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« Choisissez un travail que vous aimez et vous n'aurez pas à travailler un seul jour de

Chris Gallatin 6

Paul, I need to thank you for many things. I guess I will start with the opportunity you gave me to join your lab after the five months of internship via the Paris 7 exchange program (thank you Martine because that would never have happened without you). I remember I declined at the time. Changing everything in my life sounded a bit too complicated. It didn't take me long to realize that I was doing a mistake. And then it took me only a few days to decide to come and join your lab. This was maybe the biggest decision I made and this decision truly changed everything for me. I have no regrets. I have had a great mentor for the last four years. I enjoyed all the conversations we had (science and not science), I took note of all your advice and tried to apply them and of course I really enjoyed working on every project I was involved in. Thank you for your trust as well. I think that the most important thing I learnt from you is to not be afraid of challenges. I am also very grateful that your lab was in such an inspiring environment: St. Jude Children's Research Hospital is definitely the best place to work and I am glad you decided to move your lab here a few years ago.

Dr Triller, nous n'avons pas eu beaucoup d'occasions de communiquer pendant ces quatre ans mais j'ai beaucoup apprécié vos conseils à chaque fois que cela arrivait. Je vous remercie de m'avoir fait confiance et de m'avoir permis de partir réaliser ma thèse avec Paul alors que la situation était pour le moins inhabituelle.

Emilie, tu es celle qui m'a le plus appris, que ce soit au niveau manips ou pour l'écriture scientifique. Je suis tellement heureuse d'avoir pu travailler avec toi pendant 5 mois. Tu es passée de mentor à amie en un rien de temps et tu as réussi à toujours être présente pour moi tout au long des années dans les moments de doutes, ou pour des conseils. Je me dis que si je ne t'avais pas rencontrée dans ce cadre particulier je ne suis pas sûre que je serais là aujourd'hui à continuer dans la recherche. Alors je te remercie et malheureusement pour toi tu n'es pas encore débarrassée de moi ! Hong Joo, thank you so much for all you did for me during my PhD. You started by teaching me everything about A1 and A2 and you helped me to grow and to become independent. I enjoyed all the time spent with you and I think you are the one who had to listen to my complaints the most. I definitely could never have survived without you (or some other people wouldn't have!). And I have to say, whenever I leave, I will miss your (bad) jokes ;)

Taylor lab members (present and past), thank you for your patience and for sharing your knowledge. This lab is successful because of the diverse skills you have and I definitely learnt from each of you.

Tanja, thank you for this collaboration and the time you spent teaching me biophysics. I enjoyed all our scientific discussions and I really appreciated that you welcomed me in your lab for so long!

Muriel et Coline, vous avez réussi à distance à ce que je garde cette part de folie à travers les difficultés que peut représenter une thèse. Ce n'est pas négligeable ! Muriel, merci de m'avoir supportée ces derniers mois avec mes questions incessantes sur le déroulement de la thèse du point de vue administratif et merci d'avoir été une des rares personnes à me lire et me corriger ! Melissa, you are certainly one of the persons who helped me the most during this last year and a half of my PhD. Thank you for your help in everything (personal, science etc.). I learnt so much from you and thank you for the thesis corrections. I am glad I had a chance to meet you and become your friend.

Mamie, merci de m'avoir soutenue pendant ces 4 ans à renfort d'appels, de romans et de chocolats ! Je ne peux pas imaginer ce qu'aurait été ma vie sans ces petits moments magiques (surtout le chocolat, il faut l'avouer !).

Anthony, Benjamin, la décision de partir n'a pas été des plus faciles et je vous remercie de votre soutien ! Vous avez toujours été là pour les étapes clés de ces 4 dernières années et les moments passés ensemble ont toujours été magiques. Je vous remercie aussi d'avoir fait autant de déplacements aux US pour me voir. J'espère toujours un jour regagner le statut de soeur à part entière ;)

Papa et Maman, me laisser partir a surement été une des choses les plus difficiles pour vous et je vous remercie de m'avoir soutenue dans ce choix. Vous m'avez appris à me faire confiance et à ne pas douter de mes capacités et pour ça je vous remercie aussi. Vous avez toujours su être la dans mes grands moments de doute et me pousser à continuer quand j'avais juste envie de tout abandonner. Je n'oublierai pas non plus que vous êtes ceux qui m'ont convaincue de partir et vu toutes les belles choses que je ramènerai de ces années passées à Memphis, je ne peux que vous remercier à nouveau.

Enora, tu as été le moment magique de cette thèse. Ta venue dans ma vie m'a apporté un équilibre que je n'aurais pas réussi à trouver sans toi. Ta joie de vivre est la meilleure des distractions et je te remercie de ta patience quand je réponds à mes mails du travail au milieu d'une de nos sessions de jeux ! Sébastien, le meilleur pour la fin. S'il y a bien une chose de claire, je n'aurais jamais réussi à être là où j'en suis aujourd'hui sans toi. Merci de m'avoir fait confiance et de m'avoir suivie au bout du monde. Sans cette décision, je n'aurais jamais pu faire de thèse ici. Merci de m'avoir supportée au quotidien dans mon roller-coaster de thésarde, d'avoir accepté mes horaires de folie et d'avoir compris mes envies de réussite. Merci aussi pour la rédaction de cette thèse, je savais qu'épouser un ingénieur informatique était la clef du succès ;) Finalement, merci de me permettre de réaliser mes rêves tous les jours à tes côtés. of one or more limbs or fasciculation of the tongue (bulbar onset) occur [START_REF] Ludolph | Frontal lobe function in amyotrophic lateral sclerosis: a neuropsychologic and positron emission tomography study[END_REF][START_REF] Murakami | A novel SOD1 gene mutation in familial ALS with low penetrance in females[END_REF]. Bulbar involvement causes difficulties in swallowing and respiration and is a frequent cause of death in later stages of disease. Features that arise from dysfunction of the upper motor neurons are spasticity and brisk tendon reflexes. Loss of lower motor neurons results in muscle weakness, painful cramps, and muscle fasciculations followed by muscle atrophy and loss of tendon reflexes. When loss of both upper and lower motor neurons occurs, spasticity or weakness may predominate before the amyotrophy develops. Generally fatal within 1-5 years of onset, ALS has a prevalence of 2-3 per 100,000 individuals with its incidence peaking after 60 years of age. The causes of almost all occurrences of the disease remain unknown. In 90-95% of instances, there is no apparent genetic linkage (a form of the disease referred to as sporadic ALS), but in the remaining 5-10% of cases, the disease is inherited in a dominant manner (designated as familial ALS). to that seen in IBM [START_REF] Kimonis | VCP disease associated with myopathy, Paget disease of bone and frontotemporal dementia: review of a unique disorder[END_REF]. PDB is observed in half the patients with IBMPFD [START_REF] Kimonis | Clinical and molecular studies in a unique family with autosomal dominant limb-girdle muscular dystrophy and Paget disease of bone[END_REF][START_REF] Watts | Clinical and genetic heterogeneity in chromosome 9p associated hereditary inclusion body myopathy: exclusion of GNE and three other candidate genes[END_REF], usually with an earlier onset (mean age of 42) than is seen with isolated PDB (mean age of 59 years in those without family history).

Table of figures

The dementia associated with IBMPFD is typical of frontotemporal dementia (FTD) and is characterized by reasoning, personality, and language impairments. FTD is a degenerative condition of the brain that primarily causes atrophy and neuronal loss in the frontal and anterior temporal lobes [START_REF] Arnold | Quantitative neurohistological features of frontotemporal degeneration[END_REF][START_REF] Turner | Clinical, neuroimaging, and pathologic features of progressive nonfluent aphasia[END_REF][START_REF] Zhukareva | Loss of brain tau defines novel sporadic and familial tauopathies with frontotemporal dementia[END_REF]. Histologically, patients display a novel pattern of ubiquitin pathology distinct from other forms of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U).

Approximately 12% of individuals with IBMPFD are affected with all three disorders listed above.

50% of affected people have two of the disorders, 30% have myopathy alone, and 8% have either PDB or FTD alone (Figure 1). Similar to other neurodegenerative diseases, the degenerating neurons of ALS patients are characterized by the accumulation of protein aggregates (Forman et al., 2004). In 2006 it was discovered that the major protein component of many of these inclusions is the 43-kD TAR-DNA-binding protein (TDP-43) [START_REF] Arai | TDP-43 is a component of ubiquitin-positive taunegative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF][START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF].

IBMPFD is also characterized by cytoplasmic inclusions with approximately 55% of patients having ubiquitin-positive inclusions of TDP-43 (Figure 2) and 45% having tau-positive inclusions [START_REF] Leyton | Frontotemporal dementias: Recent advances and current controversies[END_REF]. It remains unknown how these inclusions are formed and what the consequences of such inclusions are. [START_REF] Watts | Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein[END_REF]. Later on, exome sequencing revealed VCP mutations as a cause of familial ALS as well [START_REF] Johnson | Exome sequencing reveals VCP mutations as a cause of familial ALS[END_REF]. Inherited forms of ALS and IBMPFD were also found to be often caused by missense mutations impacting heterogeneous ribonucleoproteins (hnRNPs) such as TDP-43, FUS, hnRNPA1 and hnRNPA2B1.

I.1.2.1 VCP

Valosin-containing protein (VCP), also known as p97, is a protein belonging to the ATPase Associated with diverse cellular Activities (AAA+ ATPase) protein family. This family of enzymes is associated with a wide range of cellular processes such as proteolysis, DNA replication and repair, and membrane fusion, which it carries out in an ATP-dependent manner [START_REF] Ogura | AAA+ superfamily ATPases: common structure--diverse function[END_REF]. VCP is ubiquitously expressed in the cytoplasm and the nucleus and has a tripartite structure consisting of an N-terminal domain and two central D1 and D2 AAA+ domains. It also contains linker regions that join the N-D1 and D1-D2 domains (Figure 3).

The N-terminal domain is necessary for substrate and co-factor association, whereas the D1 and D2 domains are needed for ATP binding and hydrolysis. The D1 domain is primarily responsible for VCP hexamerization but this molecular assembly is not dependent on nucleotide binding (Wang et al., 2003a;Wang et al., 2003b;[START_REF] Zhang | Structure of the AAA ATPase p97[END_REF]. The ATPase activity conferred by the D2 domain is essential for VCP's function as a molecular chaperone in various cellular processes (Esaki and Ogura, 2010;[START_REF] Song | ATPase activity of p97-valosin-containing protein (VCP). D2 mediates the major enzyme activity, and D1 contributes to the heat-induced activity[END_REF]. Six VCP monomers assemble into a macromolecular ring complex with the inner core formed by the D1/D2 domains, with the N-domains radiating outward. Structural changes can occur in the ring, allowing VCP to function as a chaperon that interacts with diverse adaptors. Notably, VCP is a ubiquitin-dependent segregase that can remove some ubiquitinylated proteins from protein complexes due to interactions with various adaptors. Through this mechanism, VCP can mediate a wide variety of essential cellular processes such as: cell cycle progression, DNA replication, protein quality control, and chromosomal decondensation. The C-terminal domain of VCP, following the D2 domain, is poorly characterized, but this region includes the major tyrosine phosphorylation site implicated in the regulation of endoplasmic reticulum (ER assembly) and cell cycle-dependent nuclear localization of VCP [START_REF] Egerton | Biochemical characterization of valosin-containing protein, a protein tyrosine kinase substrate in hematopoietic cells[END_REF][START_REF] Lavoie | Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro[END_REF][START_REF] Madeo | Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p[END_REF].

Because of its role in many cell processes, deregulation or mutations in VCP cause serious diseases. For example, VCP is upregulated in cancer while missense mutations cause a dominantly inherited degeneration of bone, brain, and muscles. Fourteen mutations in VCP have been associated with diseases including myopathy, dementia, Paget's disease of bone, ALS, and Parkinsonism. Among these mutations, R115H is the most prevalent diseaseassociated mutation and A232R causes the most serious symptoms of the diseases [START_REF] Ju | Inclusion body myopathy, Paget's disease of the bone and fronto-temporal dementia: a disorder of autophagy[END_REF]. Interestingly, the amino acids affected by the disease-causing mutations are highly conserved across species. Even though it remains unknown how mutations in VCP cause disease, some studies have shown how they affect the structure of the protein. The majority of the mutations localize to the interface of the N-and D1-domains (Figure 4). for ATP and ADP. The VCP mutations R155P and A232E, found in IBMPFD patients, show an increase in ATPase activity and increased sensitivity to heat-induced upregulation in ATPase activity [START_REF] Halawani | Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation[END_REF]. It has been shown recently that stress granule clearance was impaired when VCP was mutated [START_REF] Buchan | Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function[END_REF]). Yet, it remains unclear how the mutations lead to the pathogenesis in biological systems. sequences, and a C-terminal domain enriched in glycines (Gly-rich) that may be required for the exon skipping and splicing inhibitory activity as well as for binding to nuclear proteins (Figure 5). TDP-43 is a nuclear DNA-and RNA-binding protein with multiple functions in transcriptional repression, pre-mRNA splicing, and translational regulation [START_REF] Da Cruz | Understanding the role of TDP-43 and FUS/TLS in ALS and beyond[END_REF][START_REF] Lagier-Tourenne | TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration[END_REF]. TDP-43 is highly conserved through evolution and it associates with other members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family by interactions through its C-terminal domain [START_REF] Brundin | Prion-like transmission of protein aggregates in neurodegenerative diseases[END_REF].

TDP-43 was initially identified as the major constituent of neuronal inclusions in sporadic ALS and in the largest subset of FTD [START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]. In the degenerating neurons and glial [START_REF] Dormann | TDP-43 and FUS: a nuclear affair[END_REF]. cells of the central nervous system (CNS) of ALS patients, TDP-43 is relocated from the nucleus to large cytoplasmic aggregates. Additional studies showed that TDP-43 abnormally accumulates in the sarcoplasm of IBMPFD patients [START_REF] Salajegheh | Sarcoplasmic redistribution of nuclear TDP-43 in inclusion body myositis[END_REF]. This accumulation is accompanied by its nuclear depletion, suggesting its redistribution from the myonucleus to the sarcoplasm is similar to the nuclear to cytoplasmic redistribution seen in affected cortical regions.

Mutations in TDP-43 were identified in both sporadic ALS and familial ALS [START_REF] Sreedharan | TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF]. However, these rare mutations are associated with less than 5% of the familial cases. It is important to note that most of the disease-causing mutations are localized in the C-terminal glycine-rich region of the protein (Figure 5).

I.1.2.3 Identification of hnRNPA1 and hnRNPA2B1 as disease-causing proteins

I.1.2.3.1 Identification of VCP-mutations negative families

Some, but not all, cases of IBMPFD associated with ALS are caused by mutations in the VCP gene. Work from Kim et al. [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF]) (See annex) identified two families with dominantly inherited degeneration of muscle, bone, brain, and motor neurons. The affected members of these families had identical clinical symptoms compared to patients presenting VCP mutations.

Sequencing of the affected patients revealed that VCP was not mutated. Genetic analysis of this family by exome sequencing and linkage analysis identified mutations in two genes: hnRNPA1 and hnRNPA2B1 (Figure 6).

The two mutations described corresponded to a substitution of a valine residue in the place of a highly conserved aspartate residue in human paralogs of the hnRNP A/B family (Figure 7). The mutations identified for hnRNPA2B1 and hnRNPA1 were D290V and D262V, respectively. hnRNPA2B1 is expressed as two alternatively spliced isoforms: A2 and B1. The shorter isoform, hnRNPA2, lacks 12 amino acids in the N-terminal region and is the major isoform, accounting for ~90% of the protein in most tissues. The hnRNPA2 and hnRNPA1 proteins have two repeats of RNA-recognition motifs (RRMs) that bind RNAs. They are one of the most abundant core proteins of hnRNP complexes [START_REF] Krecic | hnRNP complexes: composition, structure, and function[END_REF]. These complexes are made of predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins function in multiple cellular activities, ranging from transcription and pre-mRNA processing in the nucleus to cytoplasmic mRNA translation and turnover . hnRNPA1 and hnRNPA2B1 are involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm, and may modulate splice site selection [START_REF] Han | Functional diversity of the hnRNPs: past, present and perspectives[END_REF]. They are mainly present in the nucleus but can also shuttle between the nucleus and the cytoplasm (Pinol-Roma and [START_REF] Pinol-Roma | Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm[END_REF]. Indeed, they are exported from the nucleus, along with other hnRNP proteins and probably bound to mRNA, to then be re-imported. Their M9 domain acts as both a nuclear localization and nuclear export signal [START_REF] Izaurralde | A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export[END_REF]. hnRNPA1 and hnRNPA2 also contain a carboxy terminal domain enriched in glycines that clusters the disease-causing mutations (Figure 8).

In muscle biopsies of healthy patients, hnRNPA2B1 and hnRNPA1 are predominantly nuclear.

In patients presenting the disease-causing mutations in these two genes, muscles biopsies reveal a relocalization of those proteins to cytoplasmic inclusions; the proteins are cleared from many nuclei [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF]. Muscle biopsies of the same patients also reveal TDP-43 pathology with a nuclear clearance and cytoplasmic inclusions. This observation is consistent with previous observations made in patients harboring mutations within VCP. hnRNPA2B1 and hnRNPA1 pathologies were also observed in VCP-related IBM [START_REF] Salajegheh | Sarcoplasmic redistribution of nuclear TDP-43 in inclusion body myositis[END_REF]. I.1.2.4 Relationship between previously described disease-implicated proteins

I.1.2.4.1 Interaction between VCP, TDP-43, hnRNPA1 and hnRNPA2

Several pieces of evidence support the conclusion that VCP, TDP-43, hnRNPA2B1 and hnRNPA1 interact with each other. First, it has been shown that the C-terminal region of TDP-43 binds directly to hnRNPA1 and hnRNPA2/B1 [START_REF] Buratti | TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing[END_REF]. This allows a cooperative regulation of RNA metabolism in cells. Drosophila melanogaster has also been used in a genetic screen to identify suppressors of VCP-related degeneration [START_REF] Ritson | TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97[END_REF]. Three genes in particular were identified: TBPH, the Drosophila homolog of TDP-43, and Hrb87F and Hrb98DE, the homologs of hnRNPA2B1 and hnRNPA1. Finally, mutations in VCP, hnRNPA2B1, hnrNPA1 and TDP-43 are shared between different neurodegenerative diseases, such as ALS, IBM, Paget's disease of bone and FTD (Figure 9).

Importantly, similarities exist in the pathology underlying muscle degeneration in ALS and IBMPFD caused by mutations in hnRNPA2B1, hnRNPA1 or VCP. Indeed, hnRNPA2B1, hnRNPA1 and TDP-43 are found in pathological cytoplasmic inclusions in sporadic and familial IBM [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF]. These proteins harbor the same structure and by analogy we could speculate that the C-terminal domain of the proteins containing the disease-causing mutations could contribute to the initiation or propagation of disease.

I.1.2.4.2 hnRNPs contain a low complexity sequence domain clustering the disease-causing mutations

Heterogeneous nuclear ribonucleoproteins or hnRNPs are defined as the set of proteins that associate with pre-mRNAs. In addition to their nuclear functions that includes regulation of mRNA splicing, a subset of hnRNP proteins also accompany mRNAs out into the cytoplasm and participate in the localization and translational regulation of the RNAs [START_REF] Dreyfuss | Messenger-RNA-binding proteins and the messages they carry[END_REF]. Here we will focus on TDP-43, hnRNPA1, hnRNPA2B1, and other hnRNPs. hnRNPs have similar structures composed of several RNA recognition motifs and a C-terminal glycine rich domain (Figure 10). This domain has relatively homogeneous amino acid content, harboring low amino acid diversity. For example, in hnRNPA1, 86% of this domain is made up of only four amino acids: glycine, serine, asparagine, and phenylalanine. Because of this low diversity, this region has been designed as a low complexity sequence domain (LCD) (Figure 10). LCDs are intrinsically disordered and are enriched in glycine as well as the uncharged polar amino acids: asparagine, glutamine, tyrosine, and serine. In the literature, several nomenclatures are used to describe it: glycine-rich domain, low complexity sequence domain (LCD) and prion-like domain (PrLD). The latter designation comes from the fact that these domains have a composition similar to the ones described for yeast prions. This domain is also extremely interesting because it clusters most of the disease-causing mutations. The mutations displayed in Figure 10 cause IBMPF and ALS.

Different hnRNPs can interact with each other through their LCDs and be recruited to a particular cellular structure: stress granules. A wealth of genetic evidence has emerged over the past 5 years implicating stress granules as a subcellular compartment that is central to the pathogenesis of degenerative diseases described earlier [START_REF] Li | Stress granules as crucibles of ALS pathogenesis[END_REF][START_REF] Ramaswami | Altered ribostasis: RNA-protein granules in degenerative disorders[END_REF]. Indeed, it is hypothesized that the cytoplasmic inclusions found in patient cells could be the result of persistent stress granules.

I.2 Stress granules as crucibles of these pathogeneses I.2.1 What are stress granules?

The central dogma of molecular biology deals with the detailed "residue-by-residue transfer of sequential information" [START_REF] Crick | Central dogma of molecular biology[END_REF]. In other terms, "DNA makes RNA and RNA makes protein". DNA is translated in the nucleus into messenger RNAs (mRNAs). In eukaryotic cells, the site of transcription (the nucleus) is usually separated from the site of translation (the cytoplasm), so the mRNA needs to be transported out of the nucleus into the cytoplasm. As mRNA exits the nucleus, the major cytoplasmic cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) recruits the translation initiation complex. This recruitment promotes protein synthesis via the assembly of polysomes (a string of translating ribosomes bound to mRNA). In cells, polysomes can be disassembled in the presence of cellular stressors such as heat shock, oxidative stress, ischemia, or viral infection. These different stresses trigger a translational arrest that leads to a process of molecular triage in which mRNA from disassembling polysomes is sorted and the fate of individual transcripts is determined.

Cytoplasmic stress granules (Figure 11) are cellular compartments that form as a direct consequence of this triage process, suggesting that these granules contain mRNAs stalled in the process of translation initiation [START_REF] Buchan | Eukaryotic stress granules: the ins and outs of translation[END_REF]. Stress granules are membraneless cytoplasmic foci ranging in size from 0.1 to 5 μm and they have been observed in yeast, protozoa, and metazoa. [START_REF] Anderson | Stress granules: the Tao of RNA triage[END_REF][START_REF] Buchan | Eukaryotic stress granules: the ins and outs of translation[END_REF]) (Figure 12). Translation initiation usually needs eIF2 in its unphosphorylated state to initiate translation, thus phosphorylation of eIF2 inhibits initiation of a further round of translation by depleting the eIF2/tRNAiMet/GTP ternary complex. In the absence of the active ternary complex, ribosomes finish their round on the translated transcript and then "run-off", as a further round cannot be initiated. The remaining 48S preinitiation complex stays bound to the 5´UTR of the mRNA [START_REF] Anderson | Stress granules: the Tao of RNA triage[END_REF]. This results in the production of a translationally stalled, noncanonical 48S complex unable to recruit the 60S ribosomal subunit. Although the next step, SG nucleation, is not yet fully understood, it is assumed that aggregation-prone SG proteins, such as TIA-1, TIAR and G3BP, associate with the 48S pre-initiation complex and form mRNP oligomers. Once this nucleation occurs, SGs assemble and contain the core SG components: eIF3, eIF4F, PABP-1 and small ribosomal subunits. The mRNA transcripts recruited to SGs are bound to multiple proteins that can interact with each other. Homotypic aggregation of the proteins assembles mRNPs into submicroscopic aggregates, which are then clustered into larger, microscopically visible SGs by heterotypic interactions between SG-nucleating proteins, and by association of some RBPs with multiple classes of transcripts [START_REF] Anderson | Stress granules: the Tao of RNA triage[END_REF]. Over time, the SGs fuse in larger and fewer structures. Some proteins lacking mRNAbinding properties can also be recruited because of their interaction with the SG nucleating proteins or with other components of SGs [START_REF] Anderson | Stress granules: the Tao of RNA triage[END_REF].

I.2.2.2 Disassembly of Stress Granules

Stress granule formation is reversible. During stress recovery, stress granules disassemble rapidly and polysomes are re-formed, leading to the translation of mRNAs (Figure 12). Because 48S pre-initiation complexes are preserved in SGs in an assembled state, translation can be rapidly reactivated upon stress recovery. Interestingly, some drugs can inhibit translational elongation and force the disassembly of pre-formed stress granules, indicating that stress granule mRNPs are in dynamic equilibrium with polysomes [START_REF] Anderson | Stress granules[END_REF].

Moreover, fluorescence recovery after photobleaching (FRAP) measurements showed a very fast turnover of RNA-binding proteins in and out of the stress granules (on the order of seconds to minutes) [START_REF] Buchan | Eukaryotic stress granules: the ins and outs of translation[END_REF]. This rapid shuttling of RBP and mRNAs within stress granules suggests that stress granules are not sites of long-term mRNP storage, unlike other types of granules such as germ granules and neuronal granules [START_REF] Anderson | Stress granules[END_REF].

I.2.3 Stress granule function

Stress granules were first described in tomato plant cell lines submitted to heat shock. These cytoplasmic aggregates were found to accumulate small heat shock proteins and translationally repressed mRNAs [START_REF] Nover | Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves[END_REF]. As mentioned above and subsequently to this discovery, a variety of cellular stresses have been shown to lead to stress granule formation in mammalian cells [START_REF] Kedersha | RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules[END_REF]. While the function of stress granules is not fully understood, some hypotheses are favored. Formation of stress granules causes a higher local concentration of their components within the granules, accompanied by a corresponding lower concentration in the diffuse cytoplasm. The reduced cytosolic concentrations of those molecules will lead to an alteration of the interactions and rates of biochemical reactions. In parallel, the higher local concentration of molecules in stress granules likely increases the rates of mRNP assembly or remodeling. This increase in mRNP assembly within stress granules promotes the assembly of translation initiation complexes during conditions when resources are limiting. Additionally, a higher local concentration of mRNP regulators is also important in order to promote the translation of specific mRNAs that are preferentially translated during stress. Therefore, an interesting possibility is that assembly of stress granules is required to allow optimal translation of stress-responsive mRNAs [START_REF] Buchan | Eukaryotic stress granules: the ins and outs of translation[END_REF]. Another important point is the dynamic nature of stress granules, which suggests that they are sites of mRNA triage wherein individual mRNAs are dynamically sorted for storage, degradation, or translation during stress and recovery.

Stress granules have also been described in tissues from stressed animals. For example, in chickens treated with the ototoxic antibiotic gentamycin, stress granules appear in cochlear cells shortly before cell death [START_REF] Mangiardi | Progression of hair cell ejection and molecular markers of apoptosis in the avian cochlea following gentamicin treatment[END_REF]. In rats, stress granule accumulation was observed in the cell bodies of hippocampal neurons following 10 minutes of global brain ischemia. Stress granules fully disassemble after 90 minutes of reperfusion [START_REF] Kayali | Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules[END_REF]). As a last example, trypanosomes isolated from the intestinal tract of insect hosts also contain stress granules, suggesting that their assembly could be a way of protecting non-translating transcripts during periods of starvation [START_REF] Cassola | Recruitment of mRNAs to cytoplasmic ribonucleoprotein granules in trypanosomes[END_REF]. This indicates that stress granules are not in vitro artifacts of cell culture but represent a cellular mechanism of sequestration in response to environmental stress.

In general, stress granule formation is considered to be a protective mechanism because it leads to the synthesis of stress protective proteins and sequesters pro-apoptotic proteins. For example, if the stress granules formation is inhibited via the use of a chemical, cold shock results in increased cell death [START_REF] Hofmann | Translation suppression promotes stress granule formation and cell survival in response to cold shock[END_REF]. Also, when transient formation of stress granules is followed by a complete disassembly (i.e. a complete recovery of translation) in neurons, they are protected from ischaemia-induced cell death [START_REF] Jamison | Persistent redistribution of poly-adenylated mRNAs correlates with translation arrest and cell death following global brain ischemia and reperfusion[END_REF][START_REF] Kayali | Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules[END_REF]. Even though stress granules may initially be protective, an over activation of this pathway or a failure to disassemble can lead to cell death [START_REF] Wolozin | Regulated protein aggregation: stress granules and neurodegeneration[END_REF].

I.2.4 Core component of stress granules

The core constituents of mammalian stress granules are components from 48S translation initiation complexes. This includes poly(A) + RNA bound to early initiation factors (such as eIF4E, eIF3, eIF4A, eIFG) and small, but not large, ribosomal subunits. Additionally, stress granules contain numerous RNA-binding proteins that vary with cell type and with the nature and duration of the stress involved.

I.2.5 Implication of disease-causing mutations in stress granule dynamics

Most, if not all, of the RNA-binding proteins linked to ALS and IBMPFD associate with stress granules in cell culture. TDP-43, TIA-1 and FUS have all been shown to co-localize with classic stress granule markers (G3BP, eIF4G, TIAR…) in cells undergoing stress [START_REF] Bosco | Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules[END_REF][START_REF] Colombrita | TDP-43 is recruited to stress granules in conditions of oxidative insult[END_REF][START_REF] Hua | Survival motor neuron protein facilitates assembly of stress granules[END_REF][START_REF] Liu-Yesucevitz | Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue[END_REF]. The strong link between these RNA binding proteins and stress granule biology led to further studies involving the disease-causing mutants. It has been shown that the mutant form of TDP-43 is enriched in stress granules compared to wild-type [START_REF] Liu-Yesucevitz | Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue[END_REF] during oxidative stress (arsenite treatment). The same phenomenon has been observed with FUS: FUS wild-type is recruited to stress granules upon stress and the disease-causing mutations significantly enhance FUS association with stress granules [START_REF] Bosco | Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules[END_REF]. Also, mutant TIA1 shows a consistent increase of stress granule incorporation compared to wild-type in both untreated and arsenite-treated cells [START_REF] Hackman | Welander distal myopathy is caused by a mutation in the RNA-binding protein TIA1[END_REF].

To test if this was happening in the case of hnRNPA1 and hnRNPA2, we examined the localization of these two proteins in cells before and after stress granule formation with arsenite ( [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF]; See Annex II). Flag-tagged versions of the wild-type and mutant proteins were expressed in HeLa cells. There was a greater accumulation of mutant hnRNPA1 and hnRNPA2 in stress granules compared to wild-type proteins (Figure 13). This difference was enhanced after oxidative stress with a more rapid incorporation into stress granules for the mutants.

Figure 13: hnRNPA1 is recruited to stress granules and this is increased by diseasecausing mutations. HeLa cells were transfected with Flag-tagged wild-type or mutant hnRNPA1 and stained with anti-Flag (green), anti-eIF4G (red) and DAPI (blue). Arrows indicate hnRNPA1-and eIF4G-positive stress granules. [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF] It seems that mutations occurring in most RNA-binding proteins involved in ALS and IBMPD disturb the normal cellular localization of these proteins. Thus, genetics and cell biology point to a disturbance in RNA granules dynamics in ALS and IBMPFD. Moreover, patient pathology is dominated by a deposition of RNA-binding inclusions. The question concerning the relationship between hyperassembly of stress granules and deposition of RNA-binding proteins aggregates has been asked several times. Our current work addresses this question in more detail (See Chapter II).

I.3 Does fibrillization mediate stress granule assembly?

Even though the molecular chain of events leading to translational arrest is known, it remains unclear how stress granule nucleation occurs, i.e. how RNA-binding proteins and RNA are recruited to form stress granules. Different studies have been trying to answer these questions.

In 2012, Kato et al. presented a novel theory [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF]. They identified a small molecule, biotinylated isoxazone (b-isox), which triggered the precipitation of a large number of proteins involved in RNA granule formation. They demonstrated that the low complexity sequence domains of these proteins were mediating this precipitation. Interestingly, in the absence of b-isox, these low complexity sequence domains underwent self-association and drove phase transitions from soluble proteins to formation of hydrogels in a concentrationdependent manner. While electron microscopy analysis showed these hydrogels were composed of uniformly polymerized amyloid-like fibers, they differed from pathological amyloid fibrils found in cytoplasmic inclusions in patients; they were reversible and dynamic, could be heterotypic and were detergent-soluble. The authors hypothesized that the hydrogel state may form the organizing principle for subcellular structures that are not membrane bound, including stress granules (Figure 14).

varying between seconds and minutes, and indeed, the assembly and disassembly of entire granules are accomplished on this same timescale [START_REF] Buchan | Eukaryotic stress granules: the ins and outs of translation[END_REF]. These rapid dynamics argue in favor of a mechanism that permits rapid assembly and disassembly. Kato I.4 Recruitment of stress signaling proteins to stress granules I.4.1 Compartmentalization of membrane-less organelles in cells Cells realize complex biochemical reactions in order to maintain their functions. Compartments and organelles are useful to create distinct chemical environments within the cells that mediate unique functions. Compartments are usually described as regions defined by a boundary that separates them from their surroundings and where the components must be able to diffuse freely, so that chemical reactions can take place inside. These compartments are often, but not always, defined as membrane-enclosed regions. Indeed, many compartments do not have membranes. Notable examples include: nucleoli, which are sites of ribosome biogenesis inside the nucleus [START_REF] Boisvert | The multifunctional nucleolus[END_REF]; centrosomes, which nucleate microtubules [START_REF] Mahen | Pattern formation in centrosome assembly[END_REF]; Cajal bodies, the site of DNA repair and telomeres [START_REF] Gall | A role for Cajal bodies in assembly of the nuclear transcription machinery[END_REF]; P-bodies, which are the place for RNA decay [START_REF] Yang | GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation[END_REF]; P-granules that are involved in germ-line establishment and maintenance [START_REF] Updike | P granule assembly and function in Caenorhabditis elegans germ cells[END_REF]; and finally stress granules [START_REF] Buchan | Eukaryotic stress granules: the ins and outs of translation[END_REF][START_REF] Decker | P-bodies and stress granules: possible roles in the control of translation and mRNA degradation[END_REF]. The structure and organization of these compartments remains unclear. Recent studies suggest that they have liquid properties and could coexist within the cell as membrane-less compartments with liquid droplet character.

I.4.2 Liquid-like behavior of cellular structures

Recent observations have recently suggested that membrane-less organelles in cells could be considered as liquid droplets. The first example was a study of P granules in Caenorhabditis elegans embryos [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF]. It was observed that these granules can flow off the nucleus, drip, and fuse into a larger drop. Moreover, they exchange components rapidly with the cytoplasm, are easily deformed by flows, and have a viscosity similar to runny honey. All of these properties imply that P granules are liquids. Another example of a liquid compartment is the nucleolus of Xenopus germinal vesicles [START_REF] Brangwynne | Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes[END_REF]. Nucleoli are the sites of ribosome production inside the nucleus and they are another example of non-membrane-bound compartment that must execute the process of making ribosomes. Nucleoli were observed to fuse and their size-distribution obeys a simple power-law, indicating the formation of liquiddroplets. Therefore they also have liquid properties [START_REF] Brangwynne | Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes[END_REF]. Other membrane-less compartments such as P-bodies [START_REF] Fromm | In vitro reconstitution of a cellular phase-transition process that involves the mRNA decapping machinery[END_REF] and FUS bodies [START_REF] Patel | A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation[END_REF], likely have the properties of liquids but this hypothesis has not yet been fully tested. The assembly and disassembly of liquid organelles within cells appear to be governed by a liquid-liquid phase separation process. Indeed, in order to make a non-membrane bound compartment, it must be separated from the liquid cytoplasm. This can be achieved through liquid-liquid demixing also called liquid-liquid phase separation (LLPS). LLPS results in demixing of one liquid from another, resulting in two separate phases [START_REF] Hyman | Liquid-liquid phase separation in biology[END_REF]. We are perhaps more used to liquids being mixtures like coffee and milk that will mix. They will mix because it is energetically more favorable than an unmixed state. However, in some cases, liquids can demix. A pretty common example happens in your kitchen when you make vinaigrette. You can often observe that oil and vinegar demix into two different phases: an oil phase and a vinegar phase. In this case, it is more favorable for the "vinegar molecules" to interact with each other than to interact with the "oil molecules". A protein solution undergoing LLPS results in protein-rich droplets dispersed in a solution depleted of protein, as was originally described for organic polymers [START_REF] Flory | Thermodynamics of High Polymer Solutions[END_REF][START_REF] Huggins | Some Properties of Solutions of Long-chain Compounds[END_REF]. The liquid nature of a condensed phase implies interaction-mediated order, i.e. regular packing of protein molecules, over short distances, whereas considerable packing disorder remains over long distances.

I.4.3.2 Phase diagrams

LLPS can be characterized by a phase diagram (an example is presented in Figure 15). For a given temperature and concentration, the diagram shows whether the solution is a one-phase mixture or whether it separates into two phases. In Figure 15, the red line defines the boundary between the one-phase regime and the two-phase regime and is defined as a coexistence curve. The way this curve shifts is dependent on different experimental conditions and these results give information about the ability of a protein's propensity to phase separate. rapid concentration of components in one place of the cell, leading to facilitated cellular reactions and increased reaction rates [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF], especially because a small increase in the concentration of molecules could allow reactions to start without other regulatory events. Also, reactions in the cytoplasm could stop itself as components from the cytoplasm segregate into the droplet phase. Finally, LLPS allows a selective sequestration or exclusion of specific biomacromolecules [START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF] I.4.3.4 Proteins harboring low complexity domain sequences can mediate LLPS Over the past year, several studies have proposed that liquid-liquid phase separation is mediated by the low complexity sequence domains present in their protein constituents. For example, RNA helicase DDX4X undergoes liquid liquid phase separation [START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF] mediated by its low complexity sequence domain. This protein can reversibly phase separate to form droplets in live cells and in vitro. The organelles formed exhibit liquid properties. Another example, LAF-1, can drive phase separation in vitro, resulting in P-granule-like liquid droplets [START_REF] Elbaum-Garfinkle | The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics[END_REF]. The study demonstrates that the intrinsically disordered low complexity N-terminal domain is necessary and sufficient for liquid-liquid phase separation. The low complexity sequence domains enable droplet formation and are found to be present in a large number of disordered proteins associated with membrane-less organelles. The class of RNA-binding proteins that we introduced earlier potentially has the same abilities and if these proteins display these properties, could explain the formation of stress granules in cells.

Chapter III: Discussion

The project I have been pursuing during my doctoral work and presented in multiple publications (Chapter II and annexes), represents progress in the understanding of neurodegenerative diseases such as ALS and IBMPFD and the molecular mechanisms underlying their pathogenicity. In this thesis manuscript, I show that more and more RNA-binding proteins harboring a low complexity sequence domain are involved in stress granule biology and disturbance. We discovered that hnRNPA1 and hnRNPA2 are two of them [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF] even though the mutations causing the disease remain rare [START_REF] Seelen | No mutations in hnRNPA1 and hnRNPA2B1 in Dutch patients with amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy[END_REF]. Important findings include: (1) the low complexity sequence domain properties that confer the ability to drive LLPS; (2) the importance of both RRM and LCD domains interacting with RNA in order to form large, cross-linked complexes; and (3) the fibrillization of the disease-causing mutants that could explain the presence of cytoplasmic inclusions in patients [START_REF] Molliex | Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization[END_REF]. One of the interesting points addressed with the characterization of LLPS is that the domains mediating this phenomenon are intrinsically disordered. The recent attribution of function of intrinsically disordered proteins has been challenging the long-standing structure-function paradigm.

Indeed, it is becoming more evident that disorder is functionally relevant.

III.1 LLPS as a mechanism commonly used by cells in order to accomplish their biological processes How do cell organize their complex biological processes in time and space? Cells divide their interiors into compartments: membrane bound and non-membrane bound organelles. There are many membrane-less organelles: the nucleoli, P-granules, P bodies, Cajal bodies and stress granules. These compartments are functionally and spatially distinct from each other even though they are not separated by membranes. They represent coherent organelles composed of thousands of molecules. For example, different tasks are accomplished in different RNA granules including RNA processing, splicing, degradation, and translational repression of mRNA. We described a mechanism, liquid-liquid phase separation, which can explain how stress granules can arise in cells as a separate compartment. It has been shown already that P granules and nucleoli were formed via the same mechanism [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF][START_REF] Brangwynne | Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes[END_REF]. We suggest that other membrane-less organelles are formed via LLPS as well. Our work is important in the sense that it provides an additional example of membrane-less organelles exhibiting liquid properties. It also shows that LLPS happens in biological conditions, making it a favored mechanism in cells. Importantly, there are other examples of cellular processes accomplished via LLPS. The nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing in order to promote rapid and context-specific increased levels of PAR at sites of DNA damage [START_REF] Altmeyer | Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)[END_REF]. A very recent study demonstrates the role of LLPS of the RNA-binding protein FUS in DNA repair [START_REF] Patel | A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation[END_REF]. In cells, FUS, which is involved in transcription, DNA repair, and RNA biogenesis, forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. Elastin-fiber assembly has also been revealed to be realized by LLPS [START_REF] Muiznieks | Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin[END_REF]. It now seems quite established that LLPS is not only a biological mechanism but that many cellular processes are made possible because of LLPS.

III.2 From stress granules to pathological inclusions: is there a unifying model?

As I presented in this thesis, mutations in the RNA-binding proteins (RBPs) hnRNPA1, hnRNPA2B1, hnRNPDL, TIA 1 and TDP 43, as well as in the segregase VCP and other regulatory factors, lead to a spectrum of related diseases. These diseases are all characterized by cytoplasmic fibrillar deposits. It is not understood how these inclusions are formed and what the consequences of such inclusions are. The favored hypothesis in the RNA field is that the inclusions arise from persistent stress granule formation. In our work we show that the mutation of a specific RBP, hnRNPA1, leads to fibril formation in liquid droplets. Another study shows that stress granule constituents undergo LLPS and that the droplets mature overtime to a more solid state [START_REF] Lin | Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins[END_REF]. Finally, it has also been demonstrated that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and that this conversion is accelerated by patient-derived mutations [START_REF] Patel | A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation[END_REF]. Our proposed model can then be generalized to many RBPs. And our results [START_REF] Molliex | Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization[END_REF], showing that a cytoplasmic increase of different RBPs causes stress granules to form spontaneously, strongly suggests that stress granules form via LLPS of different RBPs. Therefore, we present a unifying model where specific cell conditions lead to stress granule formation with recruitment of many RBPs. If the stress conditions persist due to the environment or a mutation of one or more of these RBPs, the stress granules can mature into a less dynamic state. This state could be the one described by hydrogels [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF]. Kato et al. suggest that the hydrogel stage is still reversible.

We wonder to what extent these fibrillar structures are actually reversible. It is possible that the higher order regulation of assembly is fibrillar and that some proteins can play a role to disassemble these structures (VCP could be one of them) but the longer the stress granules persist, the less reversible this phenomenon becomes. The evolution into pathological inclusions is then dependent on time. An interesting fact is that TDP-43 is always present in these inclusions, regardless of whether TDP-43 carries a mutation or not; it is possible that this protein is more aggregation-prone in general [START_REF] Molliex | Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization[END_REF] and is always found bound to other RBPs because it gets "trapped" when the liquid state turns into a gel state. Though our work provides insight into SG formation, it is still unclear what the consequences of aggregate formation are for cells and whether they directly cause the disease. The main hypotheses are:

(1) either the aggregates are toxic or (2) the proteins themselves can no longer perform their physiological function because they are trapped in a less dynamic state. These questions remain to be investigated. There is some evidence for sequence encoding with elastin [START_REF] Muiznieks | Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin[END_REF]: manipulating the sequence leads to a disturbance in droplet growth.

Assessment of the sequence composition/pattern of different LCDs found in membrane-less organelles would provide a key to understand the sequence properties encoding the ability to phase separate.

Finally, the study of these membrane-less compartments as well as the understanding of the fibril formation could eventually provide some insights into the development of novel therapeutic drugs. These drugs could act at different levels to prevent the formation of pathological inclusions in patients: either targeting the autophagy pathway in order to increase the degradation of aggregated proteins or preventing the hyperassembly of stress granules by modifying one or several of the important parameters that lead to liquid-liquid phase separation.

144

IV.3 Annex III: No mutations in hnRNPA1 and hnRNPA2B1 in Dutch patients with amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy

After mutations in hnRNPA1 and hnRNPA2B1 were discovered in the context of IBMPFD/ALS, several families were sequenced in a Dutch population. No mutations in these genes were identified in this cohort.

Figure 1 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :

 1345678910 Figure 1: Phenotypes associated with IBMPFD. ........................................................................19

I. 1

 1 .1.2 Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) Inclusion body myopathy associated with Paget's disease and frontotemporal dementia (IBMPFD) is a rare, complex, and ultimately lethal autosomal dominant disorder characterized by three main clinical features found alone or in combination in affected individuals (Nalbandian et al., 2011): myopathy, Paget's disease of bone, and frontotemporal dementia. Inclusion body myopathy (IBM) is the most common feature and is present in 80 -90% of affected individuals with an age of onset in the 30s to 40s. Patients typically demonstrate slowly progressive distal and proximal weakness and atrophy of the skeletal muscle with initial involvement of the hip girdle and shoulder girdle muscle groups. Patients exhibit an abnormal gait, inability to raise the arms, difficulty climbing stairs and mild weakness of the hands. Clinical features include a generalized reduction or absence of tendon reflexes, normal nerve conduction, and myopathic electromyogram. The serum creatine phosphokinase levels are normal to slightly elevated (Kimonis et al., 2008). Muscle biopsies reveal the presence of hypertrophic fibers, rimmed vacuoles, and cytoplasmic Valosin-containing protein-and ubiquitinpositive inclusions. In advanced cases, severe degeneration and fatty replacement of muscle fibers can be noted.Paget's disease of bone (PDB) is a chronic focal skeletal disease. The primary pathologic abnormality manifests as increased bone resorption and formation, resulting in abundant new bone, which is highly disorganized and of poor quality. The typical distribution of Paget's disease includes the spine, pelvis, scapulae, and skull. Symptoms of PDB include bone pain, localized bone enlargement, deformation of the long bones, and deafness due to eighth-nerve compression. Histological analysis shows that PDB is characterized by abnormally large, multinucleated osteoclasts with nuclear filamentous inclusions of paired helical filaments similar

Figure 1 :

 1 Figure 1: Phenotypes associated with IBMPFD. Percentage of diagnosed IBMPFD patients affected by one or more of the IBMPFD-associated clinical features. Statistics from www.genetests.org.

  Figure 2: TDP-43 histopathology. Immunostaining with an anti-TDP-43 antibody reveals staining of ubiquitinated inclusions (brown). TDP-43 inclusions (arrowheads) and nuclear clearance (arrows) are observed in neurons in the hippocampal dental gyrus.

Figure 3 :

 3 Figure 3: Schematic of VCP structure. VCP consists of 2 ATPase domains (D1 and D2) and an N-terminal domain that provides substrate specificity. These domains are separated by flexible linkers.

Figure 3 :

 3 Figure 3: Schematic of VCP structure. VCP consists of 2 ATPase domains (D1 and D2) and an N-terminal domain that provides substrate specificity. These domains are separated by flexible linkers.

  Because of this localization, a conformational change occurs, leading to impaired communication between the D1 and N-domains. This change indirectly influences the D1 nucleotide binding pocket (Fernandez-Saiz and Buchberger, 2010), altering the relative affinity

Figure 4 :

 4 Figure 4: Graphic representation of VCP crystal structure showing the distribution of the IBMPFD-linked mutations. The N domain is represented in green, the D1 domain in blue and the D2 domain in grey. Adapted from[START_REF] Niwa | The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP[END_REF]

Figure 5 :

 5 Figure 5: Domain structure of TDP-43. Known mutations are associated with ALS (shown in black) and ubiquitin-positive FTD (in blue). Adapted from (Dormann and Haass, 2011).

Figure 6 :

 6 Figure 6: Identification of previously unknown disease mutations in IBMPFD/ALS. a), Family 1 pedigree indicating individuals affected by dementia, myopathy, PDB and ALS. The causative mutation was p.D290V/D302V in hnRNPA2B1. Roman numerals denote generation and Arabic numbers denote family member within a generation. b), Family 2 pedigree indicating individuals affected by myopathy and PDB. The causative mutation was p.D262V/D314V in hnRNPA1. Adapted from[START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF]

Figure 7 :

 7 Figure 7: Sequence alignment of hnRNPA2B1 and hnRNPA1 orthologues. There is an evolutionary conservation of the mutated aspartate and surrounding residues.

Figure 8 :

 8 Figure 8: Schematic showing the structure of hnRNPA1 and hnRNPA2B1. Mutations found in ALS and IBMPDF patients are indicated.

Figure 9 :

 9 Figure 9: Diagram showing the relationship between diseases and genes. Mutations in the same genes are involved in different diseases.

Figure 10 :

 10 Figure 10: Structure comparison of several hnRNPs. The different disease-causing mutations are indicated.

Figure 11 :

 11 Figure 11: HeLa cells stressed for 15 min with sodium arsenite. The cells were stained with a marker for stress granules in red (eIF4G) and for nuclei (DAPI) in blue.

Figure 12 :

 12 Figure 12: Model of assembly and disassembly of stress granules. Under normal conditions, several ribosomes bind the circularized mRNA and translate the mRNA into newly synthesized protein. Cellular stress results in inhibition of translation initiation and starts SG formation. During SG nucleation, SG proteins (blue) bind to the 48S preinitiation complex forming mRNP oligomers. During stress recovery, SG proteins dissociate, ribosomes are recruited to the released mRNA and translation can be reinitiated. Figure modified from[START_REF] Bentmann | Stress granules in neurodegeneration-lessons learnt from TAR DNA binding protein of 43 kDa and fused in sarcoma[END_REF].

  et al. work presented a novel hypothesis but the dynamic nature of SGs is not recapitulated in hydrogels. Though this work provided insight into how these proteins interact, it is still unclear how their properties promote formation of dynamic SG assembly/disassembly. Our research led us to a different hypothesis to explain the molecular interactions promoting the formation of stress granules.

Figure 14 :

 14 Figure 14: Model presented by Kato et al. (Kato et al., 2012) for the molecular interactions promoting stress granule formation.

I. 4

 4 .3 Liquid-liquid phase separation: a mechanism that allows cellular compartmentalization I.4.3.1 Definition of liquid-liquid phase separation

I

  .4.3.3 Liquid-liquid phase separation: a mechanism for subcompartmentalization As mentioned previously, recent studies suggested that membrane-less organelles could have liquid properties and could coexist within the cell as liquid drops. Liquid-liquid phase separation provides a powerful way of thinking about cellular subcompartmentalization. LLPS allows for

Figure 15 :

 15 Figure 15: Schematic representing a phase diagram. The red line is the coexistence curve and defines the boundary between the onephase regime (the water molecules in blue and the protein molecules in green are homogeneous) and the two-phase regime (the protein molecules exclude the water molecules).

III. 3

 3 Compartmentalization via LLPS: a code in low complexity sequence domains? An important question still remains: what promotes formation of membrane-less compartments containing different proteins? Is LLPS propensity encoded in the protein sequence? The sequence compositions of LCDs in RBPs vary, and this could potentially enable sorting of protein molecules, and their associated cargo RNA, into different compartments. Careful assessment of the sequence composition and patterning of different LCDs found in membraneless organelles will provide insight into the sequence properties encoding the ability to undergo LLPS. Also, characterizing the interactions between LCDs of different proteins could provide a better understanding of molecular sorting and of compartmentalization into multiple membraneless organelles simultaneously.

  I.2 Stress granules as crucibles of these pathogeneses ...................................................33 I.2.1 What are stress granules? .......................................................................................33 I.2.2 Stress Granule dynamics ........................................................................................34 I.2.2.1 Assembly of Stress Granules ...........................................................................34 I.2.2.2 Disassembly of Stress Granules .......................................................................35 I.2.3 Stress granule function ............................................................................................37 I.2.4 Core component of stress granules .........................................................................38 LLPS as a mechanism commonly used by cells in order to accomplish their biological processes ..............................................................................................................................71 III.2 From stress granules to pathological inclusions: is there a unifying model? ................72 III.3 Compartmentalization via LLPS: a code in low complexity sequence domains? .........74 Chapter IV: Annexes ...........................................................................................................75 IV.1 Annex I: VCP is essential for mitochondrial quality control by PINK1/Parkin and this function is impaired by VCP mutations ..................................................................................75

								43
	I.4.3 Liquid-liquid	phase	separation:	a	mechanism	that	allows	cellular

I.2.5 Implication of disease-causing mutations in stress granule dynamics ......................38 I.3 Does fibrillization mediate stress granule assembly? ..................................................41 I.4 Recruitment of stress signaling proteins to stress granules .........................................43 I.4.1 Compartmentalization of membrane-less organelles in cells ...................................43 I.4.2 Liquid-like behavior of cellular structures .................................................................compartmentalization .........................................................................................................44 I.4.3.1 Definition of liquid-liquid phase separation .......................................................44 I.4.3.2 Phase diagrams ...............................................................................................45 I.4.3.3 Liquid-liquid phase separation: a mechanism for subcompartmentalization ......45 I.4.3.4 Proteins harboring low complexity domain sequences can mediate LLPS ........46 Chapter II: Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization.........................................................................47 Chapter III: Discussion ...........................................................................................................71 III.1 IV.2 Annex II: Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS .................................................................................... IV.3 Annex III: No mutations in hnRNPA1 and hnRNPA2B1 in Dutch patients with amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy ............................... References .............................................................................................................................

  .............................................................31 Figure 11: HeLa cells stressed for 15 min with sodium arsenite.. ..............................................33 Figure 12: Model of assembly and disassembly of stress granules. ..........................................36 Figure 13: hnRNPA1 is recruited to stress granules and this is increased by disease-causing mutations. .................................................................................................................................39 Schematic representing a phase diagram. ...............................................................45

	Figure 14: Model presented by Kato et al for the molecular interactions promoting stress granule formation. ..................................................................................................................................42 AAA+ ATPase ATPase Associated with diverse cellular Activities ALS Amyotrophic Lateral Sclerosis b-isox biotinylated isoxazone CNS Central Nervous System eIF4E eukaryotic translation initiation factor 4E FTLD-U Frontotemporal lobar degeneration with ubiquitin-positive inclusions Gly-rich C-terminal domain enriched in glycines hnRNPs Heterogeneous Ribonucleoproteins IBM Inclusion Body Myopathy IBMPFD Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia LCD Low Complexity sequence Domain LLPS Liquid-Liquid Phase Separation mRNAs messenger Ribonucleic Acid mRNP messenger Ribonucleoprotein NES Nnuclear Export Sequence NLS Nuclear Localization Sequence PDB Paget's Disease of Bone PrLD Prion-like Domain RBPs RNA-binding Proteins RRM RNA-Recognition Motifs SG Stress Granule TDP-43 43 kDa TAR-DNA-binding Protein VCP Valosin-Containing Protein Summary Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, inclusion body myopathy, Paget's disease of bone and frontotemporal dementia; these diseases are characterized by solid, fibrillar, cytoplasmic inclusions that are rich in RNA binding proteins (RBPs). Genetic evidence suggests a link between persistent stress granules In this thesis manuscript, I demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the folded RNA recognition motifs contribute to LLPS in the presence of RNA, potentially giving rise to several mechanisms for regulating assembly of stress granules. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. I suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease. Résumé Il a été observé que l'altération des fonctions des granules de stress, entités cytoplasmiques non-membranaires composées d'ARN et de protéines liant l'ARN (RBPs), peut conduire au développement de maladies telles que la sclérose latérale amyotrophique, la démence fronto-temporale, la myopathie à inclusions et la maladie de Paget des os. Ces pathologies sont caractérisées par un dépôt cytoplasmique d'inclusions solides enrichies en RBPs et comprenant Dans mon manuscrit de thèse, il est mis en évidence le fait que la protéine hnRNPA1, dont les mutations entrainent les maladies mentionnées plus haut, subit une séparation de phases entre deux liquides connue également sous l'appellation « Séparation de Phases Liquide-Liquide » (LLPS) dans des gouttelettes enrichies en protéines. Bien que le domaine composé d'une séquence à faible complexité (Low Complexity sequence Domains ou LCD) soit suffisant pour obtenir cette séparation de phases, les domaines de liaison à l'ARN y contribuent également en présence d'ARN. Cela a permis d'envisager l'existence de plusieurs mécanismes intervenant dans la régulation de l'assemblage de ces granules. Un autre résultat a mis en exergue le fait que la formation de fibrilles n'est pas une obligation pour permettre la séparation de phases mais que les gouttelettes, enrichies en protéines, entrainent, par ailleurs, une augmentation de la formation de ces fibrilles. La séparation de phases liquide-liquide induite par le domaine composé d'une séquence à faible complexité semble contribuer à l'assemblage des granules de stress et à leurs propriétés liquides. Finalement, cette étude propose d'établir une réelle corrélation entre la formation des granules Chapter I: Introduction I.1 Pathogenesis of different neuronal and muscle disorders I.1.1 Introduction of ALS and IBMPFD I.1.1.1 Amyotrophic lateral sclerosis (ALS) Amyotrophic lateral sclerosis (ALS) was initially described in 1869 by the famous French Figure 15: List of abbreviations FRAP Fluorescence Recovery After Photobleaching FTD stress et l'accumulation de ces inclusions pathologiques dans le cytoplasme des patients. Frontotemporal Dementia and the accumulation of pathological inclusions. des fibrilles. Une connexion génétique a été suggérée entre la persistance des granules de neurobiologist and physician Jean-Martin Charcot; therefore, this disorder first became known
	de stress qui deviennent persistants et l'accumulation d'inclusions pathologiques dans le
	cytoplasme des patients.

as Charcot's sclerosis

[START_REF] Cleveland | From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS[END_REF]

. "Amyotrophic" refers to the muscle wasting that typifies disease of the lower motor neurons. "Lateral sclerosis" refers to the observation that lateral columns of the spinal cord are hard upon palpation in autopsy specimens, wherein gliosis follows degeneration of the corticospinal tracts. In 1939, ALS ended the career of one of the most beloved baseball players of all time, Lou Gehrig. The disease is still most closely associated with his name and familiarly known in the United States of America as Lou Gehrig's disease. ALS is the most common motor neuron disease in human adults. ALS is a progressive age-dependent neurodegenerative disease. Its characteristic feature is rapidly progressive muscle weakness without sensory signs leading to limb weakness. The typical age of onset is between 50 and 60 years. At onset, fatigue, cramps, muscle weakness and wasting

Acknowledgements

First I would like to thank my committee members, Dr Séverine Boillée, Dr Martine Roussel, Dr Bertrand Cosson, Dr Mondira Kundu and Dr Fréderic Pincet. I am honored that you accepted to participate in my thesis defense, especially so close to Christmas.

 [START_REF] Ritson | TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97[END_REF]and mouse [START_REF] Custer | Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone[END_REF] to investigate the molecular pathology of the mutations.

These studies have shown that muscles containing mutations R115H and A232E in VCP were degenerated. Interestingly, we also observed a mitochondrial defect in the mutant animals, characterized by accumulation of massively swollen mitochondria.

My contribution to this work consisted on determining the importance of VCP in the clearance of damaged mitochondria (Figure 7). I also showed that mitochondrial ubiquitination by Parkin is essential for VCP recruitment (Figure 5).

107

IV.2 Annex II: Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS I was involved at the beginning of my thesis work in the project that served as the pioneer of my primary studies. I introduced the main results of this paper in the introduction of my thesis.

My contribution to this work consisted on assessing the consequences of the mutations in cells (Figure 5 and Supplementary Figure 11).