Essentielle à l'équilibre énergétique de la cellule, la mitochondrie, véritable sentinelle, joue, un rôle majeur dans le destin de la cellule, en modulant les voies de signalisation de mort cellulaire mis en jeu dans l'atrophie musculaire. L'objectif de cette thèse est de proposer des cibles thérapeutiques centrées sur la mitochondrie dans deux modèles murins dont la physiopathologie est caractérisée par une dysfonction mitochondriale associée à une atrophie musculaire : le vieillissement et la dystrophie musculaire de Duchenne (DMD). Pour lutter contre la perte de masse musculaire liée à l'âge, la déficience en myostatine (mstn), associée à un phénotype hypermusculé, est une stratégie thérapeutique prometteuse. Mais, l'altération du métabolisme mitochondrial et oxydatif induite par cette déficience réduit les effets bénéfiques d'une telle stratégie. Nous avons donc testé l'intérêt de l'utilisation de la molécule pharmacologique AICAR, activateur connu de l'AMPK, afin de « booster » la fonction mitochondriale chez la souris âgée KO mstn. Les résultats montrent chez la souris KO mstn, une amélioration du temps d'endurance de course. Au niveau signalétique, le traitement induit des effets bénéfiques mais limités sur la fonction mitochondriale. Les mécanismes restent à préciser mais tendent vers l'hypothèse d'un effet bénéfique de l'AICAR sur le stress du réticulum endoplasmique (RE). Le dysfonctionnement mitochondrial a été également largement impliqué dans la physiopathologie de la DMD. Dans notre seconde étude, ce même traitement à l'AICAR chez le modèle murin de la DMD, la souris mdx atténue le phénotype dystrophique et améliore la fonction contractile du diaphragme. Nous montrons que ces effets bénéfiques sont associés à une induction de mécanisme de survie, l'autophagie, et une limitation des phénomènes d'apoptose induit par la mitochondrie, mettant en évidence une amélioration de l'intégrité mitochondriale par stimulation de leur renouvellement dans des fibres musculaires dystrophiques. Enfin, ce travail a mis en avant pour la première fois la présence à l'état basal de stress du RE chez la mdx, propsant une nouvelle cible thérapeutique. L'impact de ce stress dans la fibre musculaire normal et pathologique est très mal connu. Nos résultats montrent que le stress du RE modifie les liens entre le réticulum sarcoplasmique et la mitochondrie, perturbe l'homéostasie calcique et active les voies de mort cellulaire associées à une dysfonction contractile. Ces résultats ouvrent une perspective de stratégie thérapeutique dans les pathologies musculaire impliquant un stress du RE, comme la DMD. Ce travail de thèse a mis en avant l'importance de développer des thérapies pharmacologiques dans les pathologies musculaires, permettant d'améliorer la fonction à la fois métabolique et de sentinelle de la mitochondrie. / Fundamental for the energetic balance of the cell, mitochondria play a key role for modulation of cell death pathway related to muscular atrophy. Thus, the purpose of this PhD is to find therapeutic strategy focus on mitochondria in two different murine models where the physiopathology is characterized by a mitochondria dysfunction associated with muscle atrophy: Aging process and Duchenne Muscular Dystrophy (DMD).To prevent loss of muscle mass associated with aging, the lack of myostatin, inducing a hypermuscular phenotype, is a promising therapeutic strategy. However, loss of myostatin is associated with a strong reduction of mitochondrial and oxidative metabolism in skeletal muscle, and this strategy need to be potentiated. In this context, we explore if mitochondrial alteration in aged wild-type mice or in aged mstn KO mice are rescued by chronic AMPK-activating treatment, using the synthetic agonist AICAR, considered as “an mimetic of exercise”. Our results show an improvement of aerobic running performance in mstn KO mice. Concerning to signaling pathways, AICAR treatment induces beneficial but limited effects on mitochondrial metabolism. Mechanisms are still under investigation but our results suggest a reduction in ER stress. Moreover, mitochondria dysfunction has been widely implicated in DMD physiopathology. This same treatment of AICAR, in the murine model of DMD, improves the diaphragm histopathology as well as maximal force generating capacity. These beneficial effects were linked with autophagy activation and apoptosis limitation, without inducing muscle fiber atrophy, and promoting the elimination of defective mitochondria.Finally, the last part of this study highlight for the first time, an increase of ER stress at basal level, suggesting a new therapeutic target. Nevertheless, ER stress impact in skeletal muscle fibers is sparsely known. The preliminary results show that ER stress decrease the link between RE and mitochondria, which have an impact on calcium homeostasis and stimulate cell death pathway with a decrease of contractile function.This study highlights the importance to develop pharmacological therapies in muscular pathology, focus on metabolic and sentinel mitochondria function.

De nombreuses maladies génétiques sont directement liées à la reconnaissance de protéines mal repliées par le contrôle qualité du réticulum endoplasmique (RE), conduisant à leur rétrotranslocation vers le cytosol puis leur dégradation. Dans le cas des glycoprotéines mutées, comme la protéine F508del-CFTR (Cystic Fibrosis Transmembrane conductance Regulator), la démannosylation extensive de leur résidus mannoses constitue une étape clé dans ce processus. L’objectif de ce travail a été d’identifier et de caractériser des molécules capables de rétablir l’expression membranaire de F508del-CFTR en ciblant cette activité enzymatique. Dans un premier temps, nous avons testé des dérivés multivalents basés sur le Deoxymannojirimycin (DMJ), un inhibiteur spécifique de la classe I des α-mannosidases et révélé leur effet correcteur puissant sur F508del-CFTR. Nous avons par la suite mis en évidence leur mécanisme d’action et tenté d’expliquer l’augmentation d’efficacité observée entre les monovalents et les multivalents. Nous nous sommes enfin focalisés sur le rôle des principales mannosidases dans le RE, l’α1,2-mannosidase I du RE (ERManI) et la famille des lectines EDEM (ER degradation-enhancing α-mannosidase-like protein). Nous avons montré par une stratégie de siRNA qu’ERManI, EDEM1 et EDEM2 sont impliquées dans la rétention réticulaire du F508del-CFTR.Notre étude ouvre ainsi de nouvelles perspectives quant à l’identification de nouveaux agents pharmacologiques ciblant ces protéines. / Numerous genetic diseases are directly associated to the recognition of misfolded proteins by the endoplasmic reticulum (ER) quality control, leading to their retention and subsequently their retrotranslocation to the cytosol for degradation. In the case of mutated glycoproteins such as F508del-CFTR (Cystic Fibrosis Transmembrane conductance Regulator), causing CF pathology, mannose trimming is a key step of this process. Our objective was to identify and characterize molecules targeting ER-mannosidases activity, with the goal to restore F508del-CFTR to the plasma membrane.First, we tested multivalent derivatives, based mainly on Deoxymannojirimycin (DMJ), a specific inhibitor of α-mannosidasesI and revealed their better corrective effect on F508del-CFTR and their mechanism of action. Then we explored the mechanism explaining the higher efficiency of the multivalents compared to the monovalent. Finally, we focused on the role of key players of mannose trimming, ER-α1,2-mannosidase I (ERManI) and EDEMs (ER degradation-enhancing α-mannosidase-like protein) proteins on F508del-CFTR trafficking defect. We showed the implication of ERManI, EDEM1 and EDEM2 in F508del-CFTR retention, using a siRNA strategy. In conclusion, our study highlights these proteins as potential pharmacological targets to develop correctors for the F508del-CFTR trafficking defect.

A via Unfolded Protein Response (UPR) é uma via de sinalização ativada pelo estresse do Retículo endoplasmático (ER). Anteriormente descrevemos um Paciente com Imunodeficiência Comum Variável (CVID) que apresenta um atraso na ativação da via UPR associado com o acumulo de imunoglobulinas dentro do ER e uma taxa de proliferação diminuída. Nossos resultados demonstram que a ativação crônica da UPR interrompe o ciclo celular de EBV-B através da quebra da natureza cíclica da ciclina A1. Essa parada é depende da linhagem EBV-B estudada e da droga utilizada. Além disso, a ativação crônica da UPR aumenta a apoptose através da ativação do braço da PERK da via UPR. Células ex-vivo e EBV-B do Paciente P apresentaram uma taxa metabólica muito baixa e numero aumentado de células em apoptose. A deficiência da resposta do paciente P frente a ativação da via UPR parece ser somente no reconhecimento de proteínas não dobradas. Nossos resultados sugerem que a proliferação deficiente observada em diversos paciente com CVID pode ser resultado de uma ativação deficiente da via UPR. / The unfolded protein response (UPR) is a signaling pathway activated by endoplasmic reticulum (ER) stress. Previously we described a patient (Patient P) with Common Variable Immunodeficiency (CVID) whose delayed activation of the UPR associates with accumulation of immunoglobulins and slower rate of proliferation. Our results showed that chronic UPR stress interrupted cell cycling of EBV-B cells through dysruption of the cyclic nature of cyclin A1. This interrption is depend of the cell type and drug. Furthermore, chronic ER stress triggered early apoptosis through activation of the PERK branch of the UPR. EBV-B and ex vivo cells from patient presented low metabolic rate and a high apoptosis rate even in the absence of ER stressors.. We noted that the deficiency of UPR pathway activation by Patient P apears to be on the recognition of unfolded proteins. Our results support the hypothesis that deficient proliferation observed in some CVID patients might be the result of deficient UPR activation.

O transplante de ilhotas pancreáticas é uma terapia promissora para o tratamento da diabetes mellitus tipo 1 (DM1). No entanto, ilhotas transplantadas estão sujeitas à rejeição pelo sistema imune dos pacientes receptores, portanto faz-se necessário o desenvolvimento de mecanismos moleculares que protejam essas células. Estudos mostraram que o hormônio prolactina (PRL) é capaz de inibir a apoptose desencadeada por citocinas pró-inflamatórias sobre células beta pancreáticas e que este processo citoprotetor depende da presença da chaperona HSPB1. Foi observado que durante o desenvolvimento do DM1, as células beta pancreáticas sofrem estresse de retículo endoplasmático e que isso contribui para desencadear apoptose. O estresse de retículo endoplasmático é caracterizado pelo acúmulo de proteínas mal dobradas nessa organela resultando na ativação da resposta a proteínas mal dobradas (UPR) que tem como finalidade recuperar a homeostase celular. No presente estudo mostramos, pela primeira vez, que PRL foi capaz de proteger células beta pancreáticas contra estresse de retículo endoplasmático promovido tanto por citocinas pró-inflamatórias (TNFα, IFNγ e IL1β) quanto pelos estressores de retículo endoplasmático: tunicamicina e tapsigargina; e que HSPB1 é essencial nesse mecanismo de citoproteção. No contexto do DM1, esse hormônio parece ter um efeito modulador da UPR aumentando os níveis de BiP, antecipando a ativação de ATF6 e PERK, mantendo a via de PERK ativa por mais tempo, inibindo a via de IRE1α, e diminuindo os níveis de CHOP em tempos maiores. Coletivamente, os resultados aqui apresentados aprofundam os conhecimentos sobre a função de HSPB1, conduzindo para o desenvolvimento de estratégias que visam à atenuação da morte de células beta por meio da modulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico. / The islet transplantation is a promising therapy for the treatment of type 1 diabetes mellitus (T1DM). However, transplanted islets are subject to rejection by the immune system of the recipient patients, therefore the development of molecular mechanisms that protect these cells is necessary. Studies have shown that the hormone prolactin (PRL) is capable of inhibiting apoptosis triggered by pro-inflammatory cytokines on pancreatic beta cells and that this cytoprotective process depends on the presence of the chaperone HSPB1. It was observed that during the development of type 1 diabetes, pancreatic beta cells undergo endoplasmic reticulum stress and that this contributes to trigger apoptosis. The endoplasmic reticulum stress is characterized by accumulation of misfolded proteins in this organelle resulting in the activation of unfolded protein response (UPR) that aims to restore cellular homeostasis. In the present study, we show for the first time that PRL was able to protect pancreatic beta cells against endoplasmic reticulum stress promoted by both pro-inflammatory cytokines (TNFα, IFNγ and IL1β) as the endoplasmic reticulum stressors: tunicamycin and thapsigargin; and HSPB1 is essential that cytoprotective mechanism. In the context of T1DM, PRL appears to have a modulating effect of the UPR by increasing the levels of BiP, anticipating the activation of ATF6 and PERK, keeping the PERK pathway active for longer, inhibiting the pathway IRE1α, and decreasing the levels of CHOP for longer times. Collectively, the results presented here deepen the knowledge of the HSPB1 function, leading to the development of strategies inducing attenuation of beta cells death through modulation of endogenous protection means, which are independent of the modulation of the immune system.

L'un des enjeux majeurs de la recherche anti-cancéreuse est de développer de nouvelles thérapies en direction des tumeurs réfractaires aux traitements conventionnels. Dans ce contexte, l'identification récente de l'activité anti-tumorale PPARgamma-indépendante des thiazolidinediones ouvre de nouvelles perspectives thérapeutiques. Au sein du laboratoire, il a été montré qu'un analogue inactif de la TGZ, la delta2-TGZ, induit une dégradation protéasome-dépendante du récepteur alpha aux oestrogènes de manière PPARgamma-indépendante. A partir de ces données, les objectifs de ma thèse ont été 1) de participer à la caractérisation de nouveaux composés à activité anti-cancéreuse 2) de mieux comprendre les mécanismes PPARgamma-indépendants mis en jeu dans l'effet anti-cancéreux des TZD. Lors de ce travail de thèse, nous avons caractérisé de nouveaux composés plus efficaces que la delta2-TGZ pour l'inhibition de la prolifération des cellules cancéreuses mammaires MCF-7 et MDA-MB-231 et faiblement toxiques sur des cultures primaires d'hépatocytes humains. De plus, nous avons montré que la delta2-TGZ est capable d'induire un stress du réticulum endoplasmique à des temps précoces dès 3 heures et une apoptose à des temps plus tardifs 48 heures. Cependant, nous n'avons pas pu conclure à l'existence d'un lien entre les deux mécanismes. Enfin, nous avons montré que la biotinylation d'un ligand naturel de PPARgamma, la 15d-PGJ2, accroît son effet anti-prolifératif sur les cellules cancéreuses mammaires MCF-7 et MDA-MB-231 et conduit à un stress du réticulum endoplasmique et à une mort par apoptose. Ces effets sont partiellement dépendants de PPARgamma pour le stress du réticulum endoplasmique mais strictement PPARgamma-indépendants pour l'apoptose. Ce travail pourrait permettre de constituer de nouveaux outils thérapeutiques dans le traitement du cancer du sein / One of the main goals of the anti-cancer research is to develop new therapeutic option for resistant tumor. In this context, the recent discovery of thiazolidinedione devoid of PPARgamma activity with a strong anti-cancer effect opens new perspectives. In a previous study, the laboratory showed that an inactive derivative of PPARgamma, the delta2-TGZ, induce a proteasome-dependent degradation of estrogen receptor alpha in PPARgamma-independent way. Thus, during my thesis we aimed 1) to participate in the study of new compounds less toxic and more effective to inhibit the proliferation of mammary cancer cells, 2) to better understand PPARgamma-independent mechanisms involved in the anti-cancer effect of the TZD. In the present work, we characterise new compounds more effective than delta2-TGZ to inhibit the proliferation of the breast cancer cells MCF-7 and MDA-MB-231 and are less toxic on primary culture of human hepatocytes. We pursued the study on mechanisms involved in PPARgamma-independent anti-proliferative activity of delta2-TGZ. We showed that delta2-TGZ is able to induce endoplasmic reticulum stress as soon as 3 hours and apoptosis in later times 48 hours of treatment. Nevertheless we could not conclude of the existence of a link between these two pathways. Finally, we studied the effect of the biotinylation of a natural ligand of PPAR?: the 15d-PGJ2. This modification drive to an increased effect of the 15d-PGJ2 on the anti-proliferative effect of breast cancer cells, reticulum endoplasmic stress and death by apoptosis. These effects are partially PPARgamma-dependent for reticulum endoplasmic stress and only PPARgamma-independent for apoptosis. This work highlighted new promising tools of breast cancers treatment

Na gestação há aumento da proliferação e redução da apoptose das células b pancreáticas. Prolactina (PRL) desencadeia estas mudanças, que são revertidas após o parto mesmo na presença de PRL. In vitro, dexametasona (DEX) se contrapõe a PRL. Avaliamos se o estresse do retículo endoplasmático (ERE) está envolvido na apoptose do pós-parto e se os glicocorticóides (GC) participam deste mecanismo. A fragmentação do DNA aumenta no 3° dia pós-parto (L3), em paralelo com a diminuição de pAKT e aumento do TRB3, indutor da apoptose por ERE. BiP, ATF4, CHOP, e a ligação de CHOP e CHOP-ATF4 no promotor do TRB3 aumentam em L3. O inibidor do ERE PBA restaurou os níveis de pAKT e CHOP e inibiu a apoptose. Células RINm5F tratadas com DEX (24h) têm aumento de BiP e ATF4, de p-eIF2 e do XBP-1 ativo. DEX também induz TRB3, mas inibe a ligação de CHOP ao TRB3. O tratamento por 72h não altera p-eIF2a, diminui XBP-1 ativo e promove apoptose, único evento revertido pela PRL. Concluímos que a apoptose das ilhotas em L3 é desencadeada por ERE, mas os GC não induzem este mecanismo. / During gestation occurs increase on the proliferation and apoptosis reduction of pancreatic b cells. Prolactin (PRL) promotes these changes which are reverted after delivery. Dexametasone (DEX) in vitro opposed to PRL. We evaluate whether endoplasmatic reticulum stress (ERS) was involved on post-delivery apoptosis and glycocorticoids (GC) participate on this mechanism. DNA fragmentation increased on the 3rd day post-delivery (L3), in parallel with pAKT diminution and inductor of apoptosis-TRB3 augment by ERS. BiP, ATF4, CHOP along with binding of CHOP and CHOP-ATF4 to the TRB3 promoter increased in L3. ERS inhibitor-PBA restored pAKT, CHOP levels and inhibited apoptosis. RINm5F cells with DEX (24h) showed increase in BiP, ATF4, p-eIF2 and in active XBP-1. DEX induced TRB3, but inhibited the binding of CHOP to TRB3. The 72h treatment did not alter p-eIF2a, diminished active XBP-1 and promoted apoptosis; the unique event reverted by PRL. We concluded that apoptosis of islets in L3 is generated by ERS; nevertheless this mechanism is not induced by GC.

O fator 2 derivado de células estromais, SDF2 (do inglês Stromal cell derived fator 2) é um gene de função ainda desconhecida, conservado em mamíferos e descrito primeiramente por Hamada et al. (1996). Neste estudo, observamos que a proteína Sdf2 de camundongo possui a alta similaridade de sequência em relação ao SDF2-like(L)1 humano e murino e de estrutura preditiva também similar em relação ao SDF2-like de Arabidopsis thaliana. A proteína mostrou-se sublocalizada no retículo endoplasmático e apresentou ampla distribuição nos tecidos e órgãos de camundongos. O mapeamento da expressão de Sdf2 ao longo da gestação humana e de camundongo nos mostrou que a proteína está presente em todas as etapas e compartimentos da placenta, com expressão em diversos tipos celulares. Nossos resultados sugerem que o SDF2 participa dos processos de diferenciação de células trofoblásticas humanas e murinas de maneira oposta. Em humanos, onde o processo é dependente da ativação de caspase-8 observou-se um aumento da expressão de SDF2. Em camundongos, onde o processo é por endoreduplicação, houve diminuição da expressão da proteína. A participação do SDF2 na via de estresse de retículo endoplasmático (RE) nas células trofoblásticas também foi analisada. Fatores adversos que podem levar à perda da homeostase do RE e levar a um acúmulo de proteínas mal enoveladas geram o fenômeno conhecido como Estresse de RE. O estresse de RE ativa a via de Resposta a Proteínas Mal Enoveladas (UPR, em inglês Unfolded Protein Response), que atua em diferentes vias de sinalização para aumentar a produção de chaperonas, a degradação das proteínas mal enoveladas e a diminuição da produção de novas proteínas. Estas respostas aumentam as chances de sobrevivência celular. Se, no entanto, a célula não recuperar sua homeostase, vias que levam à apoptose serão ativadas. A geração de estresse de RE pelo agente tunicamicina alterou significativamente a expressão de SDF2. Além disso, o silenciamento do gene SDF2 alterou a expressão dos principais fatores de controle de sobrevivência e apoptose da UPR. Desta forma, estes achados sugerem que o SDF2 desempenha um papel na regulação de sobrevivência/apoptose das células trofoblásticas pela via UPR. / The stromal cell derived factor 2 (SDF2) was first described by Hamada et al.(1996), is well conserved in mammals but its function is still unknown. In this study, we observed the predicted aminoacid sequence os Sdf2 is similar to human and mouse SDF2L1 sequence and the predicted Sdf2 structure is similar to SDF2-like from Arabidopsis thaliana. The protein is sublocalizes in the ER and it is widely expressed in mouse tissue and organs. The expression of Sdf2 throughout human and mouse gestation showed the protein is present in all gestational phases and compartments analysed and it is expressed by several cell types in the placenta. In trophoblast functional assays, SDF2 showed opposite expression patterns in human and mouse differentiation processes. In humans, where the process is dependent of caspase-8 activation, the protein is upregulated. Im mouse, the process is dependent of endoreduplication, the protein is downregulated. The participation of SDF2 in Endoplasmic Reticulum (ER) stress in trophoblastic cells was also evaluated. Adverse environmental conditions may lead to disruption of ER homeostasis causing accumulation of unfolded/misfolded proteins in ER, a phenomenon known as ER stress. ER stress activates the Unfolded Protein Response (UPR) that acts in several signaling cascades to improve chaperone production, misfolded protein degradation and to downregulate new protein production. These responses increase the capacity of cells to maintain alive even in stress conditions. Whether cells fail on restore homeostasis, the UPR activates apoptosis. We were able to observed that when gene silencing assays was used for SDF2, modifications in UPR cell survival/apoptosis markers were observed. In conclusion, we propose that SDF2 is playing a role in ER stress cell survival/apoptosis control in trophoblast cells.

Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.

La signalisation calcique joue un rôle crucial dans la réponse des cellules aux signaux extracellulaires. Ces dernières années, les progrès des techniques de microscopie ont permis de montrer que l’organisation spatio-temporelle du signal calcique était un élément fondamental de la réponse des cellules. L'organisation temporelle du signal calcium se caractérise ainsi par l'observation d'oscillations de calcium dont la fréquence varie en fonction du stimulus. Pour de nombreux types cellulaires, et notamment pour les hépatocytes, modèle cellulaire de notre étude, l’amplitude et la fréquence des oscillations de calcium sont très régulières et finement régulées. Les nombreux facteurs qui interviennent pour assurer cette régulation rendent l'étude de ce processus difficile. Ceci explique que l’étude des oscillations de calcium est un des domaines dans lequel l’aspect expérimental est communément complété avec la modélisation mathématique.Parmi les nombreux éléments qui participent à la régulation du signal calcique, les mitochondries qui ont longtemps été considérées comme ayant un rôle passif, pourraient jouer un rôle important. En effet il a été établi que les mitochondries étaient capables d’accumuler de façon active le calcium libéré par le reticulum endoplasmique. Notre objectif a donc été d’étudier l’importance des mitochondries dans la régulation des oscillations de calcium dans les hépatocytes, et grâce à nos résultats expérimentaux, de mettre au point un modèle mathématique. Pour cela nous avons choisi de modifier la prise de calcium par les mitochondries en altérant le moins possible leur fonctionnement normal. Nous avons utilisé une protéine altérant directement la prise de calcium par les mitochondries (HINT 2) et une autre protéine altérant l’interaction entre le reticulum endoplasmique et les mitochondries (R-1). Nos résultats expérimentaux et computationnels démontrent que la protéine HINT 2 augmente l’activité de la chaine respiratoire, ce qui a pour effet d’accélérer l’accumulation de calcium par les mitochondries et d’augmenter la fréquence des oscillations de calcium cytosolique dans les hépatocytes. En effet le modèle élaboré permet à la fois de reproduire les observations expérimentales mais également de prédire avec succès que l’absence de la protéine Hint 2 rend les mitochondries plus sensibles à l’ouverture du pore de transition mitochondrial (mPTP). Nous avons également démontré que le R-1 n’affecte pas significativement la prise de calcium par les mitochondries mais que sa surexpression aboutit à une élévation de la concentration calcique de base des cellules hépatocytaires. A la lumière de nos résultats, cette élévation de la concentration de calcium basale semble être due à son interaction physique avec la pompe ATPase SERCA 2. Nos résultats nous ont permis de mettre en évidence à la fois la contribution des mitochondries dans la régulation des oscillations de calcium dans les hépatocytes grâce à la protéine Hint 2, mais aussi le lien direct entre la surexpression du R-1 et l’élévation de la concentration en calcium de base dans les cellules HepG2. / Calcium signaling plays a crucial role in cell response to external stimuli. These last years, the progress of the microsocopy techniques allowed demonstrate that spatio-temporal organisation of the calcium signal is fundamental for cell physiology. This spatio-temporal organization is characterized by the observation of calcium oscillations whose frequency varies according to the stimulus. For many cell types in particular hepatocytes, calcium oscillations amplitude and frequency are regular and finely regulated. The large range of actors involved in this regulation explains the complexity of studying this process. This explains why the calcium oscillations studies are one of the area in which the experimental approach is commonly associated with computational approaches.Among all the elements participating in the regulation of calcium signaling, mitochondria that have been thought for many years to play a passive role may have a greater role. Indeed it has been shown that mitochondria are able to accumulate actively calcium from the endoplasmic reticulum.Our goal was to study the importance of mitochondria in calcium oscillations regulation in hepatocytes and to elaborate a model based on these experimental results. For that purpose we choose to change the calcium uptake by mitochondria using techniques that didn’t kill them. We used a protein that alters directly mitochondrial calcium uptake (Hint 2), and a protein involved in the interaction between endoplasmic reticulum and mitochondria (R-1).Our experimental and computational results show that HINT 2 enhances the electron transport chain activity, which lead to faster mitochondrial calcium uptake and faster calcium oscillations in the cytosol of hepatocytes. The model allows us not only to reproduce experimental data but also to successfully predict that the loss of HINT 2 lead the mitochondria to be more sensitive to the mitochondrial transition pore opening (mPTP).We also demonstrate that R-1 doesn’t change mitochondrial calcium uptake, but its over expression lead to a higher calcium concentration in resting cells. In the light of our results, this higher calcium concentration seems to be the result of R-1 interaction with ATPase pump SERCA 2.Our results allowed us to show not only the role of mitochondria in the regulation of hepatocytes calcium oscillations by HINT 2, but also the link between R-1 over expression and a higher calcium concentration in the cytosol of HepG2 cells.

Despite the tremendous advances of the last century, the cause of Alzheimer disease (AD) remains unclear. Genetic analysis of families with Alzheimer disease has revealed a disease-associated variant of the APOE gene, which encodes apolipoprotein E, a transporter of lipids in the blood and central nervous system. The effect of the AD-associated isotype, termed ApoE-E4, on disease risk has been validated, though it is unclear by what mechanism apoE-E4 confers AD risk. Mitochondria have long been implicated in AD pathogenesis, as the canonical histopathological findings of amyloid plaques and tau tangles occur in the setting of mitochondrial dysfunction. The disrupted processes include calcium homeostasis, cholesterol metabolism, phospholipid synthesis, and mitochondrial dynamics, and are all regulated by a subcompartment of the ER that is in physical contact with mitochondria. This compartment, called the mitochondria-associated ER membrane, or MAM, has been found to be overactive in AD patient cell lines and cell models of AD. Given that MAM is dysfunctional in AD and that ApoE-ε4 is the most important risk factor for AD, this dissertation examines if ApoE4 contributes to the MAM dysfunction seen in AD. The MAM dysfunction seen in AD patients and in cell models of AD has been best characterized in the context of familial AD, and it is the purpose of this study to extend those findings to the more common, sporadic, form of the disease. Familial AD is the result of autosomal dominant mutations in one of three genes, amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2). APP is the protein from which amyloid-beta, the component of amyloid plaques, is cleaved. The presenilins constitute the enzymatic core of the γ-secretase complex, which cleaves amyloid-beta from a precursor APP molecule. Both PSEN1 (PS1) and PSEN2 (PS2) localize at the MAM, and their action is speculated to influence MAM activity. Fibroblasts from familial AD patients, which contained mutations in APP, PSEN1 or PSEN2, showed a marked increase in MAM activity when compared to that of age-matched controls. In mouse embryonic fibroblasts, one can recapitulate this increased MAM activity by knocking out presenilins 1 and 2. In these Psen1/2 double knockout (DKO) cells, the typical measures of MAM function, i.e. increased cholesteryl ester and phosphatidylethanolamine synthesis, calcium transport from ER to mitochondria, and co-localization of ER and mitochondria by confocal and electron microscopy, mimicked the same phenotype found in fibroblasts obtained from familial AD patients, which suggests that the presenilins are negative regulators of ER-mitochondrial apposition. The precise mechanism by which they regulate the ER-mitochondria interface, whether directly as part of a tethering complex, or indirectly though the metabolism of APP-derived substrates, is unclear. While the effect of familial AD mutations on MAM has been characterized, the mechanism of mitochondrial dysfunction seen in the more common sporadic form of the disease remains obscure. Sporadic AD patients harbor no mutations in APP, PSEN1, or PSEN2, but rather inherit mutations in other genes which do not guarantee the development of the disease, but are instead considered risk factors. The most important of these risk factors, in terms of both amount of AD risk conferred and prevalence in the population, is ApoE. Embedded in the phospholipid monolayer of lipoproteins, ApoE is involved in the transport of phospholipids, cholesterol, and cholesteryl esters in plasma and the central nervous system (CNS). In the CNS, it is the most abundant apolipoprotein, and is secreted primarily by astrocytes and taken up by neurons. Once endocytosed, ApoE can follow three different pathways: degradation by the lysosome, intracellular retention in early endosomes, or re-lipidation and re-secretion out of the cell. Our approach takes advantage of the physiological role of ApoE as part of a high densitylike lipoprotein particle (HDL). Using astrocytes from ApoE targeted gene replacement mice in which murine APOE has been replaced by either human APOE-E3 or human APOE-E4, cultured media containing ApoE3 and ApoE4-lipoproteins can be produced and applied to target cells that do not express ApoE, such as neurons or fibroblasts. These target cells can then be analyzed for MAM activity. To examine the contribution of ApoE towards MAM dysfunction, target cells, either neurons or fibroblasts, were grown in the presence of astrocyte conditioned media (ACM) from ApoE targeted gene replacement mice. Several measures of phospholipid and cholesteryl ester synthesis were performed to analyzed MAM function. To confirm that the alterations in phospholipid synthesis were the result of altered MAM activity, the same assay was performed in cells in which a protein tethers that bind mitochondria and ER were genetically ablated. Finally, to confirm that the effects seen were the result of the HDL particles and not the result of other components of the ACM, lipoproteins were extracted from ACM by density ultracentrifugation and applied to fibroblasts. In all of the assays performed, ApoE4 conditioned media or ApoE4 isolated lipoproteins were able to induce a significant increase in MAM activity, whereas ApoE4 from recombinant sources did not. These data suggest a contribution of ApoE4 towards MAM dysfunction seen in AD. The mechanism of these ApoE4 induced MAM alterations remains to be deduced. One may speculate that given the role of ApoE in cholesterol transport outside of the cell, its intracellular retention may impact the distribution of cholesterol within the cell. MAM is a cholesterol rich subdomain with lipid raft-like properties, and any change in the cholesterol content or lipid nature of this membrane may alter its activity. To test this hypothesis, MAM was biochemically extracted from ApoE3 and ApoE4 treated cells and analyzed for cholesterol and lipidomic content. The results described in this thesis demonstrate an AD-like effect in wildtype cells when treated with ApoE-E4, and that the mechanism for these alterations may be due to disturbances in cholesterol distribution in the MAM.