In the present study, we investigated the functional differences between cluster of differentiation (CD)14++CD16- and CD14+CD16+ monocytes during anti-Candida host defense. CD14++CD16- are the "classical" monocytes and represent the majority of circulating monocytes in humans, while CD14+CD16+ monocytes patrol the vasculature for maintenance of tissue integrity and repair. Both monocyte subsets inhibited the germination of live Candida albicans, and there was no difference in their capacity to phagocytose and kill Candida. Although production of IL-6 and IL-10 induced by C. albicans was found to be similar between monocyte subsets, IL-1β and prostaglandin E2 (PGE2) production was higher in CD14++CD16- compared with CD14+CD16+ monocytes. In line with the increased production of IL-1β and PGE2, central mediators for inducing Th17 responses, CD14++CD16- monocytes induced greater Th17 responses upon stimulation with heat-killed C. albicans yeast. The percentage of cells that expressed mannose receptor (MR) was higher in the CD14++CD16- monocyte subset, and MR-specific stimulation induced higher Th17 responses only in co-cultures of CD14++CD16- monocytes and CD4 lymphocytes. In conclusion, both monocyte subsets have potent innate antifungal properties, but only CD14++CD16- monocytes are capable of inducing a potent Th17 response to C. albicans, an important component of antifungal host defense.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18198号 / 人健博第15号 / 新制||人健||2(附属図書館) / 31056 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 齋藤 邦明, 教授 三谷 章, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

No description available.

Une nouvelle voie d’immunomodulation, l’induction de l’arginase par les trypanosomes chez leurs hôtes, a été identifiée et caractérisée. Pour éviter la réponse cytotoxique de l’activation « classique » M1 des macrophages et bénéficier de leur activation « alternative » M2, les parasites induisent l’arginase, qui produit la L-ornithine, indispensable à leur développement. Cette voie d’immunomodulation mise en évidence chez la souris infestée par son parasite naturel, Trypanosoma musculi, est également présente dans d’autres trypanosomoses, en particulier la trypanosomose humaine africaine (THA). Une augmentation de l’arginase, retrouvée dans le sérum de patients trypanosomés, se normalise après un traitement efficace. T. brucei gambiense, parasite de l’homme, induit l’arginase au niveau des macrophages murins et des leucocytes humains. T. lewisi, parasite du rat, induit également l’arginase. Au cours de leur longue coévolution avec leurs hôtes, les trypanosomes extracellulaires ont sélectionné un procédé favorisant leur croissance, l’induction de l’arginase, par des facteurs d’excrétion/sécrétion. Nous avons produit un anticorps monoclonal dirigé contre ce facteur inducteur. Il bloque l’induction de l’arginase par T. musculi in vitro et in vivo. Chez la souris infectée, son injection diminue considérablement la parasitémie. Il a permis l’identification du facteur inducteur, une kinésine orpheline. Cet anticorps, inhibant l’induction de l’arginase par différents trypanosomes, reconnaîtrait une région conservée de la kinésine induisant l’arginase. Cette kinésine se lie à des récepteurs de la membrane des macrophages. In vitro, l’addition de mannose à des co-cultures macrophages-parasites bloque l’induction de l’arginase et la multiplication des parasites. Chez la souris infestée par T. musculi, l’injection de mannose diminue la parasitémie, qui est également réduite chez les souris Mrc1-/-, KO pour le récepteur mannose. L’utilisation de molécules ciblant la voie inductrice de l’arginase et/ou ce récepteur peut représenter une nouvelle approche thérapeutique dans les trypanosomoses. / Arginase induction, a mechanism of immunomodulation elaborated by trypanosomes has been identified. To avoid cytotoxic classical M1 macrophage activation, trypanosomes induce alternative M2 macrophage activation, which leads to L-ornithine production, essential for parasite growth. This immunomodulation pathway has been evidenced in a natural murine trypanosomiasis provoked by Trypanosoma musculi. This mechanism is also evidenced in human African trypanosomiasis (HAT). An increase in serum arginase is measured in HAT patients. A return to normal values is obtained after an efficacious treatment. Trypanosoma brucei gambiense, the causative agent of HAT, induces arginase in mouse macrophages and human leucocytes. T. lewisi, a rat parasite, also induces macrophage arginase.During host-parasite co-evolution, extracellular trypanosomes have selected a growth promoting mechanism, macrophage arginase induction by excreted secreted factor (ESF). We have produced a monoclonal antibody which inhibits trypanosome-induced arginase. This antibody blocks in vitro and in vivo T. musculi-induced arginase. Its injection into infected mice provokes a decrease in parasite load. This monoclonal antibody has allowed the identification of an orphan kinesin as the arginase inducing factor. The arginase inducing region of kinesin seems conserved among extracellular trypanosomes. Kinesin binds to macrophage membrane receptors. In vitro, addition of mannose to macrophage-parasite cocultures blocks arginase induction and parasite multiplication. Mannose injection decreases parasite load in infected mice. Compared to WT mice, parasite load is highly reduced in infected Mrc1 -/- KO mice. In trypanosomiasis, molecules targeting arginase pathway and/or mannose receptor, highly conserved in evolution, might represent new therapeutic approaches.

Les maladies lysosomales forment un groupe hétérogène d’une cinquantaine d’affections qualifiées de « rares ». Actuellement, seulement 9 maladies lysosomales disposent d’un traitement spécifique, principalement par enzymothérapie substitutive, mais les effets bénéfiques sont souvent limités. Le manque de ciblage pour le Récepteur du Mannose 6-Phosphate (RM6P), responsable de l’internalisation dans les lysosomes, expliquerait en partie cette efficacité modérée des enzymes thérapeutiques. Dans ce contexte, nous avons développé une approche de ciblage innovante basée sur des Analogues synthétiques du Mannose 6-Phosphate fonctionnalisés sur l’Aglycone (appelés AMFA) afin de répondre aux besoins non satisfaits par les traitements actuels.Les travaux de cette thèse portent principalement sur la maladie de Pompe, myopathie causée par la déficience d’une enzyme lysosomale, l’Alpha Glucosidase Acide (GAA), responsable de la conversion du glycogène en glucose. Afin d’améliorer l’adressage de l’enzyme thérapeutique aux lysosomes via le RM6P, nous avons fonctionnalisé la GAA recombinante humaine (rhGAA) avec les AMFA. Nos études sur la forme adulte de la maladie ont démontré une augmentation significative de l’internalisation et pour la première fois, chez des souris âgées modèles de la maladie, une restauration de la santé musculaire et une amélioration significative de la fonction motrice ont été observées (article 1). Nous nous sommes ensuite intéressés aux propriétés de la rhGAA-AMFA. Nous avons démontré que l’efficacité de la rhGAA-AMFA n’était pas uniquement due à une meilleure internalisation mais également à une meilleure maturation intracellulaire de l’enzyme (article 2). En effet, nos résultats ont démontré que chez les patients atteints de la maladie de Pompe, il existe une surexpression des phosphatases acides ACP2 et ACP5. Ces phosphatases peuvent détruire le signal mannose 6-phosphate (M6P) naturellement présent sur l’enzyme, ce qui interrompt sa maturation en forme active. L’AMFA, contrairement au M6P, est insensible à cette dégradation et assure donc la stabilité de l’adressage de l’enzyme in vitro, mais également in vivo.L’ensemble de ces résultats suggèrent que le greffage des AMFA sur des enzymes recombinantes représente une nouvelle solution thérapeutique pour le traitement de la maladie de Pompe et potentiellement pour le traitement d’autres maladies lysosomales. / Lysosomal diseases form a heterogeneous group of about fifty rare diseases. At present, only 9 lysosomal diseases have a specific treatment, mainly by enzyme replacement therapy but the beneficial effects appear often limited. The lack of targeting for the Mannose 6-Phosphate Receptor (M6PR), responsible for internalization into the lysosomes, would partly explain this moderate efficiency of the therapeutic enzymes. In this context, we have developed an innovative targeting approach based on Mannose 6-Phosphate Synthetic Analogues Functionalized at the Aglycone position (called AMFAs) to address the unmet needs of current treatments.The work of this thesis focuses mainly on Pompe disease which is a myopathy caused by the deficiency of a lysosomal enzyme, Acid Alpha Glucosidase (GAA), responsible for the conversion of glycogen into glucose. In order to improve the targeting of the therapeutic enzyme to lysosomes via the M6PR, we have functionalized the human recombinant GAA (rhGAA) with the AMFAs. Our studies on aged mice model of the adult form of the disease have demonstrated a significant increase of the enzyme internalization and for the first time, the restoration of muscle health and the significant improvement in motor function (article 1). We then investigated the properties of rhGAA-AMFA. We have proved that the effectiveness of rhGAA-AMFA is not only due to a better cell uptake but also to a more complete intracellular processing of the enzyme (article 2). Indeed, our results demonstrated that in myoblasts of patients affected by Pompe disease there is an overexpression of ACP2 and ACP5 acid phosphatases. These phosphatases can destroy the mannose 6-phosphate signal (M6P) naturally present on the enzyme, therefore possibly interrupting its processing into the active form. AMFA, unlike M6P, is insensitive to this degradation and thus ensures the stability of enzyme addressing in vitro, but also in vivo.All together, these results suggest that the grafting of the AMFAs on recombinant enzymes represents a new therapeutic solution for the treatment of Pompe disease and potentially for other lysosomal diseases.

A Lectina Ligadora de Manose (MBL) é uma proteína que reconhece carboidratos na superfície microbiana levando à ativação do sistema complemento. Este processo é mediado por Serino Proteases tal como a MASP-2. O complexo MBL/MASP-2 é responsável pela formação da C3 convertase C4bC2b. Os níveis séricos de MBL e a MASP-2 (genes MBL2 e MASP-2, respectivamente) são geneticamente determinados, e podem ser influenciados pela presença de polimorfismos em um único nucleotídeo SNPs em genes codificadores destas proteínas. OBJETIVO: Determinar os níveis séricos e polimorfismos gênicos da MBL e MASP-2 em uma amostra da população brasileira. MÉTODOS: 294 amostras de doadores de sangue [mediana = 36,51 ± 10,56; 18-63 anos; 91/294 (30,95%) sexo feminino, 203/294 (69,05%) sexo masculino] foram genotipadas para os SNPs do éxon 1 (MBL2): SNPs localizados nos códons 52 (ArgCys), 54 (GlyAsp) e 57 (GlyGlu) e SNP Asp371Tyr (D371Y, A>C ) do gene da MASP-2 (éxon 9). Foi utilizado o ensaio de temperatura de dissociação para éxon 1 (MBL2) e sequenciamento direto dos promoters (H/L, X/Y e P/Q, nas posições -550, -221 e +4, respectivamente). A combinação das variantes do éxon 1 MBL2 foram agrupadas e denominadas alelo O e o genótipo selvagem foi denominado A. O éxon 9 da MASP-2 foi genotipado através da plataforma TaqMan. RESULTADOS: MBL2: 58,5% A/A, 36,39% A/O e 5,1% O/O; promoters: 13% H/H, 39% H/L, 48% L/L; 2% X/X, 26% X/Y, 72% Y/Y; 52% P/P, 37% P/Q, 11% Q/Q; haplótipos encontrados: 15% LXPA, 28% HYPA, 8% LYQO, 12% LYPO, 11% LYPA, 22% LYQA e 4% HYPO. Quanto à produção, 56,12% produziram altos níveis de MBL, 30,61% níveis médios e 13,27% níveis baixos ou insuficientes de MBL. Para MASP-2: 38,78% A/A, 44,56% A/C e 16,67% C/C. CONCLUSÃO: A prevalência (5,1%) SNP O/O do éxon 1 (MBL2) está de acordo com a literatura brasileira, é semelhante à européia (4%) e japonesa (5%), menor que a africana (10-14%). Níveis séricos de MBL corresponderam aos genótipos determinados. Esta é a primeira avaliação da frequência do SNP D371Y do gene MASP-2 em uma população brasileira. Os resultados deste trabalho fornecem subsídios para estudos sobre repercussão de MBL e MASP-2 em situações clínicas / BACKGROUND: Mannose-binding lectin (MBL) is a protein that recognizes carbohydrates on microbial surface leading to complement activation. This process is mediated by MBL-associated serine proteases, such as MASP-2. MBL/MASP-2 complex is responsible for generating the C3 convertase C4bC2b. Both MBL and MASP-2 levels are genetically determined, and can be influenced by the presence of single nucleotide polymorphisms (SNPs) in the genes encoding for these proteins (namely MBL2 and MASP-2). OBJTECTIVE: to determine MBL and MASP-2 serum levels and the frequencies of MBL2 and MASP-2 gene polymorphisms in a Brazilian population sample. METHODS: 294 blood donor samples [median age = 36.51 ± 10.56 years, range 18-63, 91/294 (31%) females and 203/294 (69%) males] were genotyped for MBL2 exon 1 SNPs: single point mutation in codon 52 (ArgCys), 54 (GlyAsp) and 57 (GlyGlu), and MASP-2 polymorphism Asp371Tyr (D371Y, A>C) (exon 9). A melting temperature assay was used to perform the genotyping of MBL2 SNPs. The combination of variants of MBL2 were grouped together as allele O, wild types were indicated as A. Exon 1 promoters were evaluated by direct genotype sequencing- alleles H/L, X/Y and P/Q (positions -550, -221 and +4, respectively). MASP-2 exon 9 genotyping was performed by using TaqMan pre-developed assay. RESULTS: MBL2: 58.5% A/A, 36.39% A/O, 5.1% O/O; promoters: 13% H/H, 39% H/L, 48% L/L; 2% X/X, 26% X/Y, 72% Y/Y; 52% P/P, 37% P/Q, 11% Q/Q; haplotypes: 15% LXPA, 28% HYPA, 8% LYQO, 12% LYPO, 11% LYPA, 22% LYQA and 4% HYPO. MASP-2: 38.78% A/A, 44.56% A/C and 16.67% C/C. CONCLUSION: The prevalence (5.1%) of O/O genotype of MBL2 exon 1 SNPs in our population is in accordance with Brazilian reports, similar to European (4%) and Japanese (5%); lower than Africans (10-14%). There is a correlation between MBL serum levels and genotyping. Moreover, this is the first report of D371Y MASP-2 polymorphism frequency in a Brazilian population. Our data may contribute to new insights on the role of MBL and MASP-2 in clinical conditions

A Lectina Ligadora de Manose (MBL) é uma proteína que reconhece carboidratos na superfície microbiana levando à ativação do sistema complemento. Este processo é mediado por Serino Proteases tal como a MASP-2. O complexo MBL/MASP-2 é responsável pela formação da C3 convertase C4bC2b. Os níveis séricos de MBL e a MASP-2 (genes MBL2 e MASP-2, respectivamente) são geneticamente determinados, e podem ser influenciados pela presença de polimorfismos em um único nucleotídeo SNPs em genes codificadores destas proteínas. OBJETIVO: Determinar os níveis séricos e polimorfismos gênicos da MBL e MASP-2 em uma amostra da população brasileira. MÉTODOS: 294 amostras de doadores de sangue [mediana = 36,51 ± 10,56; 18-63 anos; 91/294 (30,95%) sexo feminino, 203/294 (69,05%) sexo masculino] foram genotipadas para os SNPs do éxon 1 (MBL2): SNPs localizados nos códons 52 (ArgCys), 54 (GlyAsp) e 57 (GlyGlu) e SNP Asp371Tyr (D371Y, A>C ) do gene da MASP-2 (éxon 9). Foi utilizado o ensaio de temperatura de dissociação para éxon 1 (MBL2) e sequenciamento direto dos promoters (H/L, X/Y e P/Q, nas posições -550, -221 e +4, respectivamente). A combinação das variantes do éxon 1 MBL2 foram agrupadas e denominadas alelo O e o genótipo selvagem foi denominado A. O éxon 9 da MASP-2 foi genotipado através da plataforma TaqMan. RESULTADOS: MBL2: 58,5% A/A, 36,39% A/O e 5,1% O/O; promoters: 13% H/H, 39% H/L, 48% L/L; 2% X/X, 26% X/Y, 72% Y/Y; 52% P/P, 37% P/Q, 11% Q/Q; haplótipos encontrados: 15% LXPA, 28% HYPA, 8% LYQO, 12% LYPO, 11% LYPA, 22% LYQA e 4% HYPO. Quanto à produção, 56,12% produziram altos níveis de MBL, 30,61% níveis médios e 13,27% níveis baixos ou insuficientes de MBL. Para MASP-2: 38,78% A/A, 44,56% A/C e 16,67% C/C. CONCLUSÃO: A prevalência (5,1%) SNP O/O do éxon 1 (MBL2) está de acordo com a literatura brasileira, é semelhante à européia (4%) e japonesa (5%), menor que a africana (10-14%). Níveis séricos de MBL corresponderam aos genótipos determinados. Esta é a primeira avaliação da frequência do SNP D371Y do gene MASP-2 em uma população brasileira. Os resultados deste trabalho fornecem subsídios para estudos sobre repercussão de MBL e MASP-2 em situações clínicas / BACKGROUND: Mannose-binding lectin (MBL) is a protein that recognizes carbohydrates on microbial surface leading to complement activation. This process is mediated by MBL-associated serine proteases, such as MASP-2. MBL/MASP-2 complex is responsible for generating the C3 convertase C4bC2b. Both MBL and MASP-2 levels are genetically determined, and can be influenced by the presence of single nucleotide polymorphisms (SNPs) in the genes encoding for these proteins (namely MBL2 and MASP-2). OBJTECTIVE: to determine MBL and MASP-2 serum levels and the frequencies of MBL2 and MASP-2 gene polymorphisms in a Brazilian population sample. METHODS: 294 blood donor samples [median age = 36.51 ± 10.56 years, range 18-63, 91/294 (31%) females and 203/294 (69%) males] were genotyped for MBL2 exon 1 SNPs: single point mutation in codon 52 (ArgCys), 54 (GlyAsp) and 57 (GlyGlu), and MASP-2 polymorphism Asp371Tyr (D371Y, A>C) (exon 9). A melting temperature assay was used to perform the genotyping of MBL2 SNPs. The combination of variants of MBL2 were grouped together as allele O, wild types were indicated as A. Exon 1 promoters were evaluated by direct genotype sequencing- alleles H/L, X/Y and P/Q (positions -550, -221 and +4, respectively). MASP-2 exon 9 genotyping was performed by using TaqMan pre-developed assay. RESULTS: MBL2: 58.5% A/A, 36.39% A/O, 5.1% O/O; promoters: 13% H/H, 39% H/L, 48% L/L; 2% X/X, 26% X/Y, 72% Y/Y; 52% P/P, 37% P/Q, 11% Q/Q; haplotypes: 15% LXPA, 28% HYPA, 8% LYQO, 12% LYPO, 11% LYPA, 22% LYQA and 4% HYPO. MASP-2: 38.78% A/A, 44.56% A/C and 16.67% C/C. CONCLUSION: The prevalence (5.1%) of O/O genotype of MBL2 exon 1 SNPs in our population is in accordance with Brazilian reports, similar to European (4%) and Japanese (5%); lower than Africans (10-14%). There is a correlation between MBL serum levels and genotyping. Moreover, this is the first report of D371Y MASP-2 polymorphism frequency in a Brazilian population. Our data may contribute to new insights on the role of MBL and MASP-2 in clinical conditions

Le caroubier est considéré comme l'un des arbres fruitiers et forestiers qui présente le plus grand potentiel de valorisation puisque toutes les parties de cette plante sont utilisables dans plusieurs applications industriels. Le caroubier est cultivé dans plusieurs régions du Liban mais peu d'études sont disponibles sur les voies de valorisation et sur les propriétés fonctionnelles et structurales des graines et des gousses. Ainsi, dans le cadre d'une démarche qui vise à promouvoir et contribuer à une meilleure valorisation et gestion de cette ressource renouvelable, nous avons entrepris dans cette thèse des travaux consacrés à la caractérisation et à l'évaluation de la valeur nutritive et fonctionnelle des gousses de caroube issues de plusieurs régions libanaise. Ces travaux ont porté notamment sur la caractérisation morphologique et physico-chimique des gousses et sur la purification des gommes extraits des graines. Ces gommes ont fait l'objet d'une étude approfondie portant sur leur comportement rhéologique et la relation entre ce comportement, la variété et le lieu géographique. Nous avons aussi déterminé les isothermes de sorption de ces gommes pour prédire les conditions idéales de leur conservation. Les résultats obtenus à l'issue de cette étude ont démontré clairement la présence des corrélations morphologie, composition chimique et coordonnées géographiques de différents cultivars étudiés. De même, les études structurales et rhéologiques ont montré des différences significatives entre les gommes purifiées. Cette différence semble être liée à une variation du rapport galactose/mannose et de la masse molaire observée selon les variétés. Par ailleurs, les études des isothermes de sorption ont permis de fournir des informations complémentaires sur leurs hygroscopicités et par conséquence sur les conditions idéales de leur conservation / The carob plant is considered as one of the fruit and forester tree that has the greatest potential of valorization since all the parts of this plant can be used in several food and cosmetic applications. The carob is cultivated in several areas of lebanon but few studies are available on the ways of valorization and the functional and structural properties of the different fractions of this tree (seeds and pods). Thus, in view to develop a strategy aims to promote and contribute to a better valorization and management of this renewable resource, we undertook in this thesis of work devoted to the characterization and the evaluation of nutritive and functional properties of the carob pods resulting from several lebanese areas. This work focused particularly on the morphological and physicochemical characterization of the pods and the purified fraction of gum. These gums were the subject of a thorough study relating to their rheological behavior and the relation between this behavior, the variety and the geographical localization of the studied varieties. We also determined the isotherms of sorption of these gums to predict the ideal conditions of their conservation. The obtained results clearly showed the presence of the correlations between morphology, chemical composition and geographical localization of the different studied cultivars. In the same way, the structural and rheological studies showed significant differences between purified gums. This difference seems to be related to a variation of the galactose /mannose ratio and molar mass according to the varieties. These interesting rheological properties open the carob gums a new niches of valorization with added values. In addition, the studies of the isotherms of sorption provided additional information on their hygroscopicities and by consequence on the ideal conditions of their conservation

L'avancée technologique dans les nanosciences a permis le développement d'une large gamme de matériaux nanostructurés aux applications biomédicales. Ces outils, constitués de matériaux différents, ont été développés à des fins de diagnostic et de thérapie pour réaliser notamment le ciblage, le marquage cellulaire, l'imagerie médicale et pour concevoir des systèmes de délivrance de médicaments pour le traitement de cancers ou de maladies infectieuses. La création de nano-objets regroupant l'ensemble de ces propriétés de type théranostique constitue une étape essentielle vers un traitement personnalisé et non invasif des cancers solides de petite taille. Dans cette thèse, une première partie est consacrée à la mise au point et à l'utilisation de nanoparticules de silice mésoporeuse pour le traitement des rétinoblastomes. Ce travail visait à améliorer la thérapie photodynamique en augmentant la biodisponibilité de molécules actives dans les cellules cancéreuses par deux stratégies : leur vectorisation par un nano-objet et le ciblage spécifique des cellules cancéreuses. Pour cela, les profils d'expression des récepteurs du mannose ont été analysés et les récepteurs MRC2 et CD209 se sont révélés être de bons candidats pour une thérapie ciblée du rétinoblastome. La deuxième partie des recherches réalisées s'oriente vers l'élaboration de différents nanosystèmes pour le traitement des cancers et l'imagerie médicale. Premièrement, des nanotransporteurs de principe actif à relargage pH-sensible, structurés à partir de nanoparticule de silice mésoporeuse, ont été étudiés. Ces systèmes de délivrance, sous l'effet de stimuli internes, se sont révélés être très efficaces in vitro et ex vivo pour le traitement du cancer du côlon. Deuxièmement, le potentiel de délivrance contrôlée de molécules anticancéreuses renfermées dans des nanomachines soumises à une activation externe biphotonique, a été démontré sur des cellules de cancer du sein. Enfin, les propriétés de nanoparticules magnétiques de polymères de coordination cyano-pontés se sont révélées très prometteuses pour une utilisation en tant que nouvel agent de contraste intravasculaire pour l'imagerie par résonance magnétique in vivo. / The technological advance in nanoscience has allowed the development of a wide range of nanostructured materials for biomedical applications. These tools, composed of different materials, have been developed for diagnosis and therapy, in particular to achieve targeting, cellular labeling, medical imaging and to design drug delivery systems for the treatment of cancer or infectious diseases. The elaboration of nano-tools possessing these theranostic properties would be a major step towards personalized and non-invasive treatments of small solid cancers.In this thesis, the first part is devoted to the development and the application of mesoporous silica nanoparticles for the treatment of retinoblastoma. The aim of this work was to improve photodynamic therapy by increasing the bioavailability of active molecules in cancer cells following two strategies: their vectorization through nanodevice and the specific targeting of cancer cells. For this, expression profiles of mannose receptors were analyzed and both MRC2 and CD209 receptors were found to be interesting candidates for targeted therapy of retinoblastoma.The second part corresponds to a multidisciplinary approach focused on the research of different nanosystems designed for cancer treatment and medical imaging. We first studied pH-operated hybrid silica nanocarriers designed for drug release. Under internal stimuli, these delivery systems have shown to be very efficient in vitro and ex vivo against colon cancer. Then, we demonstrated the potential of nanoimpellers designed for anticancer drug delivery mediated by external two-photon activation on breast cancer cells. Finally, we report the promising use in vivo of new magnetic cyano-bridged coordination polymer nanoparticles as an efficient intravascular magnetic resonance imaging contrast agent.

Sur la cinquantaine de maladies rares lysosomales, seules 8 peuvent être traitées par enzymothérapie substitutive avec plus ou moins d’efficacité. Il y a donc un réel besoin de développer de nouveaux traitements mais aussi de mieux caractériser les causes de ces maladies. Durant cette thèse nous nous sommes intéressés à la maladie de Pompe qui résulte de l’absence ou de la carence en enzyme lysosomale alpha-glucosidase acide (GAA) responsable de la dégradation du glycogène en glucose dans de nombreux tissus. Actuellement seule la forme infantile de cette maladie peut être traitée alors que la forme juvénile/adulte est faiblement améliorée par le traitement Myozyme®. Cette thèse a eu pour but de développer une nouvelle enzymothérapie qui, à terme, permettrait d’empêcher la progression de la maladie et de soigner, de manière satisfaisante, les formes juvéniles et adultes de la maladie de Pompe. Dans ce but, nous avons utilisé des dérivés monosaccharidiques « Analogues du Mannose-6-phosphate (M6P) Fonctionnalisés sur l’Aglycone (AMFA) », qui sont ensuite greffés sur la GAA recombinante humaine (rhGAA) afin d’améliorer son adressage aux lysosomes obtenant la rhGAA-AMFA.Une première étude in vitro, sur des fibroblastes de patients atteints de la forme adulte de la maladie, a démontré que le greffage des AMFA sur la rhGAA produite en cellules d’insectes Sf9 améliorait significativement l’affinité pour le récepteur du M6P (RM6P), l’internalisation et l’activité de l’enzyme et lui conférait une efficacité sur les souris GAA-/-, modèles de la maladie de Pompe, par rapport au traitement actuel (Article 1). Puis nous avons pu démontrer, pour la première fois, l’efficacité de la rhGAA-AMFA produite en cellules CHO sur des souris GAA-/- âgées. Ces résultats suggèrent, ainsi, la possibilité d’utiliser cette néo-enzyme dans le traitement de la forme adulte de la maladie (Article2). Enfin, le greffage des AMFA permet d’obtenir une maturation intracellulaire complète de la rhGAA sous forme active dans des myoblastes et myotubes de patients adultes et dans les quadriceps de souris âgées modèles Pompe, ce qui n’a pas été observé pour Myozyme® (Article 3). Lors de cette thèse, nous avons également démontré que de nouveaux analogues disaccharidiques, ayant une meilleure affinité que les monosaccharides pour le RM6P, peuvent efficacement cibler la rhGAA pour le traitement de la maladie de Pompe. Un brevet a été déposé sur ces résultats (Brevet PCT/FR2016/052339).En conclusion, ce travail a permis de développer une nouvelle technologie de ciblage plus efficace des enzymes lysosomales par des analogues synthétiques. La désignation de médicament orphelin pour l’alpha glucosidase acide conjuguée aux analogues du mannose-6-phosphate a été obtenue suite à ces travaux auprès de l’Agence Européenne du Médicament pour le traitement de la maladie de Pompe (EMA/OD/098/16).Mots clés: maladies lysosomales, maladie de Pompe, enzymothérapie, récepteur du mannose 6-phosphate / On 53 known rare lysosomal diseases, only 8 can be treated by enzyme replacement therapy with more or less efficiency. There is therefore a need to develop new treatments but also to better characterize these diseases. During this thesis, we focused on Pompe disease which results from the absence or deficiency of the lysosomal enzyme alpha-glucosidase acid (GAA), responsible for the degradation of glycogen in glucose in many tissues. Currently only the infantile form of this disease can be treated while the juvenile/adult form is slightly improved by Myozyme® treatment. This thesis aimed to devel a new enzyme replacement therapy which could prevent the progression of the disease and satisfactorily treat the late onset form of the disease. To do that, we used monosaccharide derivatives “Mannose-6-phosphate analogues (M6P) Functionalized on Aglycone (AMFA)”, which were grafted onto human recombinant GAA (rhGAA) in order to improve its lysosome addressing obtaining the rhGAA-AMFA.A first in vitro study on adult patient fibroblasts showed that the addition of AMFA to rhGAA, produced in Sf9 insect cells, significantly improved its affinity for the M6P receptor (RM6P), its internalization and activity. It was also more efficient on the GAA-/- Pompe mouse model compared to current treatment (Article 1). Then, we demonstrated for the first time the efficiency of rhGAA-AMFA produced in CHO cells in aged mice model. These results suggest the possibility to use this neo-enzyme in the treatment of the adult form that still resists to treatment (Article 2). Finally, the addition of AMFA allows a complete maturation of rhGAA into its active form in myoblasts and myotubes of adult patients and in the quadriceps of aged mice Pompe model. This was not observed for Myozyme® (Article 3). In this thesis we have also demonstrated that novel disaccharide analogues with a better affinity than monosaccharides for RM6P can efficiently target GAA for the treatment of Pompe disease. A patent has been filed on these results (Patent PCT / FR2016 / 052339).In conclusion, this work has led to the development of a new technology more efficient in targeting lysosomal enzymes by mean of new synthetic analogues. An orphan drug designation for the recombinant human acid alpha-glucosidase conjugated with mannose-6-phosphate analogues was obtained on the basis of this work at the European Medicines Agency for the treatment of Pompe disease (EMA/OD/098/16).Key words: lysosomal diseases, Pompe disease, enzyme replacement therapy, mannose 6-phosphate receptor