Cell Cycle Proteins and Retinal Degeneration: Evidences of New Potential Therapeutic Targets.

During different forms of neurodegenerative diseases, including the retinal degeneration, several cell cycle proteins are expressed in the dying neurons from Drosophila to human revealing that these proteins are a hallmark of neuronal degeneration. This is true for animal models of Alzheimer's, and Parkinson's diseases, Amyotrophic Lateral Sclerosis and for Retinitis Pigmentosa as well as for acute injuries such as stroke and light damage. Longitudinal investigation and loss-of-function studies attest that cell cycle proteins participate to the process of cell death although with different impacts, depending on the disease. In the retina, inhibition of cell cycle protein action can result to massive protection. Nonetheless, the dissection of the molecular mechanisms of neuronal cell death is necessary to develop adapted therapeutic tools to efficiently protect photoreceptors as well as other neuron types.

Bevacizumab, Pemetrexed, and Cisplatin, or Bevacizumab and Erlotinib for Patients With Advanced Non-Small-Cell Lung Cancer Stratified by Epidermal Growth Factor Receptor Mutation: Phase II Trial SAKK19/09.

OBJECTIVE: The goal was to demonstrate that tailored therapy, according to tumor histology and epidermal growth factor receptor (EGFR) mutation status, and the introduction of novel drug combinations in the treatment of advanced non-small-cell lung cancer are promising for further investigation. METHODS: We conducted a multicenter phase II trial with mandatory EGFR testing and 2 strata. Patients with EGFR wild type received 4 cycles of bevacizumab, pemetrexed, and cisplatin, followed by maintenance with bevacizumab and pemetrexed until progression. Patients with EGFR mutations received bevacizumab and erlotinib until progression. Patients had computed tomography scans every 6 weeks and repeat biopsy at progression. The primary end point was progression-free survival (PFS) ≥ 35% at 6 months in stratum EGFR wild type; 77 patients were required to reach a power of 90% with an alpha of 5%. Secondary end points were median PFS, overall survival, best overall response rate (ORR), and tolerability. Further biomarkers and biopsy at progression were also evaluated. RESULTS: A total of 77 evaluable patients with EGFR wild type received an average of 9 cycles (range, 1-25). PFS at 6 months was 45.5%, median PFS was 6.9 months, overall survival was 12.1 months, and ORR was 62%. Kirsten rat sarcoma oncogene mutations and circulating vascular endothelial growth factor negatively correlated with survival, but thymidylate synthase expression did not. A total of 20 patients with EGFR mutations received an average of 16 cycles. PFS at 6 months was 70%, median PFS was 14 months, and ORR was 70%. Biopsy at progression was safe and successful in 71% of the cases. CONCLUSIONS: Both combination therapies were promising for further studies. Biopsy at progression was feasible and will be part of future SAKK studies to investigate molecular mechanisms of resistance.

Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling.

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.

Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations.

OBJECTIVES: The objective of this study was to characterize the underlying molecular mechanisms in consecutive clinical Candida albicans isolates from a single patient displaying stepwise-acquired multidrug resistance. METHODS: Nine clinical isolates (P-1 to P-9) were susceptibility tested by EUCAST EDef 7.2 and Etest. P-4, P-5, P-7, P-8 and P-9 were available for further studies. Relatedness was evaluated by MLST. Additional genes were analysed by sequencing (including FKS1, ERG11, ERG2 and TAC1) and gene expression by quantitative PCR (CDR1, CDR2 and ERG11). UV-spectrophotometry and GC-MS were used for sterol analyses. In vivo virulence was determined in the insect model Galleria mellonella and evaluated by log-rank Mantel-Cox tests. RESULTS: P-1 + P-2 were susceptible, P-3 + P-4 fluconazole resistant, P-5 pan-azole resistant, P-6 + P-7 pan-azole and echinocandin resistant and P-8 + P-9 MDR. MLST supported genetic relatedness among clinical isolates. P-4 harboured four changes in Erg11 (E266D, G307S, G450E and V488I), increased expression of ERG11 and CDR2 and a change in Tac1 (R688Q). P-5, P-7, P-8 and P-9 had an additional change in Erg11 (A61E), increased expression of CDR1, CDR2 and ERG11 (except for P-7) and a different amino acid change in Tac1 (R673L). Echinocandin-resistant isolates harboured the Fks1 S645P alteration. Polyene-resistant P-8 + P-9 lacked ergosterol and harboured a frameshift mutation in ERG2 (F105SfsX23). Virulence was attenuated (but equivalent) in the clinical isolates, but higher than in the azole- and echinocandin-resistant unrelated control strain. CONCLUSIONS: C. albicans demonstrates a diverse capacity to adapt to antifungal exposure. Potentially novel resistance-inducing mutations in TAC1, ERG11 and ERG2 require independent validation.

Epigenetic Activation of SOX11 in Lymphoid Neoplasms by Histone Modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.

Proteomics and chronic inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are relatively frequent in developed countries. Physiopathological events involved in the etiology of IBDs include activation of immune, mesenchymal and epithelial cells. This review gives an overview of the currently applied proteomics technologies. It describes metabolic changes and goes into the approaches using this methodology to understand the molecular mechanisms implicated in the development of the disease.

MicroRNAs and the functional β cell mass: For better or worse.

Insulin secretion from pancreatic β cells plays a central role in the control of blood glucose levels. The amount of insulin released by β cells is precisely adjusted to match organism requirements. A number of conditions that arise during life, including pregnancy and obesity, can result in a decreased sensitivity of insulin target tissues and a consequent rise in insulin needs. To preserve glucose homoeostasis, the augmented insulin demand requires a compensatory expansion of the pancreatic β cell mass and an increase in its secretory activity. This compensatory process is accompanied by modifications in β cell gene expression, although the molecular mechanisms underlying the phenomenon are still poorly understood. Emerging evidence indicates that at least part of these compensatory events may be orchestrated by changes in the level of a novel class of gene regulators, the microRNAs. Indeed, several of these small, non-coding RNAs have either positive or negative impacts on β cell proliferation and survival. The studies reviewed here suggest that the balance between the actions of these two groups of microRNAs, which have opposing functional effects, can determine whether β cells expand sufficiently to maintain blood glucose levels in the normal range or fail to meet insulin demand and thus lead, as a consequence, towards diabetes manifestation. A better understanding of the mechanisms governing changes in the microRNA profile will open the way for the development of new strategies to prevent and/or treat both type 2 and gestational diabetes.

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.

Role of immune and angiogenic markers in the pathology of endometriosis

Background: Endometriosis is an estrogen-dependent, pro-inflammatory, pro-angiogenic condition that affects 5 to 10% of women of reproductive age. Its defining feature is the presence of endometrium-like tissue in sites outside the uterine cavity, primarily on the pelvic peritoneum and ovaries. The main clinical features are chronic pain, pain during intercourse and infertility. In patients with endometriosis, inflammatory and immune responses, angiogenesis and apoptosis are altered in favour of the survival and replenishment of endometriotic tissue. These basic pathological processes depend on the excessive formation of estrogen and prostaglandins. Recently, new cellular and molecular mechanisms for the resolution of inflammation have been discovered, revealing key roles for lipid mediators such as lipoxins, resolvins and protectins. It is possible that disequilibrium in the expression of these molecules exists in endometriosis. Objective: To compare the expression of two proteins involved in the synthe sis and in the function of lipid mediators; the Arachidonate 15-lipoxygenase (ALOXI5), implicated in the synthesis of lipoxins A4 and B4 and the Formyl peptide receptor 1 (FPRLI), the specific receptor for Lipoxin A4 and B4, between women who suffer from endometriosis and a control group. We wish to demonstrate the cellular localisation of these two molecules and to investigate if their expression is alteted in this pathology. Methods and Materials Using immunohistochemistry we will compare ALOXI5 and FPRLI staining, in endometrium, normal peritoneum and endometriotic lesions. The samples are being collected in the department of Gynaecology and Obstetrics at the Centre Hospitalier Universitaire Lausanne (CHUV). Women attending the department for laparoscopic investigation of pain/infertility, suspected endometriosis or for a hysterectomy, are invited to participate. Approval of the ethics committee (Commission d'Ethique de la recherché clinique) was obtained in March 2009. Clinical samples will only be obtained from subjects having consented. Expected results and interpretation: No published studies investigating the expression of these two molecules in endometriotic lesions exist. A better understanding of the mechanisms underlying this disease will result in the development of new medical therapies and new diagnostic tests, with the aim of ameliorating the quality of life of endometriosis patients.

Gibberellic acid signaling is required for ambient temperature-mediated induction of flowering in Arabidopsis thaliana.

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature-dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process.

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases.

Functional impact of genetic variation on gene expression

Numerous links between genetic variants and phenotypes are known and genome-wide association studies dramatically increased the number of genetic variants associated with traits during the last decade. However, how changes in the DNA perturb the molecular mechanisms and impact on the phenotype of an organism remains elusive. Studies suggest that many traitassociated variants are in the non-coding region of the genome and probably act through regulation of gene expression. During my thesis I investigated how genetic variants affect gene expression through gene regulatory mechanisms. The first chapter was a collaborative project with a pharmaceutical company, where we investigated genome-wide copy number variation (CNVs) among Cynomolgus monkeys (Macaca fascicularis) used in pharmaceutical studies, and associated them to changes in gene expression. We found substantial copy number variation and identified CNVs linked to tissue-specific expression changes of proximal genes. The second and third chapters focus on genetic variation in humans and its effects on gene regulatory mechanisms and gene expression. The second chapter studies two human trios, where the allelic effects of genetic variation on genome-wide gene expression, protein-DNA binding and chromatin modifications were investigated. We found abundant allele specific activity across all measured molecular phenotypes and show extended coordinated behavior among them. In the third chapter, we investigated the impact of genetic variation on these phenotypes in 47 unrelated individuals. We found that chromatin phenotypes are organized into local variable modules, often linked to genetic variation and gene expression. Our results suggest that chromatin variation emerges as a result of perturbations of cis-regulatory elements by genetic variants, leading to gene expression changes. The work of this thesis provides novel insights into how genetic variation impacts gene expression by perturbing regulatory mechanisms. -- De nombreux liens entre variations génétiques et phénotypes sont connus. Les études d'association pangénomique ont considérablement permis d'augmenter le nombre de variations génétiques associées à des phénotypes au cours de la dernière décennie. Cependant, comprendre comment ces changements perturbent les mécanismes moléculaires et affectent le phénotype d'un organisme nous échappe encore. Des études suggèrent que de nombreuses variations, associées à des phénotypes, sont situées dans les régions non codantes du génome et sont susceptibles d'agir en modifiant la régulation d'expression des gènes. Au cours de ma thèse, j'ai étudié comment les variations génétiques affectent les niveaux d'expression des gènes en perturbant les mécanismes de régulation de leur expression. Le travail présenté dans le premier chapitre est un projet en collaboration avec une société pharmaceutique. Nous avons étudié les variations en nombre de copies (CNV) présentes chez le macaque crabier (Macaca fascicularis) qui est utilisé dans les études pharmaceutiques, et nous les avons associées avec des changements d'expression des gènes. Nous avons découvert qu'il existe une variabilité substantielle du nombre de copies et nous avons identifié des CNVs liées aux changements d'expression des gènes situés dans leur voisinage. Ces associations sont présentes ou absentes de manière spécifique dans certains tissus. Les deuxième et troisième chapitres se concentrent sur les variations génétiques dans les populations humaines et leurs effets sur les mécanismes de régulation des gènes et leur expression. Le premier se penche sur deux trios humains, père, mère, enfant, au sein duquel nous avons étudié les effets alléliques des variations génétiques sur l'expression des gènes, les liaisons protéine-ADN et les modifications de la chromatine. Nous avons découvert que l'activité spécifique des allèles est abondante abonde dans tous ces phénotypes moléculaires et nous avons démontré que ces derniers ont un comportement coordonné entre eux. Dans le second, nous avons examiné l'impact des variations génétiques de ces phénotypes moléculaires chez 47 individus, sans lien de parenté. Nous avons observé que les phénotypes de la chromatine sont organisés en modules locaux, qui sont liés aux variations génétiques et à l'expression des gènes. Nos résultats suggèrent que la variabilité de la chromatine est due à des variations génétiques qui perturbent des éléments cis-régulateurs, et peut conduire à des changements dans l'expression des gènes. Le travail présenté dans cette thèse fournit de nouvelles pistes pour comprendre l'impact des différentes variations génétiques sur l'expression des gènes à travers les mécanismes de régulation.

Characterization of a MAF1 knockout mouse model

L'ARN polymérase 3 transcrit un petit groupe de gènes fortement exprimés et impliqués dans plusieurs mécanismes moléculaires. Les ARNs de transfert ou ARNt représentent plus ou moins la moitié du transcriptome de l'ARN polymérase 3. Ils sont directement impliqués dans la traduction des protéines en agissant comme transporteurs d'acides aminés qui sont incorporés à la chaîne naissante de polypeptides. Chez des levures cultivées dans un milieu jusqu'à épuisement des nutriments, Maf1 réprime la transcription par l'ARN polymérase 3, favorisant ainsi l'économie énergétique cellulaire. Dans un modèle de cellules de mammifères, MAF1 réprime aussi la transcription de l'ARN polymérase 3 dans des conditions de stress, cependant il n'existe aucune donnée quant à son rôle chez un mammifère vivant. Pendant mon doctorat, j'ai utilisé une souris délétée pour le gène Maf1 afin de connaître les effets de ce gène chez un mammifère. Etonnamment, la souris Maf1-­‐/-­‐ est résistante à l'obésité même si celle-­‐ci est nourrie avec une nourriture riche en matières grasses. Des études moléculaires et de métabolomiques ont montré qu'il existe des cycles futiles de production et dégradation des lipides et des ARNt, ce qui entraîne une augmentation de la dépense énergique et favorise la résistance à l'obésité. En plus de la caractérisation de la souris Maf1-­‐/-­‐, pendant ma thèse j'ai également développé une méthode afin de normaliser les données de ChIP-­‐sequencing. Cette méthode est fondée sur l'utilisation d'un contrôle interne, représenté ici par l'ajout d'une quantité fixe de chromatine provenant d'un organisme différent de celui étudié. La méthode a amélioré considérablement la reproductibilité des valeurs entre réplicas biologiques. Elle a aussi révélé des différences entre échantillons issus de conditions différentes. Une occupation supérieure de l'ARN polymérase 3 sur les gènes Pol 3 chez les souris Maf1 KO entraîne une augmentation du niveau de précurseurs d'ARNt, ayant pour effet probable la saturation de la machinerie de maturation des ARNt. En effet, chez les souris Maf1 KO, le pourcentage d'ARNt modifiés est plus faible que chez les souris type sauvage. Ce déséquilibre entre le niveau de précurseurs et d'ARNt matures entraîne une diminution de la traduction protéique. Ces résultats ont permis d'identifier de nouvelles fonctions pour la protéine MAF1, comme étant une protéine régulatrice à la fois de la transcription mais aussi de la traduction et en étant un cible potentielle au traitement à l'obésité. -- RNA polymerase III (Pol 3) transcribes a small set of highly expressed genes involved in different molecular mechanisms. tRNAs account for almost half of the Pol 3 transcriptome and are involved in translation, bringing a new amino into the nascent polypeptide chain. In yeast, under nutrient deprivation, Maf1 acts for cell energetic economy by repressing Pol 3 transcription. In mammalian cells, MAF1 also represses Pol 3 activity under conditions of serum deprivation or DNA damages but nothing is known about its role in a mammalian organism. During my thesis studies, I used a Maf1 KO mouse model to characterize the effects of Maf1 deletion in a living animal. Surprisingly, the MAF1 KO mouse developed an unexpected phenotype, being resistant to high fat diet-­‐induced obesity and displaying an extended lifespan. Molecular and metabolomics characterizations revealed futile cycles of lipids and tRNAs, which are produced and immediately degraded, which increases energy consumption in the Maf1 KO mouse and probably explains in part the protection to obesity. Additionally to the mouse characterization, I also developed a method to normalize ChIP-­‐seq data, based on the addition of a foreign chromatin to be used as an internal control. The method improved reproducibility between replicates and revealed differences of Pol 3 occupancy between WT and Maf1 KO samples that were not seen without normalization to the internal control. I then established that increased Pol 3 occupancy in the Maf1 KO mouse liver was associated with increased levels of tRNA precursor but not of mature tRNAs, the effective molecules involved in translation. The overproduction of precursor tRNAs associated with the deletion of Maf1 apparently overwhelms the tRNA processing machinery as the Maf1 KO mice have lower levels of fully modified tRNAs. This maturation defect directly impacts on translation efficiency as polysomic fractions and newly synthetized protein levels were reduced in the liver of the Maf1 KO mouse. Altogether, these results indicate new functions for MAF1, a regulator of both transcription and translation as well as a potential target for obesity treatment.

No Accumulation of Transposable Elements in Asexual Arthropods.

Transposable elements (TEs) and other repetitive DNA can accumulate in the absence of recombination, a process contributing to the degeneration of Y-chromosomes and other nonrecombining genome portions. A similar accumulation of repetitive DNA is expected for asexually reproducing species, given their entire genome is effectively nonrecombining. We tested this expectation by comparing the whole-genome TE loads of five asexual arthropod lineages and their sexual relatives, including asexual and sexual lineages of crustaceans (Daphnia water fleas), insects (Leptopilina wasps), and mites (Oribatida). Surprisingly, there was no evidence for increased TE load in genomes of asexual as compared to sexual lineages, neither for all classes of repetitive elements combined nor for specific TE families. Our study therefore suggests that nonrecombining genomes do not accumulate TEs like nonrecombining genomic regions of sexual lineages. Even if a slight but undetected increase of TEs were caused by asexual reproduction, it appears to be negligible compared to variance between species caused by processes unrelated to reproductive mode. It remains to be determined if molecular mechanisms underlying genome regulation in asexuals hamper TE activity. Alternatively, the differences in TE dynamics between nonrecombining genomes in asexual lineages versus nonrecombining genome portions in sexual species might stem from selection for benign TEs in asexual lineages because of the lack of genetic conflict between TEs and their hosts and/or because asexual lineages may only arise from sexual ancestors with particularly low TE loads.

The role of raptor in cell death

Selon les statistiques, les maladies cancéreuses sont en augmentation dans les pays en développement ainsi que dans les pays industrialisés. Ceci peut s'expliquer largement par les habitudes alimentaires, le tabagisme, les infections, le manque d'activité physique, la pollution et le stress, entre autres. Ainsi, l'Organisation Mondiale de la Santé (OMS) prévoit une augmentation de la fréquence des cancers avec 15 millions de nouveaux cas par an en 2020. La transformation d'une cellule normale en une cellule cancéreuse se déroule en plusieurs étapes avec, au niveau moléculaire, différentes mutations ciblant des protéines régulant la croissance cellulaire. Un des exemples de protéines qui participent au contrôle des voies cellulaires impliquées lors de la prolifération des cellules sont les complexes de protéines mTORCl et mTORC2 (« mammalian target of rapamycin complex 1 and 2 »). Ces complexes mTORCl et mTORC2 activent des processus anaboliques (la synthèse de protéines et de lipides, le métabolisme énergétique, entre autres) et inhibent en même temps des voies de catabolismes cellulaires (autophagie et synthèse de lysosomes). Ils sont souvent mutés dans de nombreux cas de cancers, c'est pourquoi ils sont la cible de nombreux traitements anti-cancéreux. Pour ces raisons, nous nous sommes intéressés aux mécanismes d'actions moléculaires des drogues qui ciblent les complexes mTORCl et mTORC2. Nous avons ainsi découvert qu'une molécule présente uniquement dans le complexe mTORCl, raptor, était clivée en un fragment plus petit lors du traitement de cellules cancéreuses avec des drogues. Des molécules activées durant la mort cellulaire programmée par apoptose, les caspases, se sont révélées responsables du clivage de raptor. Nous avons ensuite décrit de façon précise les sites de clivage de raptor par les caspases durant la mort cellulaire. Il s'est avéré que le clivage de raptor affaiblissait son interaction avec mTOR au sein du complexe mTORCl, ce qui participe à l'inactivation de mTORCl lors de traitements avec des molécules anti-cancéreuses. Ces résultats nous ont permis de mieux comprendre les mécanismes d'actions de différentes drogues anti-cancéreuses au niveau du complexe mTORCl, ce qui peut être utile pour la synthèse de nouvelles molécules ciblant mTORCl ainsi que pour lutter contre les mécanismes de résistance chimiothérapeutiques. -- La protéine « mammalian target of rapamycin » (mTOR) est une sérine/thréonine kinase qui est hautement conservée des protistes à l'être humain. Deux complexes mTOR existent : le complexe 1 mTOR (mTORCl) et le complexe 2 mTOR (mTORC2). Ils régulent positivement des processus anaboliques (synthèse de protéines et de lipides, le métabolisme énergétique, l'organisation du cytosquelette, la survie cellulaire) et négativement des voies cataboliques (autophagic, biogenèse de lysosomes). Les complexes mTORCl et mTORC2 sont sensibles aux signaux mitogéniques tels que les acides aminés, le glucose, les facteurs de croissance, l'état énergétique (ATP) et les niveaux d'oxygène et induisent des voies de croissance cellulaire essentielles. La voie cellulaire regulée par mTORCl peut être hyperactivée dans de nombreux cancers humains. Puisque plusieurs voies cellulaires convergent et régulent les complexes mTORCl et mTORC2, des mutations dans les kinases en amont peuvent mener à une dérégulation de l'activation de mTOR. Des stratégies thérapeutiques ont été développées pour cibler les complexes mTORCl et mTORC2, ainsi que les kinases en amont qui régulent mTOR. Plusieurs drogues ciblant mTORCl, telles que la rapamycine et la curcumine, affectent l'interaction entre mTOR et un composant spécifique de mTORCl, raptor. Dans cette étude, nous nous sommes intéressés aux mécanismes moléculaires des drogues qui ciblent mTORCl, ainsi que leur effet déstabilisant sur l'interaction entre mTOR et raptor dans des lignées cellulaires de lymphomes. Nous avons démontré que raptor était clivé en un fragment de lOOkDa après traitement avec la rapamycine, la curcumine, l'étoposide, la cisplatine, la staurosporine et le ligand Fas (FasL). Etant donné que ces drogues ont été décrites comme induisant I'apoptose, l'utilisation d'un inhibiteur de caspases (z- VAD-fmk) a révélé que le clivage de raptor, lors de la mort cellulaire, était dépendant des caspases. Des essais caspases in vitro ont permis d'identifier la caspase-6 (ainsi que probablement d'autres caspases) comme étant une protéase impliquée dans le clivage de raptor. La séquence protéique de raptor a montré potentiellement plusieurs sites de clivage de caspases aux extrémités amino-terminale et carboxy-terminale. La mutagénèse a permis d'identifier les sites de clivages de raptor par les caspases comme étant DEAD LTD (acides aminés 17-23) et DDADD (acides aminés 939¬943). De plus, le clivage de raptor corrèle avec l'inhibition de l'activité de mTORCl envers ces substrats (S6K et 4E-BP1). Nous avons aussi observé que le clivage de raptor affaiblissait l'interaction entre mTOR et raptor, ce qui indique que ce clivage est une étape critique dans l'inhibition de mTORCl durant I'apoptose. Pour terminer, la mutagénèse du site de clivage de raptor DDADD a montré une résistance à la mort cellulaire de cellules cancéreuses. Notre travail de recherche a révélé un nouveau mécanisme moléculaire qui module l'organisation et l'activité de mTORCl, ce qui peut être d'un grand intérêt pour les recherches dans le domaine de mTOR ainsi que pour la découverte de molécules ciblant mTORCl. -- The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, which is highly conserved from yeast to humans. Two different mTOR complexes exist: the mTOR complex 1 (mTORCl) and the mTOR complex 2 (mTORC2). They positively regulate anabolic processes (protein and lipid synthesis, energy metabolism, cytoskeleton organization, cell survival) and negatively regulate catabolic pathways (autophagy, lysosome biogenesis). The mTORCl and mTORC2 respond to mitogenic stimuli such as amino acids, glucose, growth factors, energy levels (ATP) and oxygen levels and drive essential cellular growth pathways. The mTORCl pathway can be found hyperactivated in numerous human cancers. As various cellular pathways converge and regulate mTORCl and mTORC2, mutations in upstream protein kinases can lead to a deregulated mTOR activation. Different therapeutic strategies have been developped to target mTORCl, mTORC2, as well as upstream protein kinases regulating mTOR pathways. Various drugs targeting mTORCl, such as rapamycin and curcumin, affect the interaction between mTOR and a specific mTORCl component, raptor. In this study, we investigated the molecular mechanisms of drugs targeting mTORCl, as well as their destabilizing effect on the mTOR-raptor interaction in lymphoma cell lines. We demonstrated that raptor was processed into a lOOkDa fragment after treatment with rapamycin, curcumin, etoposide, cisplatin, staurosporine and FasL. As these drugs were reported to induce apoptosis, the use of a pan-caspase inhibitor (z-VAD-fmk) revealed that the cleavage of raptor under cell death was caspase-dependent. In vitro caspase assays were performed to identify caspases-6 (and probably other caspases) as an important cysteine protease implicated in the cleavage of raptor. Analysis of raptor protein sequence showed several putative caspase-specific cleavage sites at the N-terminal and the C-terminal ends. Mutagenesis studies allowed us to identify the DEADLTD (amino acids 17-23) and the DDADD (amino acids 939-943) as the caspase-dependent cleavage residues of raptor. Furthermore, the cleavage of raptor correlated with inhibition of mTORCl activity towards its specific targets (4E-BP1 and S6K). We also highlighted that raptor processing weakened the interaction between mTOR and raptor, indicating that raptor cleavage is a critical step in the mTORCl inhibition process during apoptosis. Finally, mutagenesis of raptor C-terminal cleavage site (DDADD) conferred resistance to the chemotherapeutic-mediated cell death cascade of cancer cell. Our research work highlighted a new molecular mechanism modulating mTORCl organization and activity, which can be of great interest in the mTOR field research and for designing drugs trageting mTORCl.