200 000 nouveaux cas de mélanome et 65 000 décès sont dus à ce cancer de la peau chaque année. Le mélanome représente donc un véritable problème de santé publique. Actuellement, plus de 50 % des patients sont toujours en échec thérapeutique malgré le développement des thérapies ciblées et des immunothérapies, et il est donc nécessaire de créer de nouvelles thérapies contre ce cancer.Dans notre laboratoire, nous avons montré que la Metformine, un antidiabétique, induit la mort des cellules de mélanomes par autophagie et apoptose. De plus, ce composé inhibe les capacités invasives de ces cellules. Un essai clinique a été effectué au CHU de Nice sur l’effet de la Metformine sur des mélanomes métastatiques mais les résultats ont été plutôt décevants. De ce fait, et en collaboration avec l’institut de chimie de Nice, notre équipe a développé des composés dérivés de la Metformine et a identifié le CRO15. Durant ma thèse, j’ai d’abord déterminé que CRO15 possédait diminuait la viabilité des cellules de mélanomes par différents mécanismes moléculaires. Tout d’abord, l’activation de la voie AMPK, comme ce qui est observée avec la Metformine, mais également l’inhibition de la kinase MELK, un oncogène très souvent surexprimé dans le cadre du mélanome. L’inhibition de MELK par CRO15 est responsable de l’activation de la voie p53. Ces deux voies, AMPK et p53, augmentent l’expression de REDD1 qui active alors les processus d’autophagie et d’apoptose pour induire la mort des cellules de mélanomes. Finalement, j’ai pu montrer que le CRO15 est également capable d’induire une diminution de la croissance tumorale de mélanomes dans différents modèles de souris. / Every year, 200 000 new cases of melanoma and 65 000 deaths occur as a result of skin cancer, making melanoma a real public health problem. Currently, more than 50 % of patients fail treatment despite new targeted therapies and immunotherapies being developed. As a result, there is an urgent need to develop new anti-melanoma compounds to treat this aggressive disease. In our laboratory, we shown Metformin, an antidiabetic, to induce melanoma cell death by autophagy and apoptosis. Furthermore, this compounds inhibits invasive capability of these cells. A clinical trial has been performed in collaboration with the Nice hospital looking at Metformin’s effects on metastatic melanoma treatment, however these results have been disappointing. In this context, and in collaboration with the chemical institute of Nice, our team developed new Metformin-derived compounds and identified CRO15 as a promising new lead. During my PhD, I determined that CRO15 decreases melanoma cells viability by different molecular mechanisms. First, just like Metformin, CRO15 activates AMPK pathway, however it also inhibits MELK kinase activity, a protein described as an oncogene in melanoma. This inhibition is responsible for p53 activation. The two pathways, AMPK and p53, lead to increased REDD1 expression which involves autophagy and apoptosis to induce melanoma cell death. Finally, I shown that CRO15 decreases melanoma tumoral volume in different mice models. These promising results highlight a novel compound that can now be investigated for its potential use in clinic.

Les organophosphorés (OPs) sont des composés neurotoxiques qui sont largement utilisés comme pesticides. Cette utilisation intensive a conduit à une importante pollution des sols et des effluents agricoles et sont retrouvés jusque dans les aliments. Ces pesticides sont responsables de 300 000 morts à travers le monde. Les OPs ont également été développés comme agents neurotoxiques de guerre tel que le sarin. Actuellement, il n’existe pas de méthode de décontamination externe satisfaisante pour dégrader les OPs, c’est pourquoi l’utilisation d’enzymes est une stratégie attractive. Parmi les enzymes capables de dégrader les OPs, les phosphotriestérases (PTEs) sont les plus actives mais sont peu stables ce qui limite leurs applications. Les enzymes hyperthermostables ont donc été considérées. Ainsi l’enzyme SsoPox, isolée de l’archée Sulfolobus solfataricus ayant une activité lactonase et une activité de promiscuité phosphotriestérase, a été plus particulièrement étudiée. SsoPox est extrêmement robuste mais son activité phosphotriestérase est en revanche plus faible. Une stratégie d’ingénierie protéique a été réalisée afin d’obtenir un compromis entre l’activité d'une PTE et la stabilité de SsoPox. En utilisant les similarités structurales entre ces deux enzymes, une base de données mutationnelle a été réalisée pour transférer le site actif hautement performant d’une PTE dans la structure hyperstable de SsoPox. Cette stratégie a permis d’obtenir des variants de SsoPox améliorés jusqu’à 2000 fois. L'efficacité de ces variants a été démontrée in vivo chez un modèle animal, la planaire, permettant d'améliorer la survie ainsi que la mobilité et la capacité de régénération. / Organophosphates (OPs) are neurotoxic compounds widely used as pesticides. Over the years, utilization of OP led to a considerable environmental contamination of soils and agricultural wastewaters, this pollution is furthermore a major health issue as these insecticides can be found in food. OP are highly toxic and are responsible for 300,000 deaths in the world every year. OPs were also developed as chemical warfare nerve agents such as sarin. Currently, no satisfying method for external decontamination is available, therefore bioremediation with enzymes is highly appealing. Among OP degrading enzymes, phosphotriesterases (PTEs) are the most active biocatalysts but are poorly stable what hinders their potential for bioremediation. Hyperthermostable enzymes from extreme environments were thus considered to circumvent this limitation. In particular, SsoPox isolated from the archaeon Sulfolobus solfataricus, displaying a lactonase activity and a promiscuous phosphotriesterase activity was deeply investigated. SsoPox is extremely robust but its activity for OP degradation is from far lower. A protein engineering strategy was started in order to reach a compromise between PTE activity and SsoPox robustness. Using structural similarities between PTEs and SsoPox, a mutational database was designed in order to transfer the highly performant active site of PTE into the hyperstable scaffold of SsoPox. This strategy led to variants displaying up to 2,000-fold increase against OPs as compared to wild-type enzyme. The variants efficiency was demonstrated in vivo using an original animal model planarian, allowed to enhance survival rate as well as mobility and regeneration capacity.

La légumineuse Medicago établie une interaction symbiotique avec des bactéries du sol fixatrices d’azote, les rhizobia. Cette interaction provoque la formation d’un nouvel organe racinaire, la nodosité, au sein de laquelle les bactéries infectent de manière massive et chronique les cellules de la plante. Malgré cette invasion, aucune réaction de défense n’est observée ce qui suggère l’existence de mécanismes symbiotiques locaux de contrôle de l’immunité. Les gènes de Medicago DNF2 et SymCRK codant une phospholipase C-like et un récepteur-like kinase riche en cystéines, semblaient intervenir dans ces mécanismes peu connus. Mon travail de thèse a consisté à mieux caractériser les mécanismes de tolérance aux rhizobia notamment ceux faisant intervenir ces deux gènes. Nos résultats indiquent que dnf2 et symCRK forment des nodosités non-fixatrices, nécrotiques, présentant une activation des défenses et une perte de viabilité des bactéroïdes (forme intracellulaire des bactéries). Par ailleurs, l’utilisation de mutants bactériens nous a permis de montrer que, chez la plante sauvage, la perte de viabilité des bactéroïdes et l’absence de fixation d’azote ne sont pas suffisantes pour stimuler les défenses. Nos résultats indiquent également que dnf2 et symCRK agissent successivement lors du processus symbiotique et que la nécessité de dnf2 pour l’établissement de la symbiose peut être contournée dans certaines conditions de culture. Enfin, nous avons réalisé une analyse du protéome de symCRK et des expériences de physiologie végétale qui ont mis en évidence la nécessité, pour le maintien d’une symbiose efficace, de réprimer la voie éthylène après internalisation des rhizobia dans les cellules végétales. Ensemble, nos données améliorent la compréhension du phénomène de tolérance observée dans les nodosités de Légumineuses. / The legume plant Medicago establishes symbiotic interaction with nitrogen fixing bacteria, called rhizobia. This interaction leads to the formation of root organs, the nodules. A massive and chronic infection of nodule cells is observed without induction of any plant defense suggesting that a symbiotic mechanism controls immunity in the nodules. The two Medicago genes, DNF2 and SymCRK encoding a phospholipase C-like protein and a cysteine-rich receptor-like kinase respectively were identified as potentially involved in the prevention of defenses during the late steps of the symbiosis. However, this phenomenon was poorly characterized. Herein we improved the characterization of the Legume tolerance to intracellular rhizobia with an emphasis on the role of DNF2 and SymCRK. Our results indicate that dnf2 and symCRK produce necrotic nodules that do not fix nitrogen, that develop defenses and in which bacteroids, the intracellular form of rhizobia, rapidly loose viability. Using bacterial mutants, we show that reduced bacteroid viability and/or nitrogen fixation defect are not per se enough to trigger defenses in wild type plants. Our results also indicate that DNF2 and SymCRK act successively during the symbiotic process and that artificial culture conditions can bypass DNF2 requirement for symbiosis. Finally, symCRK proteome analysis and physiological studies together indicate that the ethylene pathway has to be repressed after rhizobia internalization within the plant cells to maintain efficient symbiosis. Together our data improve the knowledge on the basis of legume tolerance to rhizobia.

Hematopoiesis is a vital process in which red blood cells and cells of the immune system are formed. It is initiated during early embryonic development when we find hematopoietic progenitors in separate anatomical sites. Embryonic hematopoiesis comprises three successive and partly overlapping waves of progenitors with a different hematopoietic potential. The primary anatomical place where hematopoiesis takes place shortly before the birth is the bone marrow (BM). Since at this time point of development BM is already populated by hematopoietic stem cell (HSCs) progenitors, it becomes also the site of hematopoiesis in adulthood. However, the bone marrow is not the only place where hematopoietic progenitors emerge and develop. The Yolk sac (YS) and the Aorta-Gonad-Mesonephros (AGM) region are the initial sites of the appearance of the three waves of progenitors in the early embryogenesis. These progenitors and their descendants play an indispensable role during the development of an individual. Because there are no specific markers that would unambiguously characterize progenitors of these individual waves, their physical separation and hence also functional characterization is still incomplete. Recent studies have shown that Toll-like receptors (TLRs) are expressed on adult HSCs. The stimulation of...

Introduction: Diabetes mellitus is a polygene disease and on its manifestation have influence also enviromental factors. We have studied the role of extrapituitary prolactin (PRL) and toll-like receptors (TLR) 2 and 4 in the etiopathogenesis of autoimmune diabetes. PRL is mainly produced by hypophysis, but in small concentrations also in the periphery, where it participates in the immune reactions. Therefore, we investigated the influence of the levels of monocytic PRL mRNA on the development of diabetes, and also the influence of G allele of the -1149 G/T polymorphism in the extrapituitary promotor, which has already been associated with other autoimmune diseases. TLRs are receptors of the immune cells that recognize patogenes entering into the body. They play an important role in the iniciation of the immune response. We aimed to find out their function in the pathogenesis of the autoimmune diabetes by the detection of their mRNA levels and protein levels expressed on the cell surface of the monocytes. Material and methods: In this study we included 30 T1D and 21 LADA patients. Three control groups consisted of 23 T2D patients, 23 patients with a nondiabetic disease (neDM) and 60 healthy blood donors (TO). Blood samples have been taken from the individuals. From these blood samples we isolated...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20795号 / 医博第4295号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 原田 浩, 教授 松原 和夫, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Development of the mammary gland is likely mediated by locally produced growth factors acting in concert with circulating mitogens. To investigate the importance of mammary synthesis of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBP), the initial objective was to evaluate the physiological effects of recombinant IGF-I synthesis in the mouse mammary gland. Expression of recombinant IGF-I was targeted by the mouse mammary tumor virus - long terminal repeat (MMTV-LTR) to the mammary glands of two lines (15 and 29) of transgenic mice. Mammary synthesis of recombinant IGF-I increased the frequency of appearance of mammary alveolar buds (71% vs. 21%) in transgenic compared with non-transgenic CD-1 mice. During lactation, mammary synthesis of recombinant IGF-I reduced the amount of endogenous native IGF-I secreted into milk of transgenic mice. Regardless of transgenesis, a shift in the milk IGFBP profile from predominantly IGFBP-3 to a lower molecular weight IGFBP occurred between d 8 and d 12 of lactation. The altered composition of milk from transgenic line 29 dams reduced by 27% the average daily gain of suckling litters, compared with CD-1 dams. Moreover, mammary glands of transgenic mice were less regressed after weaning than controls and were characterized by the presence of more organized secretory lobules. The second overall objective was to evaluate the regulation and physiological effects of mammary IGF-I and IGFBP synthesis in prepubertal heifers. Serum and extracts of mammary tissue at 5% concentration in media stimulated DNA synthesis 545% and 28%, respectively, in primary mammary epithelial organoids in collagen gel culture. Addition of IGFBP-3 strongly inhibited this growth response. High feeding level tended to increase IGFBP-3 levels in mammary tissue and reduced by 30% the growth response to mammary tissue extracts. Somatotropin increased the mitogenic response to mammary extracts at high feeding level and increased the tissue content of IGF-I by 46%. In summary, local synthesis of IGF-I and IGFBP is influenced by feeding level and exogenous somatotropin and contributes substantially to effects on mammary cell proliferation. Interactions of locally produced IGFBP-3 with IGF-I and other growth factors appear to be especially important when mammary growth is modulated by feeding level. / Ph. D.

The primary aim of this text is to gain insight on how cellular activation by a insulin-like growth factor (IGF-I), in the presence of insulin-like growth factor binding protein-3 (IGFBP-3), is influenced by heparan sulfate proteoglycans (HSPG). Initial research will be presented, assumptions and hypotheses that were included in the development of mathematical models will be discussed, and the future enhancements of the models will be explored. There are many potential scenarios for how each component might influence the others. Mathematical modeling techniques will highlight the contributions made by numerous extracellular parameters on IGF-I cell surface binding. Tentative assumptions can be applied to modeling techniques and predictions may aid in the direction of future experiments. Experimentally, it was found that IGFBP-3 inhibited IGF-I Bovine Aortic Endothelial (BAE) cell surface binding while p9 HS slightly increased IGF-I BAE cell surface binding. IGFBP-3 has a higher binding affinity for IGF-I (3 x 10-9 M) than p9 HS has for IGF-I (1.5 x 10-8 M) as determined with cell-free binding assays. The presence of p9 HS countered the inhibiting effect of IGFBP-3 on IGF-I BAE cell surface binding. Although preliminary experiments with labeled p9 HS and IGFBP-3 indicated little to no cell surface binding, later experiments indicated that both IGFBP-3 and p9 HS do bind to the BAE cell surface. Pre-incubation of BAE cells with either IGFBP-3 or p9 HS resulted in an increase of IGF-I BAE cell surface binding . There was a more substantial increase of IGF-I surface binding when cells were pre-incubated with IGFBP- 3 than p9 HS. There was a larger increase of IGF-I BAE cell surface binding when cells were pre-incubated with p9 HS than when p9 HS and IGF-I were added simultaneously. This suggests that IGFBP-3 and p9 HS surface binding plays key role in IGF-I surface binding, however, p9 HS surface binding does not alter IGF-I surface binding as much as IGFBP-3 surface binding seems to. Experimental work helps further the understanding of IGF-I cellular activation as regulated by IGFBP-3 and p9 HS. Developing mathematical models allows the researcher to focus on individual elements in a complex systems and gain insight on how the real system will respond to individual changes. Discrepancies between the model results and the experimental data presented indicate that soluble receptor inhibition is not sufficient to account for experimental results. The alliance of engineering analysis and molecular biology helps to clarify significant principles relevant to the conveyance of growth factors into tissue. Awareness of the effects of individual parameters in the delivery system, made possible with mathematical models, will provide guidance and save time in the design of future therapeutics involving growth factors. / Master of Science