Deux phénomènes liés au TFIIH ont été étudiés : l'influence des mutations spécifiques dans la sous-unité XPD de TFIIH sur la réponse transcriptionnelle de certains gènes après l'irradiation UV, et l'interaction entre le TFIIH et la transcription des gènes de classe III. Une analyse détaillée de la dynamique du transcriptome a été effectuée pour la réponse des cellules humaines mutantes XP-D/CS à l'UV. Il a été démontré que la dysrégulation sélective observée de l'expression des gènes était liée à l'incapacité pour la ré-initiation transcriptionnelle et à l'hétérochromatinisation suivante, où l'histonedésacétylase SIRT1 a été identifiée comme le principal facteur. Son inhibition a permis de recouvrer l'expression normale d'un nombre substantiel des gènes affectés. Une étude de la participation pangénomique du coeur de TFIIH dans latranscription a découvert son association avec les gènes actifs de classe III. Cette association a été démontrée être indépendante de Pol II. Le coeur de TFIIH a été montré participer directement à la transcription effectuée in vitro par Pol III.

The important factor in regulation of metabolic processes is regulatory proteins, which are able to react by feed-back to energetic state of the cell. Big attention is focused on the AMP activated kinase (AMPK) and NAD+ activated deacetylase SIRT1. These enzymes interact together and their stimulation increases mitochondrial biogenesis and fatty acid oxidation. Due to this it functions beneficially against the onset of obesity, insulin resistance and ageing. Fasting, exercise and some antidiabetogenic drugs act by these regulators. n-3 polyunsaturated fatty acids (PUFA) are also known because of their stimulative effects on mitochondrial biogenesis and -oxidation. Previous work of our group have showed that intake of higher dose of n-3 polyunsaturated fatty acids (PUFA) in diet lead to increase in activity of AMPK in white adipose tissue. New results presented in this thesis show that SIRT1 is essential for increase in expression of stimulators of -oxidation (PPAR etc) in response to n-3 PUFA in diet. n-3 PUFA futher improve the metabolic profile synergistically with calorie restriction probably through SIRT1.

Systemic sclerosis (SSc) is immune-mediated fibrotic disease of unknown aetiology. Among the dominant pathogenic manifestations of SSc belong vascular changes, production of autoantibodies, activation of innate and adaptive immune responses and fibrotic processes. Transforming growth factor beta (TGF-β) has been identified as a central profibrotic factor stimulating fibroblasts to produce collagen. There are, however, a number of other mediators involved in the pathogenesis of SSc. Mutual activation and amplification of these molecules and their cascades may be a central mechanism of the SSc pathogenesis. Hedgehog (Hh) canonical signalling pathway plays an important role in the development and progression of fibrotic diseases. Expression of Hh target genes can be regulated through a canonical or non-canonical signalling cascade. The non-canonical activation of GLI transcription factors by TGF-β has not yet been investigated in SSc. The substantial part of this thesis is focused on the study of the mutual interaction of TGF-β and Hh signalling pathway. In vitro analysis confirmed TGF- β/SMAD3 dependent activation of GLI2 in dermal fibroblasts. Fibroblasts specific knockout of GLI2 prevented the development of experimental fibrosis in vivo. Combined targeting of canonical and non-canonical Hh...

Abstract Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and AMP-activated protein kinase (AMPK) are major factors regulating energy homeostasis. In this study, we aimed to investigate how energy flux affects several hepatic functions mediated by these factors. We define a novel role of PGC-1α and AMPK as modulators of the immune system in the liver. We show that PGC-1α is involved in the regulation of a cluster of genes related to the immune system, most importantly Interleukin 1 receptor antagonist (IL1Rn). Since PGC-1α is responsive to energetic stress associated with fasting or physical exercise, the same stimuli promote IL1Rn in hepatocytes. We identify AMPK as an independent inducer of IL1Rn and hypothesise that it could account for the anti-inflammatory effect of the antidiabetic drug metformin. We also demonstrate that metformin reduces expression of Sirtuin 3 (SIRT3) in hepatocytes and promotes acetylation of mitochondrial protein. We suggest that this mechanism, in spite of increased mitochondrial biogenesis, contributes to reduced ATP synthesis in metformin-treated samples. In addition, we demonstrate that Pregnane X receptor (PXR) is induced in the liver during fasting and by PGC-1α in hepatocytes. Furthermore, we describe a negative regulatory mechanism involving SIRT1, activated by pyruvate and interfering with PXR signaling. We show that SIRT1 attenuates PGC-1α-mediated co-activation of PXR and its target genes, i.e. Cyp3a11, with possible implications for drug and xenobiotic metabolism. In conclusion, we demonstrate how energetic stress affects various hepatic functions mediated by PGC-1α and AMPK. Moreover, we describe SIRT1 and metformin as factors capable of modulating this response. / Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori gamman koaktivaattori 1α (PGC-1α) ja AMP:n aktivoima proteiinikinaasi (AMPK) ovat keskeisiä energiametabolian säätelijöitä. Tässä tutkimuksessa oli tavoitteena selvittää kuinka energiataso vaikuttaa useisiin, näiden tekijöiden säätelemiin maksan toimintoihin. Osoitamme että PGC-1α ja AMPK tekijöillä on ennestään tuntematon merkitys immuunijärjestelmän säätelyssä maksassa. Näytämme myös, että PGC-1α säätelee joukkoa geenejä, joiden tehtävä liittyy immuunijärjestelmään, tärkeimpänä Interleukiini 1 reseptori antagonistia (IL1Rn). Paastoon ja fyysiseen aktiivisuuteen liittyvä energiastressi aktivoi PGC-1α:aa ja näiden samojen stimuluksien havaittiin lisäävän myös IL1Rn tasoa hepatosyyteissä. Havaitsimme AMPK:n olevan itsenäinen IL1Rn indusori ja hypoteesimme mukaan tämä voi välittää diabeteslääkkeenä käytettävän metformiinin anti-inflammatorisia vaikutuksia. Osoitamme myös, että metformiini alentaa Sirtuiini (SIRT) 3:n ekspressiota maksasoluissa ja lisää mitokondriaalisten proteiinien asetylaatiota. Uskomme tämän mekanismin, huolimatta lisääntyneestä mitokondrioiden biogeneesistä, myötävaikuttavan vähentyneeseen ATP synteesiin metformiinikäsitellyissä näytteissä. Lisäksi osoitamme, että paasto ja PGC-1α indusoivat Pregnaani X reseptorin (PXR) ilmentymistä maksasoluissa. Kuvaamme myös PXR signalointiin vaikuttavan ja pyruvaatin aktivoiman, SIRT1:n välitteisen, negatiivisen säätelymekanismin. SIRT1 estää PGC-1α välitteistä PXR koaktivaatiota ja kohdegeenien, kuten Cyp3a11, aktivaatiota, millä voidaan olettaa olevan merkitystä lääkeaineiden ja vierasaineiden metaboliaan. Yhteenvetona osoitamme, että energiastressi PGC-1α:n ja AMPK:n välittämänä vaikuttaa useisiin maksan toimintoihin. Lisäksi näytämme, että SIRT1 ja metformiini voivat moduloida näitä vaikutuksia.

NAMPT (Nicotinamide phosphoribosyltransferase) catalyses the rate-limiting step in the NAD biosynthesis from nicotinamide and thereby regulates the activity of NAD-dependent enzymes. Cancer cells are highly dependent on NAD for energy and DNA repair processes and are assumed to be more susceptible to an inhibition of NAD synthesis than non-transformed cells. We aimed to investigate whether or not inhibition of NAMPT with its specific inhibitor FK866 can sensitize leukemia cells for chemotherapeutic agents. NAMPT protein abundance, enzymatic activity and NAD concentrations were significantly higher in Jurkat and Molt-4 leukemia cell lines compared to normal peripheral blood mononuclear cells. Combination of etoposide and FK866 caused increased cell death in leukemia cell lines compared to etoposide alone. Etoposide decreased protein abundance of NAD-dependent deacetylases SIRTUIN1. After combining etoposide and FK866 treatment SIRTUIN2 was further decreased and accumulation and acetylation of the downstream target p53 was further enhanced in MOLT4 cells. Concomitantly, protein abundance of p21 and cleaved BAX was increased. Targeting NAMPT could be a novel therapeutic strategy to enhance the efficacy of chemotherapeutic agents such as etoposide against leukemia.

Le stress oxydant (SO) est un mécanisme commun à l’ischémie cérébrale et au traumatisme crânien qui entraîne notamment l’hyperactivation délétère de la poly(ADP-ribose)polymérase (PARP), une enzyme NAD+-dépendante. Cette dernière est impliquée dans le déficit neurologique et la lésion cérébrale consécutifs à ces pathologies. In vitro, l’hyperactivation de la PARP diminue le taux cérébral de NAD+, son substrat, et l’activité de la sirtuin 1 (SIRT1), une enzyme également NAD+-dépendante. L’activation de la SIRT1 est bénéfique au cours d’un SO in vitro. Si les effets bénéfiques de l’inhibition de la PARP ont été démontrés in vivo au cours d’un SO cérébral, l’implication de la SIRT1 ainsi que son rôle dans les effets de l’inhibition de la PARP n’ont pas été explorés. Dans la première partie de ce travail, nous avons mis en évidence qu’un modèle de SO cérébral induit in vivo chez le rat par une injection intrastriatale de malonate entraîne un SO prolongé, un déficit neurologique et une activation de la PARP associée à une diminution du NAD+. Dans la deuxième partie de ce travail, nous avons montré que le 3-aminobenzamide (3AB), un inhibiteur de la PARP, ne permet pas de s’opposer à la chute du NAD+ dans ce modèle, ce qui suggère que le NAD+ pourrait être consommé par d’autres enzymes NAD+-dépendantes, dont la SIRT1. L’inhibition de la PARP par le 3AB a permis d’augmenter le rapport activité/expression nucléaire de la SIRT1 et a entraîné sa translocation cytoplasmique au cours du SO. Un prétraitement par le SRT1720, un activateur spécifique de la SIRT1, diminue le déficit neurologique et la lésion striatale 6 heures après le SO cérébral, ce qui suggère que l’activation de la SIRT1 est bénéfique dans les conséquences d’un SO cérébral in vivo. L’association de l’inhibiteur de la PARP avec l’activateur de la SIRT1 (3AB+SRT1720) n’a pas potentialisé les effets protecteurs de chaque monothérapie. L’EX527, un inhibiteur de la SIRT1, ne modifie pas le déficit et la lésion. En revanche, l’association de l’inhibiteur de la PARP avec l’inhibiteur de la SIRT1 (3AB+EX527) supprime la récupération neurologique ainsi que la réduction de la lésion, induites par l’inhibition de la PARP seule (3AB). Ces données suggèrent que l’activation de la SIRT1 est impliquée dans les effets bénéfiques de l’inhibition de la PARP in vivo au cours d’un SO cérébral. En conclusion, l’ensemble de ce travail a permis une meilleure caractérisation de la PARP et de la SIRT1 au cours d’un SO cérébral in vivo. La SIRT1 pourrait constituer une cible pharmacologique pour le traitement des pathologies cérébrales au cours desquelles un SO est présent. De plus, nous avons montré que les effets bénéfiques de l’inhibition de la PARP sur les conséquences fonctionnelles et histologiques induites par le SO cérébral sont liés à l’activation de la SIRT1. / Oxidative stress (OS) is involved in cerebral ischemia and traumatic brain injury and results in deleterious activation of poly(ADP-ribose)polymerase (PARP), an NAD+-dependant enzyme. PARP is implicated in neurological deficit and brain injury post-ischemia and post-trauma. In vitro, PARP overactivation reduced both brain NAD+ levels, its substrate, and activity of sirtuin 1 (SIRT1), an other NAD+-dependant enzyme. SIRT1 activation is beneficial during in vitro OS. Even if the beneficial effects of PARP inhibition have been demonstrated, SIRT1 involvement during in vivo cerebral OS and its role in the beneficial effects of PARP inhibition have not been studied.In the first part, we demonstrated that in vivo cerebral OS induced by intrastriatal injection of malonate in rat promoted prolonged OS, neurological deficit, PARP activation and NAD+ decrease. In the second part, we showed that 3-aminobenzamide (3AB), a PARP inhibitor, did not reduce NAD+ loss, suggesting that NAD+ could be consumed by other NAD+-dependant enzymes, including SIRT1. The PARP inhibitor increased the nuclear SIRT1 activity/expression ratio and induced its cytoplasmic translocation during OS. SRT1720, a specific SIRT1 activator, reduced both neurological deficit and striatal lesion 6 hours after cerebral OS, suggesting that SIRT1 activation is beneficial on in vivo OS consequences. The combination of the PARP inhibitor with the SIRT1 activator (3AB + SRT1720) did not potentiate the neuroprotective effects of each strategy. EX527, a SIRT1 inhibitor, did not affect OS-induced deficit and lesion. However, association of the PARP inhibitor with the SIRT1 inhibitor (3AB + EX527) suppressed the neurological recovery and the reduction of lesion induced by 3AB alone. Our data suggested that SIRT1 activation is involved in the neuroprotective effects of PARP inhibition during in vivo cerebral OS. In conclusion, our work led to a better characterization of PARP and SIRT1 during in vivo cerebral OS. SIRT1 is a potential pharmacological target for the treatment of brain pathologies in which OS is present. In addition, SIRT1 activation is involved in the beneficial effects of PARP inhibition on functional and histological cerebral OS consequences

SIRT1 and SIRT2 are two NAD⁺-dependent deacetylases which negatively modulate the activity of p53, a protein which is involved in cell cycle arrest, senescence and apoptosis following genotoxic stress. Part I of the thesis describes the exploration of the chemical space around a reported unselective and modest inhibitor of SIRT1 and SIRT2 with the aim of improving the selectivity and potency of the inhibitor against the two isoforms. Particular emphasis is placed upon understanding the mode of binding of the novel analogues within the active site of the enzymes. Chapter 1 reviews the physiological roles of class III NAD⁺-dependent deacetylases, also known as sirtuins. In particular, the application of SIRT1 and SIRT2 inhibitors as potential anticancer agents is described. Amongst these, only cambinol and the tenovins showed in vivo activity in a mouse xenograft model. Previously only one analogue of cambinol had been reported in the literature. Chapter 2 describes the development of a small collection of novel cambinol analogues (First Generation Studies). The role played by different substituents at the phenyl group and at the N-1 of the thiouracil core is discussed. Along with the synthesis and structure activity relationship (SAR) associated with the core structure, in-cell experiments intended to confirm the activity of the most active compounds are reported. Chapter 3 provides a rationalisation for the SAR discussed in Chapter 2. Based on computational molecular modelling studies (GOLD), the activity of the most potent and selective SIRT2 inhibitors is explained. Two series of novel cambinol analogues were designed (Second and Third Generation Analogues) in order to assess further the proposed binding mode. Chapter 4 focuses on the development of the “Second Generation” analogues, characterised by the presence of lipophilic substituents at the sulfur atom and at the N-3 position of the thiouracil core. The synthesis, biological evaluation and SAR are discussed in detail. Chapter 5 reports the development of the “Third Generation” analogues, characterised by either a benzyl group or para-alkoxy-substituted benzyl group at the N-1 position of cambinol. Once again, the synthesis, biological evaluation and SAR data are presented. An improved understanding of the mode of binding of the novel compounds is proposed based on molecular dynamics (MD) studies. Indole-based alkaloids, such as Vincristine and Vinblastine, are well known for their anticancer activity. Recently, the anticancer activity of members of the calycanthaceous family of alkaloids has been discovered. Part II of the thesis focuses on model studies aimed at developing the total synthesis of one of these compounds, perophoramidine. Chapter 7 provides an overview of the calycanthaceous alkaloid family of natural products, including their biological properties. The structural features of perophoramidine, along with the previously reported synthetic studies are outlined. Chapter 8 describes the synthesis of an advanced intermediate in the total synthesis of dehaloperophoramidine, a structural analogue of perophoramidine Problems encountered, optimisation studies and the synthesis of a re-designed intermediate are also reported in this chapter.

Les multi-thérapies actuelles permettent de maintenir l'infection au VIH-1 sous contrôle, mais malheureusement n'entraînent pas l'éradication du virus du fait de l'existence de réservoirs cellulaires, où le virus est intégré de façon latente. Les cellules microgliales, cibles privilégiées du VIH-1 dans le cerveau, sont les macrophages résidents du système nerveux central et ont été décrites comme un réservoir cellulaire avec une longue durée de vie. Ce genre de cellule, infectée de façon latente, apparaît comme un des principaux obstacles à l'éradication. Ainsi, la compréhension des mécanismes sous-jacents impliqués dans l'extinction de la transcription virale, semble une étape cruciale afin de parvenir à purger ces réservoirs. Notre laboratoire à déjà montré l'importance du répresseur transcriptionnel CTIP2 dans l'établissement et le maintien de la latence dans ces cellules. Dans le cadre de ma thèse je me suis intéressé à deux autres facteurs cellulaires, LSD1 et HIC1. Au cours de mes travaux, j'ai mis en évidence le rôle répresseur de ces protéines sur la transcription virale dans les microglies. LSD1 coopère avec CTIP2 pour promouvoir l'établissement de marques épigénétiques au niveau du promoteur viral pour induire la mise en place d'hétérochromatine. LSD1 est à l'origine du recrutement de CTIP2, mais aussi d'un autre complexe multiprotéique, COMPASS. A la différence de CTIP2 et LSD1, le suppresseur de tumeur HIC1 est un perturbateur du transactivateur viral TAT. HIC1 est préalablement modifié post-traductionnellement par la déacétylase SIRT1 et va ensuite contrecarrer l'activité de TAT afin d'empêcher la réactivation de la transcription du virus. Ainsi, tandis que LSD1 et CTIP2 favorise l'établissement de la latence, HIC1 permet quant à lui d'entretenir cet état du provirus dans les cellules microgliales. Les travaux présentés ici mettent en évidence deux nouveaux facteurs de la restriction de l'expression virale et permettent de définir de nouvelles cibles thérapeutiques potentielles pour les stratégies de purge des réservoirs.

The cerebral cortex is one of the most complex and divergent of all biological structures and is composed of hundreds of different types of highly interconnected neurons. This complexity underlies its ability to perform exceedingly complex neural processes. One of the most important questions in developmental neurobiology is how such a vast degree of diversity and specificity is achieved during embryogenesis. Furthermore, understanding the cellular and genetic basis of cortical development may yield insights into the mechanisms underlying human disorders such as mental retardation, autism, epilepsies and brain tumors. <p>During this Phd-project, we set out to identify novel transcription factors involved in cortical neurogenesis. Therefore, we initially took advantage of a model of in vitro embryonic stem cell (ESC)-derived corticogenesis that was previously established in the lab (Gaspard et al. 2008) and from several previously generated ESC lines that allow overexpression of specific transcription factors potentially involved in corticogenesis (van den Ameele et al. 2012). <p>Among the genes tested, Bcl6, a B-cell lymphoma oncogene known to be expressed during cortical development but without well-characterized function in this context, displayed a strong proneurogenic activity and thus became the main focus of this thesis. <p><p>During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is well known to be important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signalling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition.<p>We showed that Bcl6 starts to be expressed specifically during the transition from progenitors to postmitotic neurons and is required for proper neurogenesis of the mouse cerebral cortex. Bcl6 promotes this neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. Bcl6 triggers exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD+-dependent deacetylase Sirt1, which we showed to be required for Bcl6-dependent neurogenesis in vitro. The resulting epigenetic silencing of Hes5 leads to neuronal differentiation despite active Notch signalling. These findings thus suggest a role for Bcl6 as a novel proneurogenic factor and uncover Notch-Bcl6-Sirt1 interactions that may affect other aspects of physiology and disease (Tiberi et al. 2012a). <p><p>A subsequent yet unpublished part of this Phd-project focused on unraveling roles for Bcl6 in regionalization of the cerebral cortex. In all mammals, the three major areas of the neocortex are the motor, somatosensory and visual areas, each subdivided in secondary domains and complemented with species-specific additional areas. All these domains comprise of neurons with different functionality, molecular profiles, electrical activity and connectivity. Spatial patterning of the cortex is mainly under the control of diffusible molecules produced by organizing centers, but is also regulated by intrinsic, cell-autonomous programs (Tiberi et al. 2012b). <p>Since Bcl6 expression is confined to frontal and parietal regions of the developing cerebral cortex and remains high in postmitotic neurons, also after completion of neurogenesis, we hypothesized it would be involved in acquisition of motor and somatosensory identity. As expected from the neurogenesis defect in these regions, we observed a trend towards a reduced size of the frontal areas in the Bcl6 mutant cortex. Preliminary data from cDNA microarray profiling after gain- and loss-of-function of Bcl6 and from in situ hybridization on mouse cortex however do not show dramatic changes in molecular markers of different cortical areas. Similarly, the coarse-grained pattern of thalamocortical and efferent projections of motor and somatosensory neurons appears to be spared. These preliminary findings thus suggest that Bcl6 is not strictly required for proper acquisition of motor and somatosensory areal identity. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished