La biogenèse des ribosomes est un processus complexe finement régulé pour s’adapter à la disponibilité en nutriments et en facteurs de croissance ainsi qu’à la présence éventuelle de stress. Une étape clé de la régulation de la biogenèse des ribosomes se fait par la régulation de la traduction des ARNm 5’ Terminal OligoPyrimidine (5’TOP) qui codent pour les protéines ribosomiques. Bien que la voie de signalisation mechanistic Target of Rapamycin (mTOR) ait été identifiée depuis des décennies comme activatrice de cette traduction des ARNm 5’TOP, les régulateurs impliqués ainsi que leur contrôle par la voie mTOR n’ont jamais été identifiés avec précision. Dans ce travail, nous avons montré que La-related protein 1 (Larp1), une protéine de liaison à l’ARN cible de mTOR, est indispensable à l’inhibition de la traduction des ARNm 5’TOP en aval de mTOR. De plus, Larp1 semble participer à l’inhibition de la formation du complexe d’initiation de la traduction eIF4F, qui est responsable du recrutement du complexe de pré-initiation 43S sur la coiffe m7G présente à l’extrémité 5’ de tous les ARNm. Nous avons également démontré que Larp1 peut se lier à la protéine Poly(A)-Binding Protein (PABP) et à la protéine de la petite sous-unité ribosomique RPS6 et que cette dernière interaction diminue lorsque les sites de phosphorylation de Larp1 dépendants de mTOR Ser 689 et 697 sont mutés en alanine. Ces résultats représentent une avancée importante dans la compréhension de la régulation de la traduction des ARNm 5’TOP par la voie mTOR. Cependant, des études complémentaires sont nécessaires afin de comprendre plus en détail le mécanisme exact par lequel Larp1 réprime la traduction des ARNm 5’TOP. / Ribosome biogenesis is a process that is finely tuned to adapt to nutrients and growth factors availability as well as to cellular stress and insults. Ribosomal proteins, the protein component of ribosomes, are encoded by 5’ Terminal Oligopyrimidine (5’TOP) mRNAs. A key step in ribosome biogenesis is the up-regulation of the translation of 5’TOP mRNAs. Although the mechanistic Target of Rapamycin (mTOR) pathway have been known for decades to promote 5’TOP mRNAs translation, the regulators involved and their control by the mTOR pathway remains obscure. In this work we demonstrated that La-related protein 1 (Larp1), an RNA-binding protein and substrate of mTOR, is necessary for the inhibition of 5’TOP mRNAs translation downstream of mTOR. In particular Larp1 seems to interfere with the formation of the translation initiation complex eIF4F, which is responsible for the recruitment of the 43S preinitiation complex to the m7G cap present at the 5’ end of mRNAs. Furthermore we found that Larp1 interacts with the protein Poly(A)-Binding Protein (PABP) and with the small ribosomal subunit protein RPS6 and that the latter interaction is decreased by mutation to alanine of the mTOR-dependent phosphorylation sites Ser 689 and 697. These findings are an important contribution to the understanding of the regulation of the translation of 5’TOP mRNAs by the mTOR pathway. Nevertheless more studies will be needed in order to dissect the mechanism by which Larp1 represses translation of 5’TOP mRNAs.

La biogenèse des ribosomes est un processus complexe finement régulé pour s’adapter à la disponibilité en nutriments et en facteurs de croissance ainsi qu’à la présence éventuelle de stress. Une étape clé de la régulation de la biogenèse des ribosomes se fait par la régulation de la traduction des ARNm 5’ Terminal OligoPyrimidine (5’TOP) qui codent pour les protéines ribosomiques. Bien que la voie de signalisation mechanistic Target of Rapamycin (mTOR) ait été identifiée depuis des décennies comme activatrice de cette traduction des ARNm 5’TOP, les régulateurs impliqués ainsi que leur contrôle par la voie mTOR n’ont jamais été identifiés avec précision. Dans ce travail, nous avons montré que La-related protein 1 (Larp1), une protéine de liaison à l’ARN cible de mTOR, est indispensable à l’inhibition de la traduction des ARNm 5’TOP en aval de mTOR. De plus, Larp1 semble participer à l’inhibition de la formation du complexe d’initiation de la traduction eIF4F, qui est responsable du recrutement du complexe de pré-initiation 43S sur la coiffe m7G présente à l’extrémité 5’ de tous les ARNm. Nous avons également démontré que Larp1 peut se lier à la protéine Poly(A)-Binding Protein (PABP) et à la protéine de la petite sous-unité ribosomique RPS6 et que cette dernière interaction diminue lorsque les sites de phosphorylation de Larp1 dépendants de mTOR Ser 689 et 697 sont mutés en alanine. Ces résultats représentent une avancée importante dans la compréhension de la régulation de la traduction des ARNm 5’TOP par la voie mTOR. Cependant, des études complémentaires sont nécessaires afin de comprendre plus en détail le mécanisme exact par lequel Larp1 réprime la traduction des ARNm 5’TOP. / Ribosome biogenesis is a process that is finely tuned to adapt to nutrients and growth factors availability as well as to cellular stress and insults. Ribosomal proteins, the protein component of ribosomes, are encoded by 5’ Terminal Oligopyrimidine (5’TOP) mRNAs. A key step in ribosome biogenesis is the up-regulation of the translation of 5’TOP mRNAs. Although the mechanistic Target of Rapamycin (mTOR) pathway have been known for decades to promote 5’TOP mRNAs translation, the regulators involved and their control by the mTOR pathway remains obscure. In this work we demonstrated that La-related protein 1 (Larp1), an RNA-binding protein and substrate of mTOR, is necessary for the inhibition of 5’TOP mRNAs translation downstream of mTOR. In particular Larp1 seems to interfere with the formation of the translation initiation complex eIF4F, which is responsible for the recruitment of the 43S preinitiation complex to the m7G cap present at the 5’ end of mRNAs. Furthermore we found that Larp1 interacts with the protein Poly(A)-Binding Protein (PABP) and with the small ribosomal subunit protein RPS6 and that the latter interaction is decreased by mutation to alanine of the mTOR-dependent phosphorylation sites Ser 689 and 697. These findings are an important contribution to the understanding of the regulation of the translation of 5’TOP mRNAs by the mTOR pathway. Nevertheless more studies will be needed in order to dissect the mechanism by which Larp1 represses translation of 5’TOP mRNAs.

La biogenèse des ribosomes est un processus complexe finement régulé pour s’adapter à la disponibilité en nutriments et en facteurs de croissance ainsi qu’à la présence éventuelle de stress. Une étape clé de la régulation de la biogenèse des ribosomes se fait par la régulation de la traduction des ARNm 5’ Terminal OligoPyrimidine (5’TOP) qui codent pour les protéines ribosomiques. Bien que la voie de signalisation mechanistic Target of Rapamycin (mTOR) ait été identifiée depuis des décennies comme activatrice de cette traduction des ARNm 5’TOP, les régulateurs impliqués ainsi que leur contrôle par la voie mTOR n’ont jamais été identifiés avec précision. Dans ce travail, nous avons montré que La-related protein 1 (Larp1), une protéine de liaison à l’ARN cible de mTOR, est indispensable à l’inhibition de la traduction des ARNm 5’TOP en aval de mTOR. De plus, Larp1 semble participer à l’inhibition de la formation du complexe d’initiation de la traduction eIF4F, qui est responsable du recrutement du complexe de pré-initiation 43S sur la coiffe m7G présente à l’extrémité 5’ de tous les ARNm. Nous avons également démontré que Larp1 peut se lier à la protéine Poly(A)-Binding Protein (PABP) et à la protéine de la petite sous-unité ribosomique RPS6 et que cette dernière interaction diminue lorsque les sites de phosphorylation de Larp1 dépendants de mTOR Ser 689 et 697 sont mutés en alanine. Ces résultats représentent une avancée importante dans la compréhension de la régulation de la traduction des ARNm 5’TOP par la voie mTOR. Cependant, des études complémentaires sont nécessaires afin de comprendre plus en détail le mécanisme exact par lequel Larp1 réprime la traduction des ARNm 5’TOP. / Ribosome biogenesis is a process that is finely tuned to adapt to nutrients and growth factors availability as well as to cellular stress and insults. Ribosomal proteins, the protein component of ribosomes, are encoded by 5’ Terminal Oligopyrimidine (5’TOP) mRNAs. A key step in ribosome biogenesis is the up-regulation of the translation of 5’TOP mRNAs. Although the mechanistic Target of Rapamycin (mTOR) pathway have been known for decades to promote 5’TOP mRNAs translation, the regulators involved and their control by the mTOR pathway remains obscure. In this work we demonstrated that La-related protein 1 (Larp1), an RNA-binding protein and substrate of mTOR, is necessary for the inhibition of 5’TOP mRNAs translation downstream of mTOR. In particular Larp1 seems to interfere with the formation of the translation initiation complex eIF4F, which is responsible for the recruitment of the 43S preinitiation complex to the m7G cap present at the 5’ end of mRNAs. Furthermore we found that Larp1 interacts with the protein Poly(A)-Binding Protein (PABP) and with the small ribosomal subunit protein RPS6 and that the latter interaction is decreased by mutation to alanine of the mTOR-dependent phosphorylation sites Ser 689 and 697. These findings are an important contribution to the understanding of the regulation of the translation of 5’TOP mRNAs by the mTOR pathway. Nevertheless more studies will be needed in order to dissect the mechanism by which Larp1 represses translation of 5’TOP mRNAs.

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Malignant cutaneous melanoma is the deadliest form of skin cancer, and a majority of melanoma diagnoses are a result of exposure to ultraviolet (UV) radiation. UV radiation causes DNA damage, which if not repaired correctly via nucleotide excision repair (NER) can result in mutations and melanomagenesis. The melanocortin 1 receptor (MC1R) is a Gs protein coupled receptor located on melanocyte plasma membranes and is involved in protecting the skin from UV induced damage. MC1R signaling results in the activation of two protective pathways: 1) induction of eumelanin synthesis downstream of micropthalmia-associated transcription factor (MITF) and 2) acceleration of NER downstream of ataxia telangiectaseia mutated and Rad3 related (ATR). MC1R signaling, however, also promotes melanocyte proliferation, therefore, the activation of the MC1R pathway must be regulated. The overall hypothesis of this dissertation is that the pathways downstream of MC1R can be manipulated to protect against UV induced damage. Chapter 2 investigates the regulation of the MC1R neutral antagonist human β-defensin 3 (βD3). UV damage did not induce βD3 mRNA expression in ex vivo human skin explants. The induction of βD3 expression instead correlated with inflammatory cytokines including TNF. Chapter 3 investigates the interdependence and cross talk between the two protective pathways downstream of MC1R. We directly tested the effect of MITF on the acceleration of NER and the effect of ATR on the induction of eumelanin synthesis following MC1R activation. MITF was not required for the acceleration of NER as mediated by ATR, however, the induction of transcription of enzymes involved in eumelanin synthesis was dependent upon ATR kinase activity. Finally, Chapter 4 investigates the mechanism by which MC1R promoted proliferation and whether the two UV protective pathways downstream of MC1R could be selectively activated without the risk of melanocyte proliferation. MC1R signaling resulted in activation of the mechanistic target of rapamycin complex 1 (mTORC1), a major regulator of cell growth and proliferation. Inhibition of mTORC1 signaling via rapamycin prevented MC1R induced proliferation in vitro. Rapamycin, however, did not prevent MC1R induced eumelanin synthesis or the acceleration of NER in vitro or in vivo suggesting it is possible to selectively activate the beneficial signaling pathways without the risk of melanocyte proliferation. The results of this dissertation suggest that MC1R signaling could be augmented in individuals to prevent UV induced damage.