DNA methylation is one of the principal epigenetic modifications that plays an essential role in transcriptional regulation. After fertilization, mammalian embryos undergo dynamic changes in genome-wide DNA methylation patterns and the changes are essential for normal embryo development. Ten-eleven translocation (TET) methylcytosine dioxygenases are implicated in DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The three members of TET protein family, TET1, TET2, and TET3, are highly expressed in preimplantation embryos in a stage-specific manner. Previous studies demonstrated that TET proteins are involved in diverse biological processes such as gene regulation, pluripotency maintenance, and cell differentiation by mediating 5mC oxidation. My dissertation research was conducted to elucidate the mechanistic roles of TET proteins in epigenetic reprogramming of mammalian embryos using porcine embryos as a model. The first set of studies focused on the relationship between TET proteins and pluripotency. To understand the role of TET proteins in establishing pluripotency in preimplantation embryos, CRISPR/Cas9 technology and TET-specific inhibitors were applied. TET1 depletion unexpectedly resulted in an increased expression of NANOG and ESRRB genes in blastocysts, although the DNA methylation levels of NANOG promoter were not changed. Interestingly, transcript abundance of TET3 was increased in blastocysts carrying inactivated TET1, which might have had an effect on the increase of NANOG and ESRRB. When the activity of TET enzymes was inhibited to eliminate the compensatory increase of TET3 under the absence of functional TET1, the expression levels of NANOG and ESRRB were decreased and methylation level of NANOG promoter was increased. In addition, ICM specification was impaired by the inhibition of TET enzymes. These results suggest that the TET family is a critical component of the pluripotency network of porcine embryos by regulating expression of genes involved in pluripotency and early lineage specification. In the next set of studies, the presence of TET3 isoforms in porcine oocytes and cumulus cells was investigated to dissect the gene structure of TET3 that could assist in understanding mechanistic actions of TET3 in the DNA demethylation process. Among the three TET3 isoforms identified in cumulus cells, only the pTET3L isoform, which contains CXXC domain that carry DNA binding property, was verified in mature porcine oocytes. Expression level of the pTET3L isoform was much higher in mature oocytes compared to that in somatic cells and tissues. In addition, the transcript level of this isoform was significantly increased during oocyte maturation. These results suggest that pTET3L isoform is predominantly present in mature porcine oocytes and that CXXC domain may play an important role in DNA demethylation in zygotes. In a follow-up study, the role of the TET3 CXXC domain in controlling post-fertilization demethylation in porcine embryos was investigated by injecting TET3 GFP-CXXC into mature porcine oocytes. The injected CXXC was exclusively localized in the pronuclei, indicating that the CXXC domain may localize TET3 to the nucleus. The CXXC overexpression reduced the 5mC level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, the transcript abundance of NANOG and ESRRB was increased in blastocysts derived from GFP-CXXC overexpressing zygotes. These results provide an evidence that the CXXC domain of TET3 is critical for post-fertilization demethylation of porcine embryos and proper expression of pluripotency related genes in blastocysts. In the last set of studies, the impact of MBD proteins on porcine embryo development was examined under the hypothesis that competitive binding of MBD and TET proteins to 5mC contributes to the proper maintenance of DNA methylation levels in embryos. Cloning of porcine MBD1, MBD3, and MBD4 from mature oocytes indicates that the genes are highly conserved among different species, implying the involvement of porcine MBD proteins in the maintenance of DNA methylation. MBD1 overexpression in oocytes impaired preimplantation development of porcine embryos, suggesting that the MBD1 overexpression may have negatively affected porcine embryo development because proper DNA methylation levels were not preserved under the high level of MBD1. Collectively, the studies in my dissertation demonstrate that TET family proteins are important epigenetic players involved in the regulation of pluripotency and reprogramming of DNA methylation, and are thus crucial for normal embryo development. The findings in the dissertation will improve our understanding of epigenetic events occurring in mammalian embryos, and have the potential to overcome epigenetic defects that are observed in pluripotent stem cells and in-vitro derived embryos. / Doctor of Philosophy / Epigenetic modifications are heritable changes affecting the level of gene expression without changing the sequence of the genome. DNA methylation, one of the biggest epigenetic marks in mammalian genome, is often correlated to gene repression. In mammals, DNA methylation patterns are dramatically changed during preimplantation development to acquire embryonic developmental potential. Understanding of the epigenetic changes occurring in preimplantation embryos is necessary for producing healthy domestic animals in agriculture and for developing strategies for the treatment of epigenetic defects in human. Ten-eleven translocation (TET) family enzymes, TET1, TET2, and TET3, are known to function as a DNA methylation modifier by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). My dissertation research was performed to elucidate the role of TET family during preimplantation development using porcine embryos as a model. Pluripotency refers to the ability of cells to differentiate into all cell types of a mature organism. Pluripotent cells emerge in embryos as embryonic cells acquire lineage-specific characteristics. The first set of studies focused on the role of TET enzymes in regulating the pluripotency of porcine embryos. The impacts of inhibited activities of TET enzymes on the expression of pluripotency related genes were examined. We found that the inhibition of all TET enzymes leads to a decreased expression of pluripotency related genes, an altered DNA methylation level on a gene segment controlling pluripotency, and the impaired formation of pluripotent cell lineage in porcine embryos. This study demonstrates that the TET family is critical for the acquisition of pluripotency in porcine embryos. In the following sets of studies, the function of TET3 protein in the demethylation process occurring in preimplantation embryos was investigated. Fertilized mammalian embryos undergo genome-wide demethylation process to reset germ cell specific epigenetic marks into the embryonic epigenome. Previous studies indicate that TET3 is responsible for the demethylation process in mammalian embryos, although detailed mechanistic action of TET3 is still elusive. Here, we identified a predominant expression of a specific TET3 gene in porcine oocytes. The TET3 gene contained a CXXC domain, a potential DNA binding module, suggesting that TET3 may mediate DNA demethylation through its DNA binding property. To examine the function of the CXXC domain in TET3-mediated DNA demethylation, isolated CXXC domain was injected into porcine oocytes. The injection of CXXC domain facilitated DNA demethylation in embryos, demonstrating that the DNA binding property of TET3 is important for its functionality. In the last study, we investigated the importance of genes known to interact with TET enzymes in porcine embryos. Methyl-CpG-binding domain proteins (MBDs) have the ability to bind methylated region on the genome and play a critical role in mediating DNA methylation and gene repression. Our hypothesis was that a competitive binding of MBD and TET proteins to methylated regions was critical for proper DNA methylation levels in embryos. We identified that porcine MBD sequences were very similar to other species in terms of gene structure, indicating that the genes could also possess gene repressing activity by competing with TET enzymes during porcine embryo development. Injection of MBD1 mRNA to oocytes impaired normal embryo development, suggesting that the injected MBD1 may have negatively affected early embryo development in pigs by disrupting the proper maintenance of DNA methylation levels. My dissertation researches demonstrate that maintaining proper DNA methylation levels through the TET family is critical for normal embryo development in pigs. This research assists in improving our understating of epigenetic dynamics occurring in mammalian embryos and offers a potential solution to the epigenetic defects frequently observed in mammalian embryos produced through artificial reproductive technologies and pluripotent stem cells reprogrammed from somatic cells.

A miosina Va é uma proteína motora envolvida no transporte e posicionamento de vesículas, organelas e mRNA. Além disso, postulou-se que a miosina-Va atua no seqüestro do fator pró-apoptótico, Bmf, no citoesqueleto de actina. Pesquisas realizadas em nosso laboratório demonstraram que um fragmento da miosina Va (MVaf), que corresponde ao sítio ligante de DLC2-Bmf, é capaz de induzir intensa apoptose em células de melanoma e de carcinoma in vitro. O presente trabalho teve por objetivo principal avaliar o potencial do MVaf como agente antitumoral, através de abordagens de terapia gênica em modelo animal. Foram geradas linhagens estabilizadas e com expressão controlada pelo sistema Tet-ON onde a expressão de EGFP ou EGFP-MVaf é induzida com a adição de doxiciclina. Essas linhagens foram testadas quanto à porcentagem de morte por apoptose e ativação de caspases. Tumores foram induzidos em camundongos C57BL/6 por inoculação subcutânea de células tumorigênicas positivas ou não para a expressão de EGFP-MVaf. Também foram utilizadas linhagens de fibroblasto embrionário murino selvagem (MEFs WT) e nocautes para os fatores Bim/Bmf e Bax/Bak (MEFsBim-/-,Bmf-/-; MEFsBax-/-,Bak-/-) para estudos do mecanismo de ação do fragmento da miosina Va. Observou-se que a adição de butirato de sódio potencializa a expressão de EGFP-MVaf e, conseqüentemente, o efeito pró-apoptótico desse fragmento e que essas células são mais sensíveis aos quimioterápicos etoposídeo e taxol, apresentando maior susceptibilidade à apoptose. Verificou-se que a expressão de EGFP-MVaf em células de tumores de melanoma induzidos em camundongos C57BL/6J dificulta o crescimento desses tumores. Quanto ao estudo com MEFs, observou-se que células nocautes para os fatores pró-apoptóticos Bim/Bmf e Bax/Bak são menos susceptíveis à morte induzida pelo fragmento da miosina Va. Indução da expressão de MVaf desencadeia a liberação da proteína proapoptótica Smac (fusionada ao repórter fluorescente Cherry) do espaço intermembranas da mitocôndria para o citoplasma sugerindo que a morte apoptótica induzida por MVaf requer a permeabilização da membrana mitocondrial externa (MOMP). Concluindo, os dados apresentados aqui nos permitem propor o MVaf como uma molécula promissora para o desenvolvimento de novas abordagens terapêuticas contra o câncer. / Myosin Va is a motor protein involved in the transport and positioning of vesicles, organelles and mRNA. Additionally, myosin-Va has been implicated in the sequestering of a proapoptotic factor, Bmf, to the actin cytoskeleton. Research in our laboratory demonstrated that a fragment of myosin Va (MVaf), which corresponds to the binding site of DLC2-Bmf, is capable to induce intense apoptosis in melanoma and carcinoma cells in vitro. Here, our goal was to assess the potential of MVaf as antitumor agent, through gene therapy approaches in animal models. We generated Tet-ON controlled B16-F10 melanoma cells whose expression of EGFP or EGFP-MVaf is induced with the addition of doxycycline. These cells were tested for apoptotic death and activation of caspases, and were used to induce tumors in C57BL/6J mice by subcutaneous inoculation. We also used cell lines of murine embryonic fibroblasts, wild-type (MEFs WT) and knockouts for the proapoptotic proteins Bim/Bmf or Bax/Bak (MEFsBim-/-,Bmf-/-, MEFsBax-/-,Bak-/-), to study the mechanism by which MVaf induces apoptosis. We observed that addition of sodium butyrate to the cultures enhances the EGFP-MVaf expression and, consequently, the pro-apoptotic effect of this fragment. Treated cells were more sensitive to the chemotherapeutic drugs etoposide and taxol, showing a higher susceptibility to apoptosis. Moreover, in vivo induction of EGFP-MVaf expression retards growth of B16-F10 melanoma tumors in mouse model. As for the study with MEFs, we observed that cells knockout for the proapoptotic factors Bim/Bmf or Bax/Bak are less susceptible to death induced by MVaf than wild-type MEFs. Accordingly, we showed that MVaf expression triggers release of the proapoptotic protein Smac (tagged with the fluorescent protein Cherry) supporting the involvement of the mitochondrial outer membrane permeabilization (MOMP) in the MVaf-induced apoptotic death response. In conclusion, these data lead us to propose MVaf as a promising molecule for the development of new therapeutic approaches against cancer.

A miosina Va é uma proteína motora envolvida no transporte e posicionamento de vesículas, organelas e mRNA. Além disso, postulou-se que a miosina-Va atua no seqüestro do fator pró-apoptótico, Bmf, no citoesqueleto de actina. Pesquisas realizadas em nosso laboratório demonstraram que um fragmento da miosina Va (MVaf), que corresponde ao sítio ligante de DLC2-Bmf, é capaz de induzir intensa apoptose em células de melanoma e de carcinoma in vitro. O presente trabalho teve por objetivo principal avaliar o potencial do MVaf como agente antitumoral, através de abordagens de terapia gênica em modelo animal. Foram geradas linhagens estabilizadas e com expressão controlada pelo sistema Tet-ON onde a expressão de EGFP ou EGFP-MVaf é induzida com a adição de doxiciclina. Essas linhagens foram testadas quanto à porcentagem de morte por apoptose e ativação de caspases. Tumores foram induzidos em camundongos C57BL/6 por inoculação subcutânea de células tumorigênicas positivas ou não para a expressão de EGFP-MVaf. Também foram utilizadas linhagens de fibroblasto embrionário murino selvagem (MEFs WT) e nocautes para os fatores Bim/Bmf e Bax/Bak (MEFsBim-/-,Bmf-/-; MEFsBax-/-,Bak-/-) para estudos do mecanismo de ação do fragmento da miosina Va. Observou-se que a adição de butirato de sódio potencializa a expressão de EGFP-MVaf e, conseqüentemente, o efeito pró-apoptótico desse fragmento e que essas células são mais sensíveis aos quimioterápicos etoposídeo e taxol, apresentando maior susceptibilidade à apoptose. Verificou-se que a expressão de EGFP-MVaf em células de tumores de melanoma induzidos em camundongos C57BL/6J dificulta o crescimento desses tumores. Quanto ao estudo com MEFs, observou-se que células nocautes para os fatores pró-apoptóticos Bim/Bmf e Bax/Bak são menos susceptíveis à morte induzida pelo fragmento da miosina Va. Indução da expressão de MVaf desencadeia a liberação da proteína proapoptótica Smac (fusionada ao repórter fluorescente Cherry) do espaço intermembranas da mitocôndria para o citoplasma sugerindo que a morte apoptótica induzida por MVaf requer a permeabilização da membrana mitocondrial externa (MOMP). Concluindo, os dados apresentados aqui nos permitem propor o MVaf como uma molécula promissora para o desenvolvimento de novas abordagens terapêuticas contra o câncer. / Myosin Va is a motor protein involved in the transport and positioning of vesicles, organelles and mRNA. Additionally, myosin-Va has been implicated in the sequestering of a proapoptotic factor, Bmf, to the actin cytoskeleton. Research in our laboratory demonstrated that a fragment of myosin Va (MVaf), which corresponds to the binding site of DLC2-Bmf, is capable to induce intense apoptosis in melanoma and carcinoma cells in vitro. Here, our goal was to assess the potential of MVaf as antitumor agent, through gene therapy approaches in animal models. We generated Tet-ON controlled B16-F10 melanoma cells whose expression of EGFP or EGFP-MVaf is induced with the addition of doxycycline. These cells were tested for apoptotic death and activation of caspases, and were used to induce tumors in C57BL/6J mice by subcutaneous inoculation. We also used cell lines of murine embryonic fibroblasts, wild-type (MEFs WT) and knockouts for the proapoptotic proteins Bim/Bmf or Bax/Bak (MEFsBim-/-,Bmf-/-, MEFsBax-/-,Bak-/-), to study the mechanism by which MVaf induces apoptosis. We observed that addition of sodium butyrate to the cultures enhances the EGFP-MVaf expression and, consequently, the pro-apoptotic effect of this fragment. Treated cells were more sensitive to the chemotherapeutic drugs etoposide and taxol, showing a higher susceptibility to apoptosis. Moreover, in vivo induction of EGFP-MVaf expression retards growth of B16-F10 melanoma tumors in mouse model. As for the study with MEFs, we observed that cells knockout for the proapoptotic factors Bim/Bmf or Bax/Bak are less susceptible to death induced by MVaf than wild-type MEFs. Accordingly, we showed that MVaf expression triggers release of the proapoptotic protein Smac (tagged with the fluorescent protein Cherry) supporting the involvement of the mitochondrial outer membrane permeabilization (MOMP) in the MVaf-induced apoptotic death response. In conclusion, these data lead us to propose MVaf as a promising molecule for the development of new therapeutic approaches against cancer.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20195号 / 理博第4280号 / 新制||理||1615(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Les dérivés de la cytosine sont d’importantes modifications épigénétiques dont le rôle dans l’évolution de la leucémie lymphoïde chronique (LLC) n’est pas totalement exploré. Dans ce contexte, notre première étude vise à examiner le niveau global de la 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) et 5-hydroxymethyluridine (5-hmU) dans des lymphocytes B purifiés de patients LLC (n=56) et d’individus sains (n=17). Les principaux acteurs de la régulation épigénétique (DNMT1/3A/3B, MBD2/4, TET1/2/3, SAT1) ont été évalués par PCR quantitative en temps réel. L’analyse a permis de mettre en exergue trois groupes de patients. En premier lieu, un groupe de patients stables (délai médian de progression [PFS] et délai au premier traitement [TFT] >120 mois), avec un profil épigénétique similaire au groupe contrôle. Deuxièmement, un groupe intermédiaire (PFS=84; TFT=120 mois) qui présente une augmentation de la déméthylation de l’ADN expliquée par l'induction SAT1 / TET2 pendant la progression de la maladie. Troisièmement, un groupe de patients avec une forme active de la maladie (PFS=52; TFT=112 mois) qui présentent une hyperlymphocytose, une réduction du temps de doublement des lymphocytes et des modifications épigénétiques majeures. Au sein de ce groupe, une réduction est observée pour la 5-mCyt, 5-hmCyt, 5-CaCyt et serait associée à une diminution des DNMTs, TETs et MBDs au cours de la progression de la maladie. Les profils épigénétiques mis en évidence sont indépendants du statut mutationnel IGHV mais sont associés avec les anomalies cytogénétiques. Nous nous sommes également intéressés à cette association et nous avons montré dans la deuxième étude que les modifications des dérivées de la cytosine peuvent affiner le pouvoir pronostic des anomalies cytogénétiques. En conclusion, nos résultats suggèrent que les variations de la méthylation ainsi que des intermédiaires de la déméthylation de l’ADN sont impliqués dans la progression de la LLC. / Cytosine derivatives are important epigenetic modifications whose role in the pathogenesis and evolution of chronic lymphocytic leukemia (CLL) is not fully explored. In this context, our first study aims to examine the global DNA level of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluridine (5-hmU) in purified B lymphocytes of CLL patients (n = 56) and healthy individuals (n = 17). The main actors in epigenetic regulation (DNMT1 / 3A / 3B, MBD2 / 4, TET1 / 2/3, SAT1) were evaluated by quantitative real time PCR. The analysis highlighted three groups of patients. First, a group of patients with stable disease (median time to progression [PFS] and time to first treatment [TFT]> 120 months), with an epigenetic profile similar to the control group. Secondly, an intermediate group (PFS = 84, TFT = 120 months) which shows an increase in DNA demethylation explained by SAT1 / TET2 induction during disease progression. Third, a group of patients with an active form of the disease (PFS = 52, TFT = 112 months) who have hyperlymphocytosis, a short lymphocyte doubling time, and major epigenetic changes. Within this group, a reduction is observed for 5-mCyt, 5-hmCyt, 5-CaCyt which is associated with a decrease in DNMTs, TETs and MBDs during disease progression. The identified epigenetic profiles are independent of IGHV mutational status but are associated with cytogenetic abnormalities. We were also interested in this association and we showed in the second study that modifications of cytosine derivatives levels can refine the prognostic power of cytogenetic abnormalities.In conclusion, our results suggest that methylation variations as well as DNA demethylation intermediates are involved in the progression of CLL.

La protéolyse est une fonction clé de la cellule pour le maintien de l'intégrité du protéome, pour le métabolisme et pour la régulation de nombreux processus physiologiques. Le travail présenté dans cette thèse porte sur une famille de complexes peptidases cytosoliques auto-compartimentés et énergie indépendants découverts chez les Archées, les aminopeptidases TET. Chez l'Archée hyperthermophile Pyrococcus horikoshii, organisme modèle de cette étude, il existe 3 peptidases TET présentant chacune des spécificités de substrats différentes. Les caractérisations structurales des différents membres connus de cette famille de peptidases ont révélé un assemblage dodécamériques creux en forme de tétraèdre d'environ 450 kDa. Des études récentes ont montré l'existence de complexes adoptant la même conformation que les TET dans les 3 domaines du vivant. La première partie du travail présenté a permis d'identifier des marqueurs structuraux caractéristiques de l'assemblage tétraédrique afin de déterminer sans ambiguïté l'appartenance de ces complexes à la famille des TET. La seconde partie de l'étude a conduit à élucider la question de la multiplicité des TET chez les Archées hyperthermophile mise en évidence grâce à une étude phylogénétique initiée pendant la thèse. L'étude en co-expression de PhTET2 et PhTET3 révèle que ces aminopeptidases sont capable de former un hétéro-oligomère présentant une activité enzymatique accrue vis-à-vis des homo-oligomères. La dernière partie du travail porte sur les relations oligomérisation-fonction chez les peptidases TET. L'étude d'un mutant de l'oligomérisation de PhTET2 via une stratégie intégrative alliant biochimie, enzymologie, biophysique (SAXS et AUC) et des études in vivo a permis de mettre en évidence un processus d'assemblage contrôlé permettant d'augmenter l'efficacité de la peptidase. Enfin, la méthode de variation de contraste en diffusion de neutrons aux petits angles (SANS) appliqué à l'étude de l'hétéro-oligomère a permis de révéler une topologie rationalise du complexe hétéro-oligomérique favorisant la formations de poches multi-catalytique. L'ensemble de ce travail contribue à mieux comprendre l'importance et le rôle physiologique des machines TETs dans les cellules. / Proteolysis is a key function in the cell for the maintenance of the proteome integrity, the metabolism and for the regulation of many physiological processes. The thesis work is focused on a family of self-compartmentalized energy-independent cytosolic peptidases discovered in Archaea, the TET aminopeptidases. Three different TET showing contrasted enzymatic specificities co-exist in the cytosol of the hyperthermophilic archaeon Pyrococcus horikoshii, which is the model organism for this study. The structural characterization of the known members of this family shows that they self-assemble in a unique 450 kDa hollow tetrahedral structure . Recent studies have revealed the existence of peptidases complexes that adopt the same conformation in the three domains of life. The first part of this work allowed identifying structural markers to assign without any ambiguity uncharacterized peptidases to the TET family. The second objective of the work was to understand the multiplicity of TET peptidases in hyperthermophilic archaeon that was highlighted by a phylogenomic study presented in this work . The co-expression of PhTET2 and PhTET3 in E. coli revealed that the two proteins form a hetero-oligomeric complex with enhanced enzymatic activity compared to the homo-oligomers. The last part of the work addressed the question of oligomerization-function relationship in TET particles. A mutagenesis strategy was used to slow down the oligomerization process of PhTET2, and, using an integrative strategy combining biochemistry, enzymology, biophysics (SAXS and AUC) and in vivo studies we were able to dissect the oligomerization pathway of the TET particles and to demonstrate that it is a highly controlled process aim to enhance the activity of the peptidases. Finally, the contrast variation technique in small angle neutron scattering studies (SANS) allowed us to unravel the rational topology of the TET hetero-oligomers that favored the formation of multi-catalytic enzymatic pockets in the complex. All theses studies contributed to specify the biological importance of the TET molecular machines in the cells.

Dans ce mémoire de thèse sont décrits nos efforts synthétiques qui ont conduits à la première synthèse totale de deux produits naturels isolés d’éponges marines du genre Plakortis. Deux approches synthétiques des plakortolides ont été successivement étudiées pour finalement aboutir à la synthèse de la plakortolide I qui comporte un cycle endoperoxide à 6 chainons (1,2-dioxane). La première approche qui est une extension d’une méthode développée au laboratoire consistait à créer le cycle 1,2-dioxane par une ouverture intramoléculaire d’un époxyde vinylique par un groupement hydroperoxyde en β de l’époxyde. Dans un premier temps, nous nous sommes intéressés à la préparation d’un intermédiaire de synthèse, un alkoxyméthylhexa-2,5-dien-1-ol. Nous avons aussi tenté de créer le cycle 1,2-dioxane par une double ouverture d’un di époxyde 1,5 par de l’eau oxygénée. Nous avons ensuite modifié notre stratégie de synthèse en introduisant au début de la synthèse la chaine latérale de la plakortolide I en partant de la R-épichlorhydrine commerciale. Nous avons ainsi synthétisé le β-hydroperoxy vinyl époxyde trisubstitué, précurseur du cycle 1,2-dioxane. Lors de cette synthèse, nous avons mis au point une méthode efficace et chimiosélective de méthylenation d’une cétone en présence d’un ester utilisant le réactif de Nysted catalysé par Ti(OiPr)2Cl2. La seconde approche du système bicyclique peroxylactone fait appel à une addition de Michael intramoléculaire d’un hydroperoxyde sur la double liaison d’un buténolide. Cette voie fut couronnée de succès car la (-)-ent-plakortolide I et la seco-plakortolide E ont été synthétisées / In this thesis manuscript are described our synthetic efforts and the first total synthesis of two natural products isolated from the sponges of the genus Plakortis. In total, two different synthetic approaches were studied to finally accomplish the synthesis of plakortolide I. The first approach is an extension of the method developed by our group which consists in the creation of the 1,2-dioxane cycle by intramolecular opening of vinyl epoxide with β-hydroperoxy group. Firstly, we was interested in the preparation of alkoxymethylhexa-2,5-dien-1-ol. We have also tried to create the 1,2-dioxane cycle by double opening of bis-1,5-epoxide with hydrogen peroxide. Further more we have synthesised trisubstituted β-hydroperoxy vinyl epoxide, precursor of 1,2-dioxan ring, from R-epichlorohydrin. During this synthesis a procedure of chemoselective methylenation of ketone in the presence of epoxide by Nysted reagent and Ti(OiPr)2Cl2 was developed. Finally, (-)-ent-plakortolide I and seco-plakortolide E were synthesised by intramolecular Michael addition of hydroperoxide to double bond of the butenolide moiety

No description available.

Les systèmes moléculaires décrits chez les Archaea mettent en évidence un caractère primitif et une simplicité, comparativement à leurs homologues eucaryotes. Par ailleurs, leur caractère extrêmophile a pour conséquence une hyper-robustesse qui rend leur manipulation in vitro et les études structurales beaucoup plus aisées. Ainsi les Archaea représentent de bons modèles pour comprendre les fonctions cellulaires complexes, particulièrement celles qui mettent en jeu de grandes machineries moléculaires, comme celles impliquées dans la protéolyse. Mon travail de thèse a consisté à comprendre les mécanismes de résistance et l'importance des systèmes de protéolyse dans l'adaptation des Archaea halophiles aux stress environnementaux. Les Archaea halophiles accumulent des concentrations multi-molaires de KCl/NaCl dans leur cytosol (3.4M KCl / 1.1M NaCl chez Halobacterium salinarum). Ainsi, les protéines de ces organismes sont elles-mêmes halophiles et ne sont solubles et repliées que dans des conditions de salinité extrêmes (de 2 à 5M).Nous avons étudié la réponse de l'Archaea halophile stricte H. salinarum à des stress, dont les stress à basse salinité, en se focalisant en particulier sur les modifications de la dynamique moléculaire du protéome in vivo (diffusion neutronique). Au cours de ce travail, il a été mis en évidence un phénomène de survie à la basse salinité associé à des modifications morphologiques.Un autre objectif de ma thèse a été de contribuer à la compréhension du rôle dans la protéolyse intracellulaire du complexe aminopeptidasique TET, dans les conditions de stress mises en place dans les études précédentes. / Molecular systems described for Archaea show primitive and simple characteristics, compared to their homologous eukaryotes. In addition, extremophilic characteristic results in an hyper-robust which makes in vitro manipulation and structural studies much easier. Thus, Archaea represent good models for understanding complex cellular functions, particularly those that involve large molecular machines, such as those involved in proteolysis. My thesis consisted in understanding the resistance mechanisms and the importance of proteolytic systems in the adaptation of halophilic Archaea to environmental stresses. Halophilic Archaea accumulate multi-molar concentrations of KCl / NaCl in their cytosol (3.4M KCl / NaCl 1.1M for Halobacterium Salinarum). This requires a very special biochemistry that allows operation in solvents where free water is scarce. Thus, the proteins of these organisms are themselves halophilic and are soluble and folded only in extreme salinity conditions (2 to 5 M). This particular biochemistry partly explain the extraordinary ability of halophilic Archaea to resist physical and chemical stress (temperature, radiation, dehydration). We study the response of the halophilic Archaea strict H. salinarum at low-salinity stress. Indeed, beyond the osmotic shock, the fall of the environment salt concentration causes a decrease in the intracellular KCl concentration, which should have a direct effect on the folding state of intracellular protein, as in case of heat stress. In the first part of this thesis, a study was conduct to determine viability limits and cytosolic modifications, associated with a salinity decrease. These studies involve intracellular salt dosages, viability studies (microscopic counts, color live / dead), induction of chaperone proteins linked to stress response and biophysical neutron experiments, to evaluate the effect of stress on proteins folding. In this work, a phenomenon of survival at low salinity linked to morphological changes was revealed. To describe this phenomenon, this second study involves confocal microscopy experiences, fluorescence microscopy, viability tests, counting on box, scanning electron microscopy, electron microscopy by negative staining, salt intracellular dosages and proteins separation experiments, to study the overall proteome composition during low salinity stress. In this study, a fall of the intracellular K $^+$ concentration and the proteome clarification during stress was revealed. Low salt concentrations causes halophiles proteins denaturation, the accumulation of misfolded proteins in the cytoplasm involves chaperones systems and intracellular proteolysis machinery. In this context, another objective of my thesis was to contribute to the understanding of the intracellular proteolysis role in the PAN-proteasome system and in the aminopeptidase TET complex, in stress conditions established in previous studies. This part of the thesis involves experiments of endopeptidase activity assay, aminopeptidase activity assay, quantification of mRNA genic expression by Northern blot, immunoprecipitation, proteins separation by sucrose gradient and proteasome chemical inhibition (drug). We show the role of the PAN-proteasome system in stress response and we deepen our understanding of the aminopeptidase TET role in vivo. This protease appears to have an independent role of the proteasome complex. The protease TET seems to participate at the amino acids treatment in cells to maintain the metabolic activities in nutritional deficiencies.