L'autophagie est un processus d'autodigestion qui se produit dans toutes les cellules eucaryotes et conduit à la dégradation d'éléments du cytoplasme (organites, macromolécules) par le lysosome. Ce mécanisme, qui se produit de manière basale, permet le renouvellement du contenu cytoplasmique mais également la survie cellulaire lorsqu'il est induit par différents stress (carence nutritionnelle, hypoxie…). L'autophagie est alors impliquée dans diverses pathologies comme les maladies neurodégénératives et le cancer car sa dérégulation peut grandement perturber l'homéostasie cellulaire. Le but de ma thèse est de déterminer le rôle de la protéine nucléaire et cytoplasmique BAT3 dans l'autophagie et d'étudier son mécanisme de régulation. Cette protéine de 150 kDa, également appelée BAG6 ou Scythe, est composée de nombreux domaines protéiques (UBL, Prolin-rich, NLS, BAG) qui lui permettent d'interagir avec de multiples partenaires. Sa fonction majeure réside dans le contrôle qualité du cytoplasme mais BAT3 est aussi impliquée dans l'immunité ou l'apoptose. Ce travail identifie la protéine BAT3 comme essentiel pour l'autophagie basale et induite. Nous montrons que son mécanisme d'action passe par la régulation de la localisation de l'acétyltransférase p300 et l'acétylation de ces substrats : p53 et une protéine de la machinerie de l'autophagie : ATG7. En effet, BAT3 (i) limite la présence de p300 dans le cytosol en (ii) maintenant un faible et régulable niveau d'acétylation d'ATG7 et (iii) permet l'acétylation de p53 dans le noyau au cours de la carence nutritionnelle, événement indispensable à l'induction de l'autophagie. / Autophagy, literally meaning self-eating, is a highly evolutionary conserved process in eukaryotes in which parts of the cytoplasm (organelles, macromolecules) are degraded by lysosomes. Basal autophagy is a quality control mechanism allowing the renewal of the cytoplasm but autophagy is also induced by cellular stress (starvation, hypoxia…) to improve cell survival. Autophagy has been implicated in several physiopathologies such as cancer or neurodegenerative diseases. Deregulations of autophagy may profoundly affect homeostasis.The purpose of my thesis is to explore the role of the nucleo-cytoplasmic shuttling protein BAT3 in autophagy and the mechanism of BAT3-dependent autophagy.Also known as BAG6 or Scythe, this 150 kDa protein is composed of various domain (UBL, Prolin-Rich, NLS, BAG) by which BAT3 interacts with multiple partners. The major of role BAT3 seems to be the protein quality control but BAT3 is also implicated in immunity and apoptosis. Our work demonstrates that the protein BAT3 is essential for basal and starvation-induced autophagy. We show that BAT3 regulation of autophagy is mediated by the modulation of p300 acetyltransferase intracellular localization and acetylation of two subtrates: p53 and the autophagy-related protein ATG7. Indeed, Bat3 allows: (i) the limitation of p300 into cytosol resulting in (ii) the maintenance of a low level of ATG7 acetylation and (iii) the increase of the starvation-induced p53 autophagy leading to the induction of autophagy.

Renal ischaemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) which is in turn the leading cause of morbidity and mortality in hospitalized patients. The principle aim of this thesis was to evaluate potential new therapies that might afford protection against IRI in both in vitro and in vivo settings. Recent evidence suggests that nitrite (NO2-) may play an important role in protecting the myocardium from IRI. Our initial work into the role of NO2- in an in vitro model of renal IRI in proximal tubular epithelial cells provided evidence that NO2- can prevent apoptosis and preserve cell viability. This lead to an in vivo study where high NO2- concentrations (50 mg/L) were given orally to rats for 7 days prior to inducing renal IRI but no beneficial effects of this treatment were observed. Another potential treatment identified was thiamine (vitamin B1) and this, like NO2-was investigated to see if it had the potential to protect rats from AKI injury. It has been previously recognized that in renal IRI the high energy phosphate ATP is found to be severely depleted whilst is is known that thiamine can play a pivotal role in generating ATP. Furthermore, thiamine has previously been demonstrated to protect against myocardial ischaemic injury and has the ability to reduce myocardial infarct size. In vitro, thiamine was found to reduce the degree of apoptosis in cultured HK-2 cells caused by ischaemia whilst in vivo it afforded protection against AKI caused by renal IRI by anti-apoptotic, anti-inflammatory and anti-oxidant mechanisms. Finally, a study into the possible therapeutic role of gene therapy with bone morphogenic protein 7 (BMP-7) in renal IRI was undertaken. Previous work has established that i.v. BMP-7 is able to protect against renal IRI but it has also been associated with ectopic bone formation at the site of injection. Therefore another method to increase circulating BMP-7 was sought. We initially found that BMP-7 gene therapy could attenuate apoptosis and preserves cell viability in an in vitro model of renal IRI. However, whilst in vivo gene therapy with electroporation of BMP-7 plasmid DNA increased BMP-7 expression in mice serum 2 days post electroporation, it was unable to protect the animals against IRI induced AKI. In rats the direct injection of naked DNA BMP-7 plasmid systematic 2 days prior to renal IRI was able to upregulate BMP-7 expression 4 days later in kidney tissue. Despite this it was unable to afford protection against renal IRI. Apoptosis and necrosis play a crucial role in the pathogenesis of renal IRI induced AKI. In this thesis we investigated the role of three putative therapeutic agents and their role in apoptosis and necrosis in vitro in PTECs and in vivo against renal IRI induced AKI. All three therapeutic drugs were able to attenuate apoptosis in PTECs but were unable to protect against necrosis, whilst against renal IRI induced AKI only thiamine was found to be protective. Thiamine appears to hold the most promise and more work needs to be undertaken so that its potential benefit in AKI can be realised.

Ischaemic heart disease is the most common cause of mortality in the western hemisphere and it is rapidly becoming the leading cause of death globally. Moreover, therapeutic interventions by cardiologists and cardiac surgeons frequently subject the heart to acute I/R injury, which in itself can cause mortality. Recent investigations of adult stem cells have primarily focused on their regenerative potential for chronic ischaemic heart disease. In this thesis, I have investigated the hypothesis that adult bone marrow derived stem cells are cardioprotective in acute regional myocardial I/R injury. In a rat model of left anterior descending coronary artery (LAD) reversible occlusion and reperfusion, I demonstrate that an intravenous bolus of adult bone marrow derived (1) bone marrow mononuclear (BMNNC) and (2) mesenchymal stem cells (MSC) upon reperfusion can attenuate infarct size. This effect is comparable to ischaemic preconditioning (IPC), which is the gold standard for cardioprotection. Next, I demonstrated the mechanisms for adult stem cell cardioprotection are principally anti-apoptotic and depend upon stem cell secreted factors to (1) activate phosphatidylinositide 3-kinase (PI3)/Akt cell survival kinase-signaling pathway (2) inhibit glycogen synthase kinase-3β (3) inhibit p38MAPK (4) inhibit nuclear translocation of p65NF-κB. 7 Proteomic analysis of myocardium subjected to I/R and treated with either BMMNC or BMMNC derived supernatant (BMS) upon reperfusion demonstrated higher expression of a whole host of pro-survival proteins. These were notably (1) 14-3-3-ε protein (2) anti-oxidant peroxiredoxin-6 (3) heat shock protein (HSP) αB-crystallin, HSP72, HSP tumour necrosis factor receptor-1 associated protein, and HSP ischaemia responsive protein-94 (4) glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (5) mitochondrial aconitase and mitochondrial voltage-dependent anionselective channel protein-1. Thereafter, I investigated the mobilization of endogenous bone marrow stem cells and trafficking to the ischaemic myocardium by stromal cell derived factor-1 (SDF-1) /chemokine, receptor type 4 (CXCR4) signaling. I demonstrate high up-regulated expression of CXCR4 and CD26 in BMMNC following IPC, which might have a role in IPC-mediated cardioprotection. Finally, and in concordance with this finding I demonstrate that both IPC and an exogenous MSC bolus upon reperfusion can synergize to abolish acute myocardial I/R injury.

La polarité cellulaire est un processus fondamental qui contrôle les spécificités fonctionnelle et physiologique de la plupart des cellules eucaryotes. Cette asymétrie intracellulaire repose sur l’existence de compartiments membranaires distincts, à la fois dans leur composition en protéines mais également en phosphatidyl-inositols (PIs). Ainsi, la mise en place et le maintien de la localisation asymétrique de modules multi-protéiques associés notamment aux protéines PAR sont essentiels pour l’élaboration des domaines de polarité cellulaire. Durant ma thèse, j’ai étudié les relations entre les protéines de polarité et les PIs dans le contrôle de la polarité cellulaire. Plus particulièrement, en utilisant la chambre ovarienne de Drosophile, j’ai cherché à caractériser la suite d’évènements qui en amont régule l’activité de la PIP5K, Skittles (SKTL), qui produit le PI(4,5)P2 et à caractériser les mécanismes moléculaires qui lient le PI(4,5)P2, SKTL et les protéines PAR dans le contrôle et le maintien de la polarité cellulaire. J’ai contribué à caractériser l’importance de PI(4,5)P2 majoritairement produit par SKTL, dans le maintien de la polarité apico-basale et lors de la morphogenèse des cellules folliculaires de la chambre ovarienne. Le PI(4,5)P2 assure la localisation apicale de PAR3 et le maintien des jonctions adhérentes, sans affecter la localisation de PAR1. Par une méthode de quantification précise, j’ai ensuite démontré dans l’ovocyte que SKTL et le PI(4,5)P2, probablement grâce au trafic vésiculaire, étaient requis pour à la fois l’accumulation à l’antérieur de PAR3 et son exclusion au postérieur qui se fait à partir du stade 9B. L’accumulation antérieure de PAR3 est également dépendante d’un transport Dynéine dépendant et de la kinase IKKε tandis que son exclusion postérieure dépendant des phosphorylations par PAR1. Enfin, j’ai également étudié les modifications post traductionnelles de SKTL et leur importance dans la polarité cellulaire. J’ai identifié la présence de palmitoylation et de phosphorylations dont certaines impliquent la kinase PAR1 et la phosphatase PP1. Ces phosphorylations pourraient avoir un lien avec le rôle de SKTL dans le trafic vésiculaire. Ces résultats permettent donc d’élucider certains mécanismes cellulaires qui contrôlent la mise en place et le maintien de la polarité des cellules en liant les PIs et les protéines PAR / Cell polarity is a fundamental process that controls cell’s functional and physiological specificities. This process relies on membranous compartments differently composed both on proteins and on phosphatidyl-inositols (PIs). Indeed, through their asymmetric localization, polarity proteins, such as the PAR proteins, are essentials to establish and maintain polarity of the cells. During my PhD, I studied the interplay between the polarity proteins and the PIs. Using the Drosophila egg chamber, as a model, I aimed to characterized the upstream events that regulate the PI(4,5)P2 producing kinase (PIP5K), Skittles (SKTL), activity and localization. I also studied the downstream molecular process that link the PI(4,5)P2, SKTL and the PAR proteins in cell polarity. I contributed to the characterization of the importance of PI(4,5)P2, mainly produced by SKTL in maintaining the apical-basal polarity and during the morphogenesis of the follicle cells. The PI(4,5)P2 is ensuring PAR3 and adherens junctions but not PAR1 proper localizations. Next, through a precise quantification method, I showed that SKTL and the PI(4,5)P2, probably via vesicular traffic, were also ensuring PAR3 proper localizations (anterior accumulation and stage 9B posterior exclusion) in the oocyte. PAR3 accumulation also relies on a Dynein mediated transport and the IKKε kinase while its posterior exclusion relies on PAR1 phosphorylation. Finally, I studied SKTL post translational modifications and their relevance on cell polarity. I identified palmitoylation and phosphorylations that are regulated by the kinase PAR1 and the phosphatase PP1. SKTL phosphorylations seem to be related to its role on the vesicular traffic. Altogether these results clarify some mechanisms involving both PIs and PAR proteins in cell polarity maintaining and establishment

The important role of the central intermediary metabolite acetyl-coenzyme A (AcCoA)for several anabolic and catabolic pathways is well characterised. However, the role of AcCoA as the only known donor of acetyl groups for protein acetylation in regulation of enzyme activities, protein complex stability as well as epigenetic status off chromatin, is only recently emerging. Among multiple other pathways, the autophagy pathway has now been shown to be directly regulated by protein acetylation and deacetylation. Therefore, it was reasoned that the availability of AcCoA, via the modulation of AcCoA generating enzymes, may regulate autophagy. This study has focussed on the role of the acetate-mediated route to nuclear-cytosolic AcCoA synthesis, catalysed by AcCoA synthetase 2 (ACSS2), in the regulation of autophagy.

Acute kidney injury (AKI) is a major risk factor for chronic kidney disease (CKD). For patients who recover from AKI, there is a 25% increase in the risk of CKD, and a mortality rate of up to 50% after 10 years. Nuclear factor kappa-B (NF-κB) is a family of transcription factors that regulates the transcription of many proteins that play a key role in inflammation. Inhibitor of IκB kinase (IKK) is directly upstream of NF-κB. My aim was to investigate a) the role of IKK in the progression of AKI to CKD, and b) whether its inhibition attenuates renal fibrosis. In this thesis I used a model of unilateral renal ischaemia-reperfusion injury with contralateral nephrectomy, to firstly map the acute time course of AKI. From the data generated from the time course, I decided to treat the animals at 24 h post reperfusion with the IKK inhibitor, IKK16, as i) this was at the peak of renal dysfunction (24 h post reperfusion), and ii) prior to the activation of NF-κB (48 h post reperfusion). The inhibition of IKK at 24 hours post reperfusion, as a delayed treatment, successfully attenuated renal dysfunction, NF-κB activation and renal structural damage. I subsequently increased the recovery time after ischaemia-reperfusion in my rat model to 28 days to study the development of fibrosis post AKI. The inhibition of IKK at 24 hours post reperfusion successfully attenuated the development of fibrosis, formation of myofibroblasts, macrophage infiltration, the expression of pro-fibrotic markers and the deposition of extracellular matrix components at 28 days post reperfusion. In addition, the delayed inhibition of IKK at days 7-13 post unilateral ureteral obstruction in a rat model, successfully attenuated the development of fibrosis, formation of myofibroblasts, macrophage infiltration, the expression of pro-fibrotic markers and the deposition of extracellular matrix components. These data indicate that the activation of the IKK complex drives tubulointerstitial fibrosis, and suggests that the inhibition of IKK could be a useful pharmacological tool for the creation of therapies to combat AKI and the subsequent development of fibrosis, via the reduction of both inflammation and the prevention of the expression of pro-fibrotic markers.

Diseases which affect the central nervous system present a huge burden to sufferers and caregivers. In tandem with longevity, prevalences of age-related neurodegenerative diseases are increasing. However, despite the evident necessity for pharmaceutical interventions, there has been a distinct lack of drug development to combat these disorders. This is largely attributed to high financial costs of using rodent models. Thus the validation of a more cost-effective in vivo system would facilitate pharmaceutical screening. The work presented in this thesis addresses this issue by assessing the utility of zebra fish in two costly areas of translational neurobiology { lead identi cation and safety pharmacology. An aversive classical conditioning assay was developed and automated as a behavioural screening method. This robust assay allows fast assessment of cognition and cognitive decline. The effect of neurotoxin treatment on aversive learning was then assessed using this assay, demonstrating its efficacy as a screening tool for neurodegeneration research. Subsequently, a transgenic zebra fish line - expressing a mutated form of the Alzheimer's-associated human amyloid precursor protein - was assessed, demonstrating an age-related cognitive impairment. Additionally new genetic zebra fish lines were generated, which over-express genes (both endogenous and transgenic) related to Alzheimer's-like pathologies. Whilst these were not assessed within this thesis, they present promising tools for possible future investigations. Regarding safety pharmacology, regulatory bodies require all CNS-penetrant drugs be assessed for abuse potential. Zebra fish display reward responses to several common drugs of abuse (e.g. amphetamine, cocaine, morphine). Thus, the latter sections of this thesis evaluated the utility of zebra fish for assessing human abuse potential. A CPP paradigm was utilised to test a range of drugs, with the sensitivity and specificity of zebra fish compared to previous reports using rodent. Additionally, the development of a zebra fish drug discrimination assay was attempted. However the paradigms utilised failed to develop an efficacious assay.

The dynamic S-palmitoylation regulates many intracellular events, including protein trafficking, anchoring, targeting, and protein-protein interactions. Direct detection of S-palmitoylation by conventional liquid chromatography-mass spectrometry (LC-MS) methods is challenging because of the tendency of palmitoyl loss during sample preparation and gas phase fragmentation. Additionally, the high hydrophobicity of the palmitoyl group can prevent proper elution of palmitoyl peptides from the commonly used C18 column. Here, we developed a comprehensive strategy tailored for S-palmitoyl detection using three palmitoyl peptide standards. We found that S-palmitoylation was largely preserved in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent and that various fragmentation methods provided complementary information for palmitoyl localization. Moreover, S-palmitoyl peptides were efficiently analyzed using a C4 column and the derivatization of free cysteine with a hydrophobic tag allowed relative quantification of palmitoyl peptides and their unmodified counterparts. We further discovered potential complications to S-palmitoylation analysis caused by the use of ProteaseMAXTM, an MS-compatible detergent. The hydrophobic degradation products of ProteaseMAXTM reacted with the free cysteine thiols, generating artifacts that mimic S-acylation and hydroxyfarnesylation. Another MS-compatible detergent, RapiGestTM, did not produce such artifacts, and showed the ability to stabilize S-palmitoylation by preventing thioester hydrolysis and dithiothreitol-induced thioester cleavage. Moreover, we found that the palmitoyl peptide GCpalmLGNAK could undergo intermolecular palmitoyl migration from the cysteine to the peptide N-terminus or the lysine side chain during sample preparation, and this could lead to false discovery of N-palmitoylation. RapiGestTM inhibited such migration, and is thus recommended for S-palmitoyl sample preparation. We then applied the established method to analyze the regulator of G-protein signaling 4 (RGS4) which had been reported to undergo S-palmitoylation by radioactive labeling. It had also been reported that the S-palmitoylation state of RGS4 affects its GTPase activity. With LC-MS/MS analysis, we found that the addition of palmitate to the cell culture medium in metabolic labeling experiments could boost the level of S-palmitoylation, leading to false discovery of new S-palmitoylation site(s). We also noted discrepancies between the S-palmitoylation sites identified by radioactive labeling and by LC-MS/MS analysis. Further studies are needed to evaluate the reliability of S-palmitoyl detection by these two methods.

Les modifications post-traductionnelles (MPTs) d’histones sont apparues comme un acteur majeur de la régulation de l’expression des gènes. Cependant peu de choses sont connues sur le réel impact des MPTs sur la chromatine. Il a été suggéré que les MPTs d’histones (H2A, H2B, H3 et H4) ont le potentiel de moduler la fonction chromatinienne selon un « codehistone » en recrutant des protéines spécifiques de liaison. L’objectif de mon projet est d’approfondir la fonction de l’acétylation du domaine globulaire de l’histone H3 et de comparer cette modification avec celles des queues N-terminale in vivo sur une lignée ES cellulaire. Pour étudier l’impact de ces MPTs in vivo, toutes les copies endogènes du gène H3 sauvage (WT) doivent être remplacées par des copies mutées. Ainsi la première étape de mon projet est d’établir une lignée cellulaire exprimant seulement H3 mutée (e.g reproduisant une acétylation permanente) afin d’étudier les effets des modifications sur le domaine globulaire de H3 sur (a) l’expression génique, (b) l’architecture chromatinienne mais également pour étudier (c) les effets réciproques et synergiques entre les différentes modifications du domaine globulaire et (d) comparer ces effets avec les modifications sur la queue N-terminale dans un système in vivo. / Post-translational modifications (PTMs) of histones have emerged as key players in the regulation of gene expression. However, little is known to what extent PTMs can directly impact chromatin. It has been suggested that PTMs of core histones (H2A, H2B, H3 and H4) have the potential to govern chromatin function according to the so called ‘‘histone code’’ hypothesis by recruiting specific binding proteins. The goal of my project is to gain insight in the function acetylation within the globular domain of H3 and to compare these modifications with histone tail modifications, in vivo by using the CRISPR in mouse embryonic stem cells (ES). To study the impact of PTMs in vivo, all endogenous wild type (WT) H3 gene copies have to be replaced with mutant copies. Hence, the primary focus of my project is to establish cell lines that exclusively express mutated H3 (e.g. mimicking acetylation) in order to study effects of H3 globular domain modifications on (a) gene expression (b) chromatin architecture as well as to study (c) cross talks and synergisms between globular domain modifications and (d) compare the effects with tail modifications in an vivo system.

Epigenetic modifications are alterations in the cell nucleus that affect gene expression and can occur in chromatin at the level of DNA methylation or histone modifications. Such ‘epigenetic marks’ can be heritable through cell division but leave the DNA sequence unchanged. Post-­translational modifications can be found on the histone proteins associated with DNA; the majority of histone modifications are found on the lysine-­rich N-‐terminal amino acid “tails”. Histone acetylation and methylation influence the chromatin structure by loosening or tightening the packaging of DNA, respectively, in association with other chromatin modifiers. Condensed chromatin is linked to transcriptional silencing and genetic imprinting and also occurs at chromosomal centromeres, where it is linked to kinetochore binding. Heart development is well studied, but the epigenetic processes involved are not yet completely understood. While active chromatin mechanisms such as histone acetylation and chromatin remodelling have been described in the heart, the role of gene repressive epigenetic mechanisms has been poorly investigated. Cardiomyocytes are post-­mitotic cells that do not divide to regenerate a damaged heart. The regeneration of cardiomyocytes after myocardial infarction is an important topic of interest in cardiovascular science. There are various approaches to heart repair after infarction, including activating cardiomyocytes so they become mitotic once again, or growing cardiomyocytes in vitro to attach to a lesion site. An important factor in these approaches is understanding the epigenetic mechanisms controlling cell division. In this thesis, we aim to advance the current knowledge of the epigenetic repressive mechanisms involved in cardiomyocyte formation and heart development to explain their lack of regenerative capacities. We studied the epigenetic changes that occur during cardiac development leading to a non-­‐regenerative state to pinpoint the moment at which these changes arise. We found that the epigenetic process is independent of whether cardiac lineage differentiation occurs during embryogenesis or during differentiation in vitro. We discovered that cardiac heterochromatin displays a singular epigenetic signature during development as compared to brain, another post-­mitotic tissue, or liver, an actively regenerative tissue. We observed an epigenetic change in the repressive histone modification histone H3 lysine 9 trimethylation that was specific to heart development. This change involved a nuclear reorganisation of heterochromatin and a reduction of the levels of this mark in E13.5 and E14.5 embryos, as compared to E10.5 embryos. This was consistent with our observations of the histone lysine methyltransferase SUV39H1, the levels of which were lower after stage E10.5 of development. However, contradictorily, in differentiated cardiomyocytes in vitro, SUV39H1 was increased but showed low levels of H3K9me3, compared to ES cells, which had low levels of SUV39H1 and high levels of H3K9me3. We detected extremely low levels of the H3K9me3 in adult heart tissue. We observed that in adult hearts, the myocardium had maintained these major changes in H3K9me3, while this effect was not observed in the epicardium. Genomic studies were carried out to determine changes at a genomic level between the two key epigenetic stages in heart development we identified at E10.5 and E13.5. Methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation sequencing for H3K9me3 analyses were carried out to find overall changes in methylation patterns. No global changes in DNA methylation were detected between these developmental stages. These results imply that the differences observed in H3K9me3 are due to remodelling of the heterochromatin during heart development and cardiomyocyte formation, rather than quantitative changes.