Les cellules de Schwann (CS) sont les cellules gliales myélinisantes du Système Nerveux Périphérique (SNP). Il existe une communication étroite entre ces cellules et les axones auxquels elles s’associent et ce dès les stades les plus précoces de leur développement. Elles migrent tout en se divisant le long des axones; cette division migratoire est suivie d’une deuxième division post-migratoire dans le but d’établir un ratio 1:1 avec les axones pour ensuite les myéliniser. Ce travail vise à analyser, in vivo, le comportement des CS chez le poisson zèbre au cours de leurs divisions.Nous avons remarqué que les CS se divisent parallèlement aux axones le long du nerf de la Ligne Latérale Postérieure (PLL). En analysant les deux mutants has et nok, nous avons montré que les gènes de polarité apicale aPKC et pals1 ne sont pas requis pour la migration et la division des CS, ni pour leur capacité à myéliniser. Nous avons mis en évidence, en analysant le mutant cassiopeia qui présente des défauts d’organisation du fuseau mitotique et en utilisant l’agent pharmacologique le nocodazole, que l’assemblage du fuseau mitotique au cours de la division des CS est essentiel pour la myélinisation.En parallèle, nous avons analysé le rôle du gène rgs4 (regulator of G-protein Signaling 4) dans le développement du SNP chez le poisson zèbre. Nous avons généré un mutant stable rgs4 par la technique CRISPR/Cas9 et montré un rôle de ce gène dans le développement du ganglion de la PLL et des motoneurones, et ce en agissant en amont de la voie PI3K/Akt/mTOR.Contrairement à l’inhibition pharmacologique qui suggère un rôle de rgs4 dans la myélinisation périphérique, le mutant ne présente pas de défauts de myéline. / Schwann cells (SCs) are the myelinating glial cells of the Peripheral Nervous System (PNS). They derive from neural crest cells during development, then migrate and divide along the axons of the peripheral nerves. This migratory division is followed by a post-migratory division in order to radially sort the axons in a 1:1 ratio and wrap them with a myelin sheath. This work provides an analysis of the polarity of SC divisions, in vivo, in intact zebrafish embryos.We showed that SCs divide parallel to the axons along the Posterior Lateral Line nerve (PLL). By analyzing the two mutants has and nok, we revealed that the apical polarity genes aPKC and pals1, are neither required for the migration and division of SCs, nor for their capacity to myelinate. By studying the cassiopeia mutant that shows defects in mitotic spindle, we revealed that the assembly of the mitotic spindle is essential for SC myelination.We have also analysed the role of rgs4 (regulator of G-protein Signaling 4) in PNS development. We generated a stable rgs4 mutant using the CRISPR/Cas9 technology. We showed that rgs4 plays an essentiel role in PLLg and motoneurons development by acting upstream of PI3K/Akt/mTOR pathway. Pharmacological analysis suggested a role for rgs4 in peripheral myelination, however, the rgs4 mutant do not show any myelin defects.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19048号 / 農博第2126号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4930(農学部図書室) / 31999 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 植田 和光, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Phosducin-like protein (PhLP) binds G-protein beta gamma subunits and is thought to assist in assembly of the G-protein beta gamma dimer. Phosphorylation of PhLP at serine residues 18-20 by the casein kinase 2 (CK2) appears to play an essential role in this process. PhLP has also been shown to interact with the chaperonin containing TCP-1 (CCT) atop its apical domain, not entering the substrate folding cavity. However, the physiological role of the PhLP-CCT interaction in G-protein beta gamma dimer formation remains unclear. This study addresses the mechanism of G-protein beta gamma assembly by exploring the specific roles of CCT and CK2 phosphorylation of PhLP in the assembly process. Both overexpressed and endogenous Gbeta were shown to co-immunoprecipitate with CCT to a similar extent as PhLP, indicating that CCT may be involved in the folding of Gbeta. In addition, Ggamma overexpression enhanced the binding of PhLP to CCT, suggesting the formation of a ternary PhLP-Gbeta-CCT complex. In contrast, overexpression of PhLP caused the release of G-beta from CCT. This release was blocked by a PhLP S18-20A variant that lacks the S18-20 CK2 phosphorylation site. PhLP S18-20A has been previously shown to negatively affect the G-protein beta gamma dimer formation, suggesting a correlation between PhLP-mediated release of Gbeta from CCT and G protein beta gamma assembly. Experiments investigating the role of Ggamma in this process show that Ggamma does not interact with CCT nor is it the essential factor in the release of Gbeta from CCT. A new model is therefore proposed for the G-protein beta gamma subunits' assembly involving the formation of a PhLP-Gbeta-CCT ternary complex followed by the release of a phosphorylated PhLP-Gbeta complex from CCT. In the PhLP-Gbeta complex, the Ggamma binding face of Gbeta is exposed, allowing for the formation of the G-protein beta gamma dimer.

G-protein coupled receptors (GPCRs) play a key role in a broad range of biological processes by binding to a wide variety of signaling molecules, which have resulted in 34% of all FDA-approved drugs which target GPCRs. The human genome encodes for approximately 800 GPCR members of which about 140 non-olfactory receptors remain orphans with an unknown function and endogenous ligand. Despite prolonged efforts to deorphanize the unresolved receptors, they remain orphans until this day. By studying scientific publications, this thesis has clarified the challenges with the deorphanization of GPCRs to explain why there are still so many orphan GPCRs when they have confirmed involvement in so many human disorders.

Specifically addressing cell surface molecules on cancer cells facilitates targeted cancer therapies that offer the potential to selectively destroy malignant cells, while sparing healthy tissue. Thus, undesired side-effects in tumor patients are highly reduced. Peptide-binding receptors are frequently overexpressed on cancer cells and therefore promising targets for selective tumor therapy. In this review, peptide-binding receptors for anti-cancer drug delivery are summarized with a focus on peptide ligands as delivery agents. In the first part, some of the most studied peptide-binding receptors are presented, and the ghrelin receptor and the Y1 receptor are introduced as more recent targets for cancer therapy. Furthermore, nonpeptidic small molecules for receptor targeting on cancer cells are outlined. In the second part, peptide conjugates for the delivery of therapeutic cargos in cancer therapy are described. The essential properties of receptor-targeting peptides are specified, and recent developments in the fields of classical peptide-drug conjugates with toxic agents, radiolabeled peptides for radionuclide therapy, and boronated peptides for boron neutron capture therapy are presented.

No description available.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23789号 / 医博第4835号 / 新制||医||1057(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 渡邊 直樹, 教授 松田 道行, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Breast cancer is the second leading cause of death in United States. Using 2D-HPLE, a novel separation technology, G-protein coupled receptor-associated sorting protein 1(GASP-1) was identified in sera of patients with early stage cancer, while it could not be detected in sera from healthy individuals. This was the first indication that GASP-1 was positively correlated with breast cancer. However, the function of GASP-1 in breast cancer was unknown. In this study, I verified the 2D-HPLE results by quantifying the expression level of GASP-1 in sera and tissue specimens of cancer patients using specific antibodies against GASP-1. A GASP-1 specific ELISA was developed and used to quantify GASP-1 levels in cancer patient sera. Immunohistochemistry was performed to verify and localize GASP-1 expression in tumor. I also characterized the tumorigenic potential of GASP-1 andidentified the signaling pathways mediated by GASP-1 in breast cancer cells in vitro.GASP-1 expression levelsin MDA-MB-231 cells were modified by transfecting cells with anti-GASP-1 shRNA and over-expression plasmids. Stable cell lines were prepared and their tumorigenic potential was evaluated using cell proliferation, migration, and colony formation assays. These cells were analyzed for markers used to identify epithelial to mesenchymal transition (EMT) using RT-PCR and western blot. They were also analyzed for NFkappaB activity, src phosphorylation, and GPR30 expression. The results showed that GASP-1 was over-expressed in sera and tissue specimens of breast cancer patients and other cancer types including brain, lung, liver and pancreatic cancer and that it correlated with early stage disease. GASP-1 positively regulated migration, and is required for cell proliferation and colony formation. GASP-1 is also necessary for the expression of EMT marker slug, increases NFkappaB activity and GPR30 expression level, while decreases the inhibitory phospho-src Tyr 530. I conclude that GASP-1 is a nearly marker for multiple cancer types. GASP-1 promotes tumorigenesis in breast cancer, possibly through multiple cancer related signaling pathways. These findings may contribute to our understanding of the mechanism of breast cancer tumorigenesis and identify new biomarkers that can be used for diagnosis and therapy of cancer. / Biology

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Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.