While mitochondrial benefits have been established separately in both young and aged adults following chronic RE, the acute effects have not been well characterized. Methods: Sedentary young and aged adult males completed either an acute bout of fatiguing RE or endurance exercise (EE), and muscle biopsies were obtained at 3, 24 and 48 h post- exercise depending on the study. Results: Despite equivalent lean-body mass, increased age was associated with elevated mtDNA deletions, indicating potential for mitochondrial dysfunction. RE was associated with reduced mitochondrial content (transcripts, protein, and mtDNA copy number) at 48 h post-exercise, a response that did not differ with increasing age. Paradoxically, reduced mitochondrial content occurred alongside elevated total peroxisome proliferator-activated receptor γ coactivator one α (PGC-1α) mRNA; however, RE altered only the PGC-1α4 isoform post-exercise, a transcript that regulates myostatin and insulin-like growth factor one (IGF1) signalling and ultimately muscle hypertrophy and not mitochondrial adaptations. In addition, PGC-1α modulates the unfolded protein response (UPR), and RE was subsequently shown to elevate endoplasmic reticulum stress and elicit the UPR. Conclusion: PGC-1α mRNA increases regardless of exercise mode; however, differential expression or regulation of alternate PGC-1α isoforms or transcriptional binding partners co-activated by PGC-1α may dictate the specific phenotypic adaptations that occur following divergent modes of exercise. Furthermore, RE acutely decreases mitochondrial content despite elevated PGC-1α mRNA, and this response is not influenced by age.</p> / Doctor of Philosophy (Medical Science)

Tissue fibrosis occurs in the advanced stages of various chronic diseases and can account for 45% of all deaths related to chronic diseases worldwide. The extracellular matrix (ECM) components comprising the fibrotic scar are primarily derived from myofibroblasts, which are contractile fibroblasts arising from the trans-differentiation of several cellular progenitors. Disturbances in immune cell infiltration and function could lead to the uncontrolled production of pro/anti-inflammatory mediators, which may alter the phenotype, state, and function of myofibroblasts progenitors, leading to aberrant wound repair and pathological fibrosis. A great deal of knowledge has implicated macrophages in the pathogenesis and exacerbation of the fibrotic process. Nonetheless, much remains to be elucidated on the potential mechanisms regulating macrophage accumulation and pro-fibrotic polarization, and whether these mechanisms can be further investigated to modulate tissue repair. The Endoplasmic reticulum (ER) stress has recently been implicated as a key mechanism that propagates the pathogenesis of the fibrotic process. How ER stress precisely impacts the fibrotic process is still unclear. This thesis partly explored how modulating the outcome of ER stress – the unfolded protein response (UPR), would affect the severity of lung fibrosis and addressed the role of IL-6 signalling in macrophages during fibrosis. The data demonstrated that UPR activation in pro-fibrotic macrophages and partial deficiency of Grp78, the master regulator of the UPR, abrogated pulmonary fibrotic changes and reduced the accumulation of pro-fibrotic (M2-like) macrophages. These findings were later associated with high TUNEL levels, 7AAD positive cells, Chop and cleaved caspase 3 levels, which are suggestive of GRP78 mediated apoptosis in this population. On the contrary, mice lacking a terminal UPR mediator of apoptosis, called Chop, had increased ECM deposition and greater persistence of non-apoptotic macrophages. These findings suggest that UPR-mediated macrophage polarization and apoptosis may alter lung wound repair processes. As IL-6 synergized the effect of IL-4 to promote a hyper M2 macrophage state, it provided a unique and compelling model to study the dynamics of macrophage alternative programming, which has set the stage to investigate whether the UPR was implicated in the generation of a hyper pro-fibrotic macrophage phenotype. This hyper M2 macrophage model led to the identification of ER expansion program and the IRE1-XBP1 arm of the UPR in pro-fibrotic macrophage polarization, and suggested an unprecedented in vivo role of IL-6 in priming macrophages in the injured lungs to possibly potentiate pathological wound repair. Looking forward, many questions remain to be answered in order to precisely identify the vital UPR axis regulating ER expansion in macrophages during pathological wound repair and to get closer to the understanding of whether the UPR modulates the pro-fibrotic/pro-resolving capacity of macrophages. Insights on these mechanisms may facilitate the development of therapeutics that better manage chronic fibrotic diseases which pose fatal consequences and increase public concern. / Thesis / Doctor of Philosophy (PhD)

The unfolded protein response (UPR) is composed of three highly conserved pathways (ATF6, IRE1, PERK). Cellular stressors induce protein misfolding and aggregation in the endoplasmic reticulum (ER). This signaling pathway maintains protein homeostasis when there is stress in the ER. When the UPR is activated, the eukaryotic initiation factor 2 alpha (eIF2α) becomes phosphorylated, which inhibits global mRNA translation. If ER stress remains chronically unmitigated, the UPR induces apoptosis. GADD34 and CReP shift in expression when the UPR is activated and work as phosphatases and dephosphorylate eIF2α in a feedback loop, allowing protein synthesis to resume. Several human diseases, including fatty liver disease (FLD) are affected by cell stress from improper protein folding and accumulation, making the UPR a therapeutic target. Previous studies have indicated the UPR to both cause or become activated by FLD, depending on the duration of cellular stress. At least 25% of humans worldwide have steatosis, and zebrafish are a powerful model organism for FLD studies. Their embryos are easily obtained, and the liver develops quickly in their transparent larvae, which allows us to visualize the development of fat in the liver. It is unknown how exactly the UPR is involved in inducing lipogenesis in hepatocytes. We sought to better understand the link between UPR activation and steatosis. Pharmacological treatments with various drugs, some of which induce ER stress, were administered over different durations in zebrafish embryos and subsequently the expression of UPR network and lipogenesis genes were quantified through RT-qPCR. To visualize whether these drugs induced steatosis, zebrafish livers were stained with Oil Red O and imaged. Our results indicate that all chronic durations of pharmacological treatments resulted in fatty liver, and the expression of atf6 decreased in response to treatment that prevents the dephosphorylation of eIF2α. This data provides insight pertaining to the activity of the UPR network during FLD in zebrafish models.

Le stress du Réticulum Endoplasmique (RE) est impliqué dans la physiopathologie des maladies rénales, et la réponse UPR (Unfolded Protein Response), qui est activée en réponse à ce stress, joue un rôle important dans l'homéostasie des cellules tubulaires rénales et des podocytes. L’étude des mécanismes moléculaires et des conséquences de l'activation de cette voie est donc importante dans la compréhension de la physiopathologie des maladies rénales et dans la caractérisation de biomarqueurs de lésions évolutives. L’Angiogénine (ANG, appelée également RNase 5) est une ribonucléase secrétée, qui est impliquée dans la réponse à certains stress cellulaires, et permet une adaptation cellulaire et tissulaire.
L'objectif de ce travail a été de mettre en évidence les mécanismes de régulation et les fonctions biologiques de l'ANG en réponse au stress du RE. A partir d'un modèle de cellules tubulaires rénales humaines en culture, nous avons montré que le stress du RE induisait l’expression de l’Angiogénine ainsi que sa sécrétion. Cette observation a été également faite sur différents modèles murins de lésions rénales. Le facteur transcriptionel sXBP1, activé par le transducteur de la réponse UPR, IRE1a, est directement impliqué dans la régulation de l'expression de l'Angiogénine.
Nous avons mis en évidence que l'Angiogénine participait à l’inhibition de la traduction protéique en réponse au stress du RE en produisant des fragments d'ARN de transfert appelés tiRNAs (stress-induced tRNA fragments) qui répriment la traduction des protéines en interférant avec le complexe initiateur de la traduction. L'Angiogénine favorise la survie cellulaire en réduisant l'apoptose induite par le stress du RE, et des souris invalidées pour le gène codant l'Angiogénine sont plus sensibles aux lésions de nécrose tubulaire aigues induites par la Tunicamycine. Outre les propriétés cellulaires "intrinsèques" de l'Angiogénine, nous avons également caractérisé les mécanismes de sécrétion de l'Angiogénine par l'épithélium rénal en situation de stress du RE. La sécrétion épithéliale de l'Angiogénine est sous le contrôle des facteurs transcriptionnels NF-κB et sXBP1, et se produit sous un mode conventionnel, c’est-à-dire dépendant du transit par l'appareil de Golgi. A ce titre, la régulation de l'Angiogénine est similaire à celle de l'Interleukine 6. L'Angiogénine induit une polarisation des macrophages vers un phénotype pro-inflammatoire. Enfin, considérant que l'Angiogénine est secrétée par l'épithélium rénal en situation de stress, nous avons montré que l’Angiogénine peut être un marqueur non invasif de souffrance rénale. L'Angiogénine peut être quantifiée dans les urines de patients porteurs de maladies rénales, et sa concentration est corrélée à la concentration urinaire de Retinol Binding Protein (une protéine de petit poids moléculaire, marqueur de dysfonction tubulaire), mais pas avec celle de l'Albumine. En outre, la concentration urinaire d'Angiogénine est significativement plus élevée dans les urines de patients transplantés rénaux dont la biopsie rénale met en évidence des lésions de tubulite (rejet aigu cellulaire et néphropathie associée au BK virus) que dans les urines de patients indemnes de lésions tubulaires (rejet humoral, ou absence de lésions histologiques). Nous avons mis en évidence par immuno-histochimie un marquage nucléaire du facteur transcriptionnel sXBP1 dans les tubules de reins porteurs de lésions de tubulite, suggérant un lien potentiel entre sécrétion d'Angiogénine et activation du facteur transcriptionnel sXBP1 dans un environnement inflammatoire. En conclusion, nous avons intégré la régulation l'Angiogénine dans la réponse épithéliale rénale au stress du RE, et caractérisé ses fonctions biologiques intracellulaires et paracrines. Notre travail a identifié l'Angiogénine urinaire en étant que potentiel marqueur de lésions rénales tubulaires. / The Endoplasmic Reticulum (ER) stress is involved in the pathophysiology of renal diseases ; the UPR (Unfolded Protein Response), which is activated in response to that stress plays an important role in renal tubular cells and podocytes homeostasis and consequently in tissu homeostasis. Understanding the molecular mechanisms and the consequences of the activation of this pathway is important to characterize the pathophysiology of renal diseases and identification of biomarkers of ongoing lesions. Angiogenin (ANG, also known as RNase 5) is a secreted ribonuclease, which is involved in the cellular stress response, it allows cell and tissue adaptation. The goal of this work was to clarify and identify the mechanisms regulating Angiogenin’s expression and its biological functions during ER stress. Using a human renal tubular cell line, we have shown that ER stress induces the expression of angiogenin and its secretion. This observation was also made on several murine models of renal injury. The transcriptional factor sXBP1 activated by the UPR transducer, IRE1α, is directly involved in regulating the expression of angiogenin. We have shown that angiogenin participates in the inhibition of protein translation in response to ER stress by cleaving transfer RNA and generating tiRNAs (stress-induced tRNA fragments) that suppress protein translation by interfering with the translation initiation complex. Angiogenin promotes cell survival by reducing ER stress-induced apoptosis, ANG knockout mice are more sensitive to acute tubular necrotic lesions induced by tunicamycin. In addition to the cell-autonomous effects of angiogenin, we also characterized the mechanisms by which Angiogenin is secreted by the renal epithelium under ER stress. Angiogenin is secreted in a conventional manner under the control of the transcriptional factors NF-kB and sXBP1. As such, the regulation of angiogenin is similar to Interleukin-6. We also demonstrated that Angiogenin induces macrophage polarization to a pro-inflammatory phenotype. Finally, considering that angiogenin is secreted by the renal epithelium under stress, we have shown that angiogenin may be a noninvasive marker of kidney injury. Angiogenin can be quantified in the urine of patients with kidney disease, its urinary concentration is correlated to the urinary concentration of Retinol Binding Protein (a low molecular weight protein marker of tubular dysfunction), but not with that of Albumin . In addition, the urinary concentration of angiogenin is significantly higher in the urine of renal transplant patients whose renal biopsy highlights tubulitis lesions (cell acute rejection and BK virus associated nephropathy) than in the urine of patients without histological tubular damage (antibody-mediated rejection, or no visible histological lesions). We have demonstrated by immuno-histochemistry a tubular nuclear localization of the activated transcriptional factor sXBP1 in the biopsies of patients with high tubulitis score, suggesting a potential relationship between the secretion of Angiogenin and the activation of transcriptional factor sXBP1 within an inflammatory environment. To conclude, we have described Angiogenin as a new mediator of the integrated ER stress response, and characterized its cell- and non-cell-autonomous biological functions. Our study have identified urinary angiogenin as a potential marker of ongoing kidney tubular injuries.

Les connaissances actuelles en pathologie hépatique suggèrent que HCV n'est pas directement oncogénique mais expose les patients au risque de cancer du foie dans un contexte inflammatoire associé à une réponse UPR (Unfolded Protein Response) et une régénération hépatique. La nétrine-1, le ligand canonique de la famille des DRs (Récepteurs à dépendance), est une protéine anti-apoptotique impliquée dans le développement, l'inflammation et la tumorigenèse. Les DRs induisent l'apoptose en absence de leurs ligands. A ce jour, il n'existe aucune donnée reliant le concept de DR et les virus oncogènes. Au cours de ma thèse, j'ai contribué à démontrer que la fonctionnalité des DRs était altérée au cours de l'infection par HCV in vitro et in vivo. Nous avons montré que la surexpression de la nétrine-1 augmente l'infectivité des virions et promeut leur entrée via l'activation et la diminution du recyclage de l'EGFR. De son coté, HCV augmente l'expression de la nétrine-1 suite à l'activation de l'épissage de son ARN pré-messager. Nous avons aussi montré que l'expression du récepteur à la nétrine-1, UNC5A, était diminuée au cours de l'infection suite à des diminutions transcriptionnelle et traductionnelle. Dans ce cadre, la nétrine-1 joue le rôle de facteur proviral en inhibant une potentielle voie de signalisation antivirale induite par le récepteur UNC5A non lié. Nous avons ensuite voulu savoir quelles conséquences cette surexpression de nétrine-1 pourrait avoir en physiopathologie hépatique en contexte non infectieux. Un stress du RE (Réticulum Endoplasmique) est observé au cours de l'infection par HCV. Le stress du RE entraîne l'activation de la réponse UPR qui induit l'apoptose médiée par la DAPK1 en cas de stress prolongé. Le fait que le récepteur UNC5B active aussi l'apoptose via l'activation de la DAPK1 nous a conduit à étudier l'implication de la nétrine-1 dans la survie cellulaire au cours de la réponse UPR en contexte hépatique. Nous avons démontré à la fois in vitro et in vivo que l'expression de la nétrine-1 pourrait protéger les cellules contre l'apoptose induite par la réponse UPR suite à sa liaison aux récepteurs UNC5A et C qui entraîne l'inhibition de la DAPK1. De nombreuses études ont également reporté des rôles de la nétrine-1 dans l'inflammation et la néoangiogenèse. Nous avons montré que la nétrine-1 inhibe la migration transendothéliale hépatique des PBMCs (Peripheral Blood Mononucleated Cells) et accélère la tubulogenèse des cellules endothéliales intrasinusoïdales hépatiques. Dans leur ensemble, mes travaux de thèse suggèrent que la nétrine-1 via ses récepteurs UNC5s joue des rôles délétères en pathophysiologie hépatique favorables à la persistance virale et à la résistance à la mort cellulaire / Current knowledge in hepatic pathology suggests that HCV is not directly oncogenic but puts patients at risk for liver cancer in a context associated with a chronic inflammation, UPR (Unfolded Protein Response) and liver regeneration. Netrin-1, the canonical ligand of the DR (Dependence Receptor) family, is an antiapoptotic secreted factor implicated in development, cancer and cancer-associated inflammatory diseases. DRs induce cell death when unbound. No data linking the DR system to oncogenic viruses are available to date. During the first part of my PhD, I contributed to demonstrate that HCV infection alters DR functionality both in vitro and in vivo. We found that Netrin-1 conditions HCV virion infectivity and promotes virion entry by increasing the activation and decreasing the recycling of the EGFR. In turn, HCV increases Netrin-1 expression through enhanced Netrin-1 pre-mRNA splicing. The Netrin-1 UNC5A receptor expression was decreased upon HCV infection through diminished transcription and translation. In this setting, Netrin-1 acts as a proviral factor by inhibiting a putative antiviral signaling pathway conveyed by the unbound UNC5A receptor. In this context, we wanted to determine what consequences such Netrin-1 up-regulation could induce in non-infectious hepatic pathophysiology. Chronic ER (endoplasmic reticulum) stress is observed during HCV infection. ER stress leads to UPR activation which triggers apoptosis via DAPK1 activation upon prolonged stress. The fact that the UNC5B receptor induces apoptosis through DAPK1 activation led us to investigate Netrin-1 implication in cell survival upon UPR in the liver. During the second part of my PhD, I have demonstrated both in vitro and in vivo in mice that Netrin-1 translation during UPR could protect cells against UPR-related cell death after binding to UNC5A and C, in a DAPK1-mediated fashion. Several studies have also identified Netrin-1 roles in inflammation and neo-angiogenesis. We found that Netrin-1 inhibits hepatic transendothelial migration of PBMCs (Peripheral Blood Mononucleated Cells) and accelerates tubulogenesis of liver sinusoidal endothelial cells. Netrin-1’s role in a hepatic inflammation and neoangiogenesis, both events being tightly associated with viral hepatitis, remains to be thoroughly elucidated. Altogether, our results suggest that Netrin-1 plays UNC5-dependent deleterious roles in hepatic pathophysiology, leading to viral persistence as well as resistance to cell death

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.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the UK and Western world. More than 90% of sporadic CRCs harbour mutations in the multi-functional tumour suppressor gene Adenomatous polyposis coli (<i>Apc</i>). The most commonly studied function of APC is its role as a scaffold for the β-catenin destruction complex involved in Wnt signalling. However, APC binds many other proteins. For example, it directly binds to and stabilises microtubules and actin. These non-Wnt related functions of APC are poorly understood. My PhD examines non-Wnt functions of APC. To this end, I created degron-tagged APC in DT40 cells that allowed for the rapid, conditional degradation of endogenous APC. The aim was to identify the immediate effects on cellular processes. Then, to identify the contribution of different APC domains by measuring the ability to rescue any defects when reintroducing fragments of APC. However, creation of these degron-tagged <i>Apc </i>knock-in cell lines resulted in hypomorphic phenotypes and auxin-associated off-target effects. Nonetheless, I compared the response of APC^high, APC^low, and APC^minimal cells to DNA damaging agents and Taxol® but found no significant differences. Subsequently, I focused on the relationship between APC and apoptosis. Previous observations suggested that deficiency in <i>Apc </i>rendered cells less sensitive to low doses of Taxol®. However, <i>Apc </i>deficient cells were more readily killed when Taxol® was combined with the Bcl-2 inhibitor, ABT-737. One possible explanation is the increase in Bcl-2 protein upon <i>Apc </i>depletion. However, I found that ABT-737, Taxol® and <i>Apc </i>depletion each cause activation of the unfolded protein response. This suggests that these treatments elicit a stress response that can stimulate apoptosis. Moreover, the same treatments also cause changes in mitochondria. Importantly, all of these effects do not require an increase in the β-catenin protein. Together, my data reveal novel links between APC and apoptosis that could be exploited clinically.

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.

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.

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.