L’hypertension artérielle pulmonaire (HTAP) est une maladie mortelle caractérisée par un important remodelage vasculaire, principalement dû à l’hyperprolifération et à la résistance à l’apoptose des cellules du muscle lisse de l’artère pulmonaire (CML-AP). Récemment il a été montré que les CML-AP présentent un remodelage du métabolisme énergétique, avec une régulation négative de l’oxidation phosphorylante associée à une activation de la voie glycolytique, qui semble contribuer à leur phénotype particulier. La désacétylase sirtuine1 (SIRT1) est un important modulateur du métabolisme énergétique, notamment via son activation de peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), régulateur clé de la biogenèse mitochondriale. Dans cette étude, nous montrons pour la première fois que la prolifération des CML-AP de rat et humaines est caractérisée par une réduction de l’activité de SIRT1, et est augmentée suite à l’inhibition pharmacologique ou la sous-expression spécifique de SIRT1. De plus, suite à hypoxie chronique, des souris génétiquement déficientes en SIRT1 présentent un remodelage vasculaire plus important que celui observé chez les souris contrôles, ce qui est associé à une augmentation accentuée de l’hypertrophie et de la pression systolique du ventricule droit. Au contraire, l’activation pharmacologique de SIRT1 inhibe fortement la prolifération des CML-AP, et est associée à l’activation de la biogenèse mitochondriale. L’ensemble de ces résultats suggère que l'inactivation de SIRT1 joue un rôle causal dans l’hyperprolifération des CML-AP et cette enzyme pourrait être une nouvelle cible thérapeutique prometteuse pour le traitement de l’HTAP. / Pulmonary arterial hypertension (PAH) is a lethal disease characterized by an intensive vascular remodelling, mainly due to hyper-proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). Recently it has been found that PASMCs, similarly to cancer cells, demonstrate a shift in energy metabolism from oxidative phosphorylation towards glycolysis thus contributing to their particular phenotype. The deacetylase sirtuin1 (SIRT1) is an important modulator of energy metabolism, particularly via its activation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), the master regulator of mitochondrial biogenesis. Here we show for the first time that rat and human PASMC proliferation is characterised by a diminution of SIRT1 activity, and is potentiated by SIRT1 pharmacological inhibition or specific downregulation. Moreover, after chronic hypoxia exposure, SIRT1 KO mice display a more intense vascular remodelling compared to their control littermates and this is associated with an exacerbated increase in right ventricle systolic pressure and hypertrophy. Conversely, pharmacological SIRT1 activation strongly inhibits PASMC proliferation, and is associated with the activation of mitochondrial biogenesis. In general, the data obtained show that SIRT1 inactivation plays a causative role in PASMC proliferation and this enzyme could be a promising therapeutic target for PAH treatment.

in English Formation of mitochondria by the conversion of a bacterial endosymbiont is the fundamental moment in the evolution of eukaryotes. An integral part of the organelle genesis was the displacement of the endosymbiont genes to host nucleus and simultaneous creation of new pathways for delivery of proteins synthesized now in the host cytoplasm. Resulting protein translocases are complexes combining original bacterial components and eukaryote-specific proteins. In addition to these novel protein import machines, some components of the original bacterial secretory pathways have remained in the organelle. While the function of a widely distributed mitochondrial homolog of YidC, Oxa1, is well understood, the role of infrequent components of Sec or Tat translocases has not yet been elucidated. So far, more attention has been paid to their abundant plastid homologs, which assemble photosynthetic complexes in the thylakoid membrane. In the thesis, the structure and function of prokaryotic YidC, Sec and Tat machineries and their eukaryotic homologs are described. By comparing both organelles of the endosymbiotic origin, the hypothesis is drawn on why these translocases have been more "evolutionary successful" in plastids than in mitochondria.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs unique to animals that guard the germline genome integrity by regulating transposons, viruses, and genes. In mice, piRNAs are highly expressed in testis and guide one of the three PIWI proteins to regulate their targets. The purpose of the 3′ end 2′-O-methyl modification in piRNAs is unknown. It has been speculated that the modification increases stability and facilitates the function of piRNAs, but the direct evidence is lacking. My dissertation addresses two unanswered questions about mouse piRNAs: (1) how are piRNAs produced and how conserved is the piRNA pathway in all animals, and (2) why are mouse piRNAs 2′-O-methylated at their 3′ ends? How piRNAs are generated is still poorly characterized in several model organisms. Studies of these model organisms imply the mechanisms that produce piRNAs differ among animals, tissues and cell types. Here, we demonstrate that a single unified mechanism can explain piRNA production in most animals, from human to the non-bilateral animal hydra. Our analysis elucidated that, in male mouse and female fly germlines, PIWI proteins guided by the initiator piRNA slice long piRNA precursor transcripts, and this PIWI-guided slicing action starts the piRNA biogenesis. PIWI proteins also position the endonuclease to further fragment long piRNA precursor transcripts into a string of tail-to-head, phased trailing piRNAs in a stepwise manner. Our discovery shows the central role of PIWI proteins in the piRNA pathway: both initiating and sustaining the production of piRNAs. For the second question, we discovered that pre-piRNA trimming and piRNA 2′-O-methylation protect piRNAs from separate decay mechanisms. We showed that in the absence of 2′-O-methylation, mouse piRNAs with extensive complementarity to long RNAs are destabilized and destroyed by a mechanism similar to target-directed microRNA degradation (TDMD). On the other hand, untrimmed pre-piRNAs are destroyed by a different mechanism, independent of their extensive complementarity to long RNAs. In the absence of both 2′-O-methylation and trimming, the piRNA pathway collapses which supports the idea of piRNA trimming and methylation collaborating to stabilize piRNAs. Our work suggests that 2′-O-methylation and trimming are important for maintaining the steady-state abundance of piRNAs which is necessary for their function in either transposon silencing or gene regulation.

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<p>The β-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a β-barrel fold which can vary from 8 to 36 β-strands, with each serving an important role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis has remained elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. Here we present our recent work on the structures of BAM in its near native environment, characterized by cryo-EM, and study its interaction with OMP substrates. Specifically, we focused on the role of BAM-mediated EspP biogenesis, and structurally characterized crosslinked intermediates to atomic resolution, allowing for a more complete understanding of BAM-mediated OMP biogenesis. We also characterized BAM-mediated OmpT and OmpA biogenesis, which further supports a BamA-budding model for OMP biogenesis. Given its essential role in Gram-negative bacteria, BAM is an attractive target for antibiotics, and we contributed to characterizing a novel antibiotic designed against BAM called darobactin, which binds to the lateral gate of BAM, thereby disrupting OMP biogenesis and leading to programmed bacterial lysis.</p>

2-0xoglutarate/Fe(II)-dependent dioxygenases (ZOG Oxygenases) are a relatively poorly characterised enzyme family that hydroxylate biological macromolecules to regulate a variety of essential cellular processes in mammals, including; chromatin remodeling, extra-cellular matrix formation and oxygen sensing. The work in this th esis focuses on a ZOG Oxygenase termed Myc-Induced Nuclear Antigen (MINAS3). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity afits substrate(s) and their role in cancer is unknown. The aims of the resea rch that has resulted in this thesis were to; (i) develop a cell model of MINAS3 enzyme activity, (ii) apply this model to study the role of MINAS3 activity in cell transformation and cancer, and (iii) discover novel cellular processes regulated by MINA53 activity. As such, I have created an isogenic cell model consisting of K-Ras-transformed MINAS3 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINAS3. Using this model I have shown that MINAS3 activity maintains normal levels of the large ribosomal subunit (60S), and suppresses anchorage-independent growth, autophagy and gene expression. These observations suggest the existence and involvement of one or more substrates. Indeed, proteomic and biochemical analyses in collaboration with the Schofield laboratory (Chemistry, Oxford) confirmed the identity of a MINA53 substrate, the 60S ribosomal protein Rp127a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyJ hydroxylase; this represents the first discovery of a ribosomal oxygenase. The model developed here did not support a positive role for MINA53 in the transformation of MEFs. Rather it suggested that MINA53 can suppress transformation in some contexts, This prompted a wider investigation that demonstrated underexpression of MINA53 in several tumour types, and the presence of inactivating mutations in breast. ovarian and colon cancer. This thesis provides data supporting further research to understand the role of Rpl27a hydroxylation in the regulation of 60S biogenesis, autophagy and cancer. 2

Paralogs are present during ribosome biogenesis as well as in mature ribosomes in form of ribosomal proteins, and are commonly believed to play redundant functions within the cell. Two previously identified paralogs are the protein pair Ssf1 and Ssf2 (94% homologous). Ssf2 is believed to replace Ssf1 in case of its absence from cells, and depletion of both proteins leads to severely impaired cell growth. Results reveal that, under normal conditions, the Ssf paralogs associate with similar sets of proteins but with varying stabilities. Moreover, disruption of their pre-rRNP particles using high stringency buffers revealed that at least three proteins, possibly Dbp9, Drs1 and Nog1, are strongly associated with each Ssf protein under these conditions, and most likely represent a distinct subcomplex. In this study, depletion phenotypes obtained upon altering Nop7, Ssf1 and/or Ssf2 protein levels revealed that the Ssf paralogs cannot fully compensate for the depletion of one another because they are both, independently, required along parallel pathways that are dependent on the levels of availability of specific ribosome biogenesis proteins. Finally, this work provides evidence that, in yeast, Nop7 is genetically linked with both Ssf proteins. / Les paralogues sont présents lors de la biogenèse des ribosomes ainsi que dans les ribosomes matures sous forme de protéines ribosomiques, et sont généralement censées jouer des fonctions redondantes dans la cellule. Deux paralogues précédemment identifiées sont la paire de protéines Ssf1 et Ssf2 (94 % d'homologie). Ssf2 remplacerait Ssf1 en cas d’absence du dernier dans la cellule, et l’absence des deux protéines diminue la croissance cellulaire. Nos résultats révèlent que, dans des conditions normales, les paralogues Ssf s’associent à des ensembles de protéines similaires, mais avec différentes stabilités. De plus, la perturbation de leurs particules pré-rRNP à l’aide de tampons de haute stringence a révélé qu'au moins trois protéines, probablement Dbp9, Drs1 et Nog1, sont fortement associées à chaque protéine Ssf dans ces conditions, et très probablement représentent des sous-complexes distincts. Dans cette étude, les phénotypes cellulaires observés lors de la déplétion des protéines Nop7, Ssf1 et/ou Ssf2 ont révélé que les paralogues Ssf ne peuvent pas compenser entièrement pour la diminution de l'autre, car ils sont, indépendamment l’un de l’autre, nécessaires le long de voies de biogénèse ribosomale parallèles qui dépendent des niveaux de protéines impliqués dans la biogénèse des ribosomes disponibles. Enfin, ce travail fournit des preuves que, dans la levure, Nop7 est génétiquement lié aux deux protéines Ssf.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is known as the master regulator of mitochondrial biogenesis. PGC-1α holds this role by acting as a transcriptional coactivator for an array of transcription factors and nuclear hormone receptors, such as NRF-1/2 and ERRα/γ, whose downstream targets function in mitochondrial biogenesis and oxidative phosphorylation. PGC-1α is regulated both at the transcriptional and post-translational level in several signaling pathways, including p38 MAPK and AMPK. This regulation affects which transcription factor binding events can occur in a given tissue, and thus affects regulation of PGC-1α target genes. PGC-1α is downregulated in many neurodegenerative disorders as well as in muscular dystrophies, diabetes, and aging. Therefore, PGC-1α is prized as a potential therapeutic target to create novel treatments for these various diseases.However, details governing the spatio-temporal regulation of PGC-1α are not completely understood, and overexpression of PGC-1α throughout the body or even in certain tissues or subsets of cells have had detrimental effects in animal and cell models. Therefore, it is necessary to gain knowledge of how to modulate PGC-1α in a tissue-specific manner utilizing these different levels of regulation in order to develop novel therapies. In order to further understand all the functions that have been attributed to PGC-1α, the PGC-1α isoforms need to be accounted for and understood in human tissues. Several murine isoforms have been published, as well as several human brain and muscle isoforms. However, most of these isoforms have only been validated as mature transcripts, and it is not known whether they produce functional protein. Our lab has identified the isoform b transcript in human brain tissue via 5’ RACE and have developed an isoform b specific antibody. This project aimed to characterize the isoform b transcripts and also to validate and optimize this antibody for immunoblotting conditions for detection of further PGC-1α-b isoform protein variants in human tissues. Preliminary studies in our lab have shown that in postmortem frontal cortex from age-matched PD and healthy patients, isoform a transcript levels were 10-15 times more abundant than that of isoform b. These differences in regulation could be partially attributed to the isoform b promoter region being heavily methylated, as shown in this thesis through bisulphite cloning and sequencing as well as 454 bisulphite sequence analysis. The high degree of methylation, correlated with the low level of isoform b transcript in brain and it is not known whether this transcript would be translated into protein in this tissue. In order to probe for isoform b protein expression using human cell lines and tissues, however, it was necessary to create a recombinant protein in order to have a positive control with which to optimize our novel antibody. In our previous 5’ RACE studies, an alternatively spliced PGC-1α-b transcript was found which coded for an early stop codon. This truncated isoform was called PGC-1α-b-3T1, and mature transcript was found in both human skeletal muscle and brain. For this project, PGC-1a-b-3T1 was cloned from human skeletal muscle into a bacterial expression vector to create a recombinant GST fusion protein. This protein was used to validate and optimize our PGC-1α-b specific antibody as well as to determine sensitivity and specificity. The purified recombinant protein contained 3 bands of lower molecular weight that were detected via western blot with both GST and the PGC-1α-b specific antibody. These bands were trypsin cleaved and subjected to mass spectrometry analysis, which verified that all bands detected by the PGC-1a-b specific antibody contained the epitope sequence, and thus binding was specific. This protein was then used to determine western blotting conditions and sensitivity, which is 10 ng using a 1:100 dilution of the antibody. This antibody was then used to probe SH-SY5Y WCL, a human neuroblastoma cell line. Peptide competition assay confirmed 5 PGC-1α-b specific proteins in these lysates. The sizes of these proteins matched to several murine PGC-1α-b isoforms as well as putative PGC-1α-b versions of PGC-1a-a isoforms. These findings provided the putative identities of several endogenous functional human PGC-1α-b isoforms. Mammalian overexpression vectors of these isoforms are still in development. By using this antibody and these expression vectors to further characterize these isoforms, including determining tissue specificity, more knowledge of PGC-1α will be gained. This information could then be used to develop novel, tissue specific treatments for pharmacological intervention of diseases characterized by PGC-1α misregulation.