Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral. / The cytotoxic mechanisms of action of the histone deacetylase inhibitor LBH589 was investigated in association with cisplatin in two cell lines derived from non-small lung cancer (NSCLC). The results were analyzed based on the type of the cell death associated with alterations in enzymes related to energy metabolism and glycolytic pathway. To perform the study, we used two non-small lung tumor cell lines, A549 (wild type for the p53 gene) and Calu-1 (null for p53 gene) treated with LBH589 in combination with cisplatin or not. Assays for time and dose responses were performed through the cell viability analysis (MTT) for each drug separately in both cell lines to choose the best conditions for our study. The conditions of isolated treatments with reduced viability lower than the IC50 of each drug were selected for carrying out the combined treatments. Assays for apoptosis detection by flow cytometry were performed by Annexin V / PI, the expression of proteins related to apoptosis by Western Blotting. The expression of glycolytic related proteins were performed by Western blotting and their mRNA expressions by qPCR. The results demonstrated that the combined LBH589 plus CDDP was able to induce apoptosis in 70% of cells -Calu-1- and 54.9% -A549- in 24 hours, and 90% (Calu-1) and 62.1 % (A549) in 48 hours regardless of the p53 status. The expression levels of enzymes related to energy metabolism also presented changes in the studied treatments. The LBH589 increase of about 4x the HK isoforms I and II mRNA levels in both cell lines. There was also an increase in the expression isoforms I and II of HK protein. Other enzymes related to the glycolytic pathway as PFKP, PKM2 and LDHA were analyzed and we observed reduced protein expression, especially in the presence of LBH589. The combination of CDDP with LBH589 appears promising for the treatment of NSCLC inducing apoptosis via alterations in energy metabolism tumor.

Cancer cells are most clearly characterized by their abnormal and uncontrolled cell growth. One of the most notable theories that explains the vast proliferative capacity of tumorigenic cells is the Warburg effect, a significant shift in metabolism wherein cancer cells preferentially fuel cell division using aerobic glycolysis instead of aerobic respiration. This upregulation of glycolytic fermentation in aerobic environments is highly unusual - glycolysis is typically utilized in anaerobic conditions, but nonetheless dominates cancer metabolic activity in spite of the presence of oxygen. Since the discovery the Warburg effect in the 1920s, researchers have struggled to identify whether aerobic glycolysis is a cause or consequence of carcinogenesis. Interestingly, a new theory recently emerged that challenges this widely-accepted metabolic paradigm for cancer. Known as the reverse Warburg effect, this new mechanism shows that in carcinomas such as breast cancer, the Warburg effect occurs not in cancer cells, but rather in tumor-adjacent stromal fibroblasts. These cancer-associated fibroblasts (CAFs) in the greater tumor microenvironment produce lactate - a high-energy metabolite formed as a byproduct of aerobic glycolysis - to fuel aerobic respiration and rapid tumorigenesis in neighboring cancer cells. This emerging theory emphasizes the pivotal role of the tumor microenvironment in determining whether cancer cells undergo aerobic glycolysis or aerobic respiration. Central to this lactate-linked metabolic intersection are two critical enzymes that regulate a cell's metabolic commitment - lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHc). In order to clarify the mechanisms through which CAFs induce tumorigenesis in breast cancer, we plan to carry out two specific aims: (1) evaluate the enzymatic activity of LDH and PDHc, and (2) compare LDH and PDHc enzyme content. Using co-culture techniques to study the breast cancer tumor microenvironment in vitro, we will compare the enzymatic activity and enzyme content of both MCF7 breast cancer cells and CAFs to identify whether the reverse Warburg effect occurs due to post-translational enzyme activation or increased enzyme synthesis.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral. / The cytotoxic mechanisms of action of the histone deacetylase inhibitor LBH589 was investigated in association with cisplatin in two cell lines derived from non-small lung cancer (NSCLC). The results were analyzed based on the type of the cell death associated with alterations in enzymes related to energy metabolism and glycolytic pathway. To perform the study, we used two non-small lung tumor cell lines, A549 (wild type for the p53 gene) and Calu-1 (null for p53 gene) treated with LBH589 in combination with cisplatin or not. Assays for time and dose responses were performed through the cell viability analysis (MTT) for each drug separately in both cell lines to choose the best conditions for our study. The conditions of isolated treatments with reduced viability lower than the IC50 of each drug were selected for carrying out the combined treatments. Assays for apoptosis detection by flow cytometry were performed by Annexin V / PI, the expression of proteins related to apoptosis by Western Blotting. The expression of glycolytic related proteins were performed by Western blotting and their mRNA expressions by qPCR. The results demonstrated that the combined LBH589 plus CDDP was able to induce apoptosis in 70% of cells -Calu-1- and 54.9% -A549- in 24 hours, and 90% (Calu-1) and 62.1 % (A549) in 48 hours regardless of the p53 status. The expression levels of enzymes related to energy metabolism also presented changes in the studied treatments. The LBH589 increase of about 4x the HK isoforms I and II mRNA levels in both cell lines. There was also an increase in the expression isoforms I and II of HK protein. Other enzymes related to the glycolytic pathway as PFKP, PKM2 and LDHA were analyzed and we observed reduced protein expression, especially in the presence of LBH589. The combination of CDDP with LBH589 appears promising for the treatment of NSCLC inducing apoptosis via alterations in energy metabolism tumor.

La formation et le développement d'une tumeur sont provoqués par une série de défauts qui se produisent à l'intérieur de la cellule cancéreuse et dans son microenvironnement. Ces anomalies permettent à la cellule de développer ses propres stratégies de croissance, de prolifération, de différenciation et de métabolisme. Toutes ces adaptations, ainsi que la création d'un micro-environnement unique favorisent la croissance de la tumeur et inhibent la réponse immunitaire anti-tumorale. Le métabolisme des cellules cancéreuses et l'évasion immunitaire sont des points très sensibles dans le développement des cancers et peuvent être utilisés en clinique. Les études récentes suggèrent que ces deux phénomènes sont liés, et que le métabolisme des cellules cancéreuses peut amener à l'échappement immunitaire par la tumeur. Le métabolisme des cellules tumorales a tendance à éviter l'activité mitochondriale et la phosphorylation oxydative, et est principalement basée sur la glycolyse pour la production d'énergie (effet Warburg). Mon travail de thèse est divisé en deux parties. Dans la première partie nous avons proposé un concept thérapeutique novateur avec une nouvelle thérapie combinatoire pour le traitement de cancers hématologiques. Cette thérapie est basée sur l'induction de changements métaboliques par le dichloroacétate (DCA), et elle est associée avec la chimiothérapie conventionnelle (doxorubicine, vincristine) pour réactiver les fonctions de p53. Les tumeurs avec p53 mutantes sont résistantes à cette combinaison. Dans ce cas, nous avons constaté que le DCA peut coopère avec 17-AAG (l'inhibiteur de Hsp90) pour éliminer spécifiquement les cellules cancéreuses. En conséquence, une meilleure compréhension des signaux et des mécanismes par lesquels le DCA sensibilise les cellules tumorales à la chimiothérapie est nécessaire pour en comprendre le mode d'action. En outre, l'identification de ce mécanisme permettra d'élucider les voies métaboliques impliquées dans la survie des cellules cancéreuses. La deuxième partie de ma thèse se concentre sur la biologie des cellules NK. Les cellules NK sont des lymphocytes du système immunitaire inné et possèdent une cytotoxicité naturelle contre les cibles, c'est à dire les cellules tumorales. L'utilisation optimale des cellules NK en clinique nécessite leur expansion et leur activation in vitro. Les cellules NK s'activent en présence de cytokines ou par le contact avec les cellules cibles. L'activation des cellules NK induit la prolifération, mais celle-ci dépend aussi de la présence d'autres cellules immunitaires. L'activation, par les cytokines et par les cellules cibles, induit un différent ARNm/microARN profil d'expression. L'analyse détaillée des isoformes de la protéine tyrosine phosphatase CD45 a permis de caractériser de nouvelles populations de cellules NK anti-tumorales humaines. L'identification de différentes populations de cellules NK est très importante pour la compréhension de leur physiologie et pour l'amélioration de leur utilisation en immunothérapie clinique. Cela peut également donner des informations précieuses sur l'état physiologique de l'hôte. En effet, l'augmentation des cellules CD45RAdim et CD45RO + dans le compartiment des cellules NK matures identifie clairement les patients avec des hémopathies malignes. Nous pensons que leur détection peut être utilisée comme un outil de diagnostic et également pour évaluer l'efficacité des traitements anti-tumoraux, car ces populations de cellules NK spécifiques devraient diminuer lors de l'élimination de cellules tumorales cibles. Dans l'avenir, nous voulons combiner le traitement du métabolisme de la tumeur avec la thérapie anti-tumorale basée sur les cellules NK. Sur la base de nos données préliminaires, nous pouvons proposer le traitement des cellules cancéreuses par des médicaments métaboliques pour augmenter la sensibilité et la reconnaissance par les cellules NK activées. / Tumor formation and development are caused by a range of defects that occur inside the cancer cell and in the external cellular microenvironment. These abnormalities allow developing tumors to establish their own strategies of growth, proliferation, differentiation and metabolism. All these adaptations, as well as the creation of a unique microenvironment, promote tumor growth and suppress the anti-cancer immune response. Tumor cell metabolism and immune evasion are sensitive points of cancer development that can be targeted in clinic. Recent studies suggest that these two phenomena are related and that cancer cell metabolism may propel tumor immune escape. Tumor cell metabolism tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS), and largely relies on glycolysis to produce energy (Warburg effect). My thesis work is divided into two parts. The first one proposes an innovative therapeutic strategy, which is the use of different combinatorial therapy depending on the p53 status for the treatment of hematological cancers. This is based on the induction of metabolic changes by dichloroacetate (DCA), combined with conventional chemotherapy (doxorubicin, vincristine) to reactivate wild type p53 functions. Mutant p53 tumors are resistant to this combination approach. However, we found that DCA synergized with the Hsp90 inhibitor 17-AAG to specifically eliminate these cells. Therefore, a clearer understanding of the signals and mechanisms by which DCA sensitize cancer cells to chemotherapy was needed to understand its mode of action. We uncovered it in our work. In addition, identification of this mechanism will help to elucidate metabolic pathways involved in cancer cell survival.The second part of my thesis is focused on the study of NK cell biology. NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity against targets, i.e. tumor cells. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and the encounter with target cells activate NK cells, induce their proliferation, and cause clearly different mRNA/miRNA expression profile. Detailed analysis of the leucocyte-specific phosphatase CD45 isoforms allowed us to characterize new human anti-tumor NK cell populations. The identification of the different NK cell populations is important for understanding their physiology and for improving their therapeutic use in the clinic. It can also give valuable information about the host physiological status. Indeed, the increase of CD45RAdim and CD45RO+ cells in the mature NK cell compartment clearly identifies patients with hematological malignancies. We thus hypothesize that their detection could be used as a diagnostic tool, and also to assess the efficacy of antitumor treatments, because these specific NK cell populations should decrease upon removal of the targeted tumor cells. Our future goal is to use a novel combinatorial therapy in hematological cancers that will combine metabolic drugs and NK cell-based therapy. Based on our preliminary data, we propose that the treatment of cancer cells with metabolic drugs could increase their sensitivity and recognition by activated NK cells.

Background: Tumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate. Methods: Glioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed. Results: The abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate. Conclusions: Our experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.

La glycogénose de type I (GSDI) est une maladie génétique rare, due à une déficience en glucose-6 phosphatase (G6Pase), enzyme clé de la production endogène de glucose. En plus des hypoglycémies sévères, la perte de l'activité G6Pase conduit à l'accumulation de glycogène, mais aussi de lipides dans le foie et les reins. A long-terme, la plupart des patients développent des tumeurs hépatiques et une maladie rénale chronique (MRC).Le but de cette thèse a été de caractériser les mécanismes moléculaires impliqués dans la carcinogenèse hépatique et la MRC grâce à des modèles murins viables et uniques, avec une délétion de la G6Pase spécifiquement dans le foie ou les reins, reproduisant respectivement toutes les caractéristiques de la pathologie hépatique ou rénale.Au niveau du foie, notre étude a permis de mettre en évidence une reprogrammation métabolique « Warburg-like » très similaire à celle des cellules cancéreuses, associée à une perte des défenses cellulaires et des suppresseurs de tumeur. De plus, nous avons montré que les adénomes hépatocellulaires, se transformant ensuite en carcinomes, se développent en absence de fibrose, en accord avec l'absence d'activation des voies pro-fibrotiques. Au niveau des reins, l'étude de la MRC a mis en évidence le développement de kystes rénaux chez les souris atteintes de GSDI, observés aussi chez les patients à un stade avancé de la MRC. Finalement, une dernière étude portant sur l'activation de l'oxydation des lipides, par un traitement des souris au fénofibrate, a permis de suggérer le rôle délétère de l'accumulation des lipides dans le développement des pathologies hépatique et rénale / Glycogen storage disease type I (GSDI) is a rare genetic disease, due to a deficiency in glucose-6 phosphatase (G6Pase), a key enzyme in the endogenous glucose production. Besides severe hypoglycemia, the loss of G6Pase leads to the accumulation of glycogen and lipids in the liver and kidneys. On the long term, most patients develop hepatic tumors and chronic kidney disease (CKD).The goal of this thesis was to characterize the molecular mechanisms involved in hepatic carcinogenesis and CKD, thanks to viable and unique mouse models with specific deletion of G6Pase in the liver or kidneys, which exhibit all hallmarks of hepatic and renal pathologies, respectively.On a hepatic level, our study allowed us to highlight a « Warburg-like » metabolic reprogramming, very similar to what is observed in cancer cells, associated with a loss of cellular defenses and tumor suppressors. Furthermore, we showed that formation of hepatocellular adenoma, which transform later in carcinoma, occurs in the absence of liver fibrosis, due to the fact that pro-fibrotic pathways are not activated. In the kidneys, the study of CKD highlighted the development of renal cysts in mice with GSDI, as well as in the patients presenting an advanced stage of CKD. Finally, the last study on the activation of the oxidation of lipids, by treating the mice with fenofibrate, allowed us to suggest a deleterious role of lipid accumulation in the development of the hepatic and renal pathologies

In this master’s thesis, the dance artist Adorée Villany's hitherto unexplored performances in Sweden 1914–1920 have been reconstructed and interpreted. Villanys naked dance aroused both interest and a lively debate in the Swedish press. She labelled her art “Reform Dance” and thus formed a part of the emerging modern dance movement. By moving beyond the morality debate surrounding her performances, visual and performative aspects of her dances and photographs are made visible.    In her art, Villany strived to bring dance forms from ancient culture to life. Her embodiment of images from ancient Egypt can be understood employing Warburg's concepts Pathosformula and Nachleben, explaining how art works through artistic imagery and form can resurrect emotions from another time. Villany's dance piece Salome’s dance has been reconstructed and analysed through a joint reading of the source materials film, texts and photographs. Here, Warburg's concept of Denkraum was proven productive as a theory and method.

Les récepteurs couplés aux protéines G (RCPG) sont une famille de protéines hautement conservée chez les eucaryotes et constituent la plus grande famille de récepteurs. Ces récepteurs sont impliqués dans presque tous les processus physiologiques, mais leur capacité à réguler un vaste éventail de processus biologiques différents fait l'objet de recherches intenses. Bien qu'ils soient classiquement considérés comme des récepteurs de la membrane plasmique, les RCPG sont présents dans tous les organites membranaires intracellulaires et certains d'entre eux ont la capacité de transduire des signaux à partir de ces organites. La signalisation d'un RCPG à partir de ces organelles intracellulaires est appelée signalisation biaisée par la localisation et cette signalisation peut avoir un résultat fonctionnel différent de celui des événements de signalisation du récepteur localisé dans la membrane plasmique. La signalisation biaisée par la localisation est un concept émergent en biologie des RCPG et peut ajouter une couche supplémentaire à la fonction du récepteur. D'autre part, avec la détection de certains RCPG à l'intérieur de différents organites, y compris le noyau, une modalité fonctionnelle non réceptrice pour les RCPG pourrait être postulée et pourrait également expliquer les divers rôles de cette famille. Cependant, cet aspect est presque entièrement inexploré. HCAR1 (GPR81), en tant que RCPG, est activé de manière endogène par le lactate et il a été démontré qu'il favorise la malignité du cancer en favorisant un niveau plus élevé de glycolyse dû à l'effet Warburg et cela par différentes voies. Son niveau d'expression est très élevé dans de nombreux cancers et présente une corrélation négative avec le pronostic du patient. Cependant, son mécanisme d'action n'est pas bien compris. Dans cette thèse, nous avons étudié la localisation nucléaire et les rôles potentiels du HCAR1 et nous avons découvert que ce récepteur est localisé à la membrane nucléaire et à l'intérieur du noyau, en plus de sa localisation à la membrane plasmique. Le HCAR1 nucléaire (N-HCAR1) est capable d'induire une signalisation intranucléaire basée sur la localisation pour induire la phosphorylation de ERK et d’AKT dans le noyau. En utilisant des approches protéomiques et génomiques, nous avons découvert que N-HCAR1 est impliqué dans plusieurs fonctions non réceptrices régulant différents processus à travers ses interactomes nucléaires. Ce regroupement nucléaire de HCAR1, en fonction de ses facteurs de liaison, favorise la traduction des protéines, la biogenèse ribosomale et la réparation des dommages à l'ADN. De manière intéressante, N-HCAR1 interagit également avec des facteurs de remodelage de la chromatine et régule directement l'expression des gènes d'après notre séquençage ChIP à l'échelle du génome. Nous avons également effectué un séquençage de l’ARN et les résultats montrent que N-HCAR1 régule l'expression d'un réseau de gènes plus large que son homologue de la membrane plasmique. Notamment, l'exclusion nucléaire de HCAR1 s'est avérée avoir le même effet que son knockdown complet sur la croissance tumorale et les métastases in vivo. Nos données révèlent une signalisation basée sur la localisation et des fonctions non canoniques pour un RCPG dans le noyau par lesquelles HCAR1 peut réguler différents processus cellulaires. / G Protein-Coupled Receptors (GPCR) are a highly conserved protein family in eukaryotes through evolution and they are the largest receptor family. These receptors are virtually involved in every physiological processes, but their ability to regulate such a vast array of different biological processes is under intense investigation. Although classically considered a plasma membrane receptor, GPCRs are found in every intracellular membranous organelle and some of them are shown to have the capacity for signal transduction from those organelles. The signaling of a GPCR from these intracellular organelles is called location-biased signaling and this signaling could have a different functional output than the signaling events from the plasma membrane-localized receptor. Location-biased signaling is an emerging concept in the GPCR biology and can add an extra layer to the receptor function. On the other hand, with the detection of some GPCRs inside different organelles including the nucleus, a non-receptor functional modality for GPCRs could be postulated and could also account for the diverse roles of this family. However, this aspect is almost entirely unexplored. HCAR1 (GPR81), as a GPCR, is endogenously activated by lactate and has been shown to promote cancer malignancy via a higher level of glycolysis due to the Warburg effect, through different pathways. Its expression level is highly elevated in many cancers and negatively correlates with the patient’s prognosis. However, its mechanism of action is not well understood. In this thesis, we investigated the nuclear localization and potential roles of HCAR1 therein and we found this receptor is localized to the nuclear membrane and inside the nucleus, besides its plasma membrane localization. The Nuclear HCAR1 (N-HCAR1) is capable of inducing location-biased signaling intranuclearly to induce nuclear-ERK and AKT phosphorylation. Using proteomics and genomics approaches, we discovered that N-HCAR1 is involved in several different non-receptor functions regulating different processes through its nuclear interactomes. This nuclear pool of HCAR1, depending on its binding factors, promotes protein translation, ribosomal biogenesis, and DNA-damage repair. Interestingly, N-HCAR1 also interacts with chromatin remodeling factors and directly regulates gene expression based on our genome-wide ChIP-sequencing. We also performed RNA-seq, and the results show N-HCAR1 regulates the expression of a broader gene network than its plasma membrane counterpart. Notably, nuclear exclusion of HCAR1 proved to have the same effect as its complete knockdown on tumor growth and metastasis in vivo. Our data reveal location-biased signaling and non-canonical functions for a GPCR in the nucleus by which HCAR1 can regulate different cellular processes.

Background: The NKG2D ligands are a set of cell surface proteins, the expression of which can make cells susceptible to immunity mediated by NKG2D receptor expressing cells, which include NK cells, CD8⁺ αβ T cells and γδ T cells. The NKG2D ligands are known to be expressed in distinct settings, including viral infection, cancer, T cell activation, and cellular proliferation, settings also tightly associated with Warburg metabolism. The molecular events which determine NKG2D ligand expression status are unknown. Aims: We aim to enhance understanding of the deterministic molecular events that control NKG2D ligand expression. Specifically, to explore the relationship between Warburg metabolism and NKG2D ligand expression in cell line and physiological models, and second, to identify open chromatin elements at NKG2D ligand loci, and develop computational methods to analyse this data. Methods: We use a range of molecular biology techniques to delineate the role of glucose metabolism in NKG2D ligand expression in a HEK293T cell model. We develop a physiological CMV-primary fibroblast model of NKG2D ligand induction to validate our key findings. We adapt, optimise and validate a DNaseI-seq protocol, to define open chromatin sites at the NKG2D ligand loci. We develop a data analysis `pipeline', including our own peak-finding software (“PeakHunter"), to identify open chromatin sites in the data. Key results: Glucose drives NKG2D ligand expression. This effect requires cellular uptake and metabolism of glucose. Purine nucleotides are a key glucose metabolite for this effect, and purine nucleosides are sufficient to induce NKG2D ligand expression in our HEK293T model. We have identified the open chromatin sites at the NKG2D loci in MCF7 breast cancer cells, and optimised and validated this protocol. Finally we have developed “PeakHunter" a multifunctional software tool for mapped DNaseI-seq data analysis. Conclusions: Glucose and its contribution to purine metabolism play a central role in the induction of NKG2D ligand expression in physiological settings. The influence of glucose leads to significant alterations in cellular NKG2D-dependent immunogenicity. PeakHunter is a useful tool for analysis of mapped DNaseI-seq data.

L’étude des concepts fondamentaux de l’iconologie permet de dégager la conception de l’art, de l’histoire et de la création qu’elle implique. L’examen de l’analyse iconographique, des formations par analogie et de la critique du formalisme de Wölfflin permet de réviser l’idée selon laquelle l’intellectualisme de Panofsky le conduirait à méconnaître la spécificité du visuel. La mise au jour des torsions que l’iconologue fait subir au concept de forme symbolique clarifie la question du néo-kantisme et pose celle de l’usage de concepts (philosophiques) par l’historien. Le propre de la démarche historienne ressort aussi de l’analyse du concept rieglien de Kunstwollen qui engage la théorie de la création, la définition de l’art, son autonomie et la légitimité de l’iconologie. Celle-ci passe également par la résolution d’une tension entre le souci d’écarter toute approche esthétique ou normative de l’objet d’art et la nécessaire prise en compte de sa spécificité épistémologique et pratique. Enfin le concept d’habitus et la refonte des notions d’influences et de tradition lèvent l’antinomie entre liberté créatrice et nécessité historique. L’usage du modèle dialectique (hégélien) de l’histoire relève d’un choix de méthode et d’une prise de distance par rapport à Warburg dont les concepts centraux sont détournés. / The study of the fundamental concepts of iconology enables us to identify the idea of art, of history and of creation it presupposes and also to rethink the traditional interpretation which is made of it. The analysis of the iconographical moment, of the creation by analogy and of the criticism of Wölfflin’s formalism challenges the idea that Panofsky supposedly fails to recognize the implacability of the visual aspect. As for Panofsky’s thesis of Neo-Kantianism, it is based on resorting to the concept of symbolic form. But the twisting it undergoes is relevant of the unique way in which the historian resorts to (philosophical) concepts. The distinguishing feature of the historical approach also emerges from the study of the Rieglian Kunstwollen concept which involves the theory of creation, the definition and the autonomy of art, and the legitimacy of iconology. This legitimacy also emerges from the solving of the tension between the desire to turn down any esthetical or normative approach of the objet d’art, and the necessity to take its epistemological and practical specificity into account. The historian’s will to be objective and the transformation of the taste-based judgment into the knowledge-based judgment it seems to imply are also questioned. Finally, the concept of habitus and the transformation of the notions of influence and tradition remove the antinomy between creative freedom and historical necessity. The use of the dialectic (Hegelian) model of history is a choice of method and implies that Panofsky voluntarily stands back from Warburg’s philosophy, the key concepts of which are thus embezzled.