Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Medical Biochemistry. / Typescript bound. Includes bibliographical references: (leaves 211-230) Also available online.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [76]-87).

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 75-100) Also available online.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 113-124).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005. / On t.p. "2+" is superscript. Includes bibliographical references (leaves 126-133). Also available in electronic version.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 85-97).

Thesis (Ph.D.) - University of St Andrews, February 2010.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / On t.p. "2̳+̳" is superscript. Includes bibliographical references (leaves 126-139). Also available in electronic version.

Les cancers de l’ovaire représentent la première cause de décès par cancer gynécologique. L’échec des traitements, lié à l’apparition d’une chimiorésistance, implique de trouver de nouvelles stratégies thérapeutiques. Dans ce cadre, le laboratoire a mis en évidence la coopération de deux molécules anti-apoptotiques surexprimées dans les cancers de l’ovaire, Mcl-1 et Bcl-xL, qui empêchent alors le déclenchement de la mort cellulaire par apoptose. L’inhibition de ces cibles est alors mise en place. Un BH3-mimétique inhibiteur de Bcl-xL a été développé par la société Abbvie, l’ABT-737, qui possède un dérivé administrable par voie orale l’ABT-263 en essai clinique. En revanche, aucun inhibiteur de Mcl-1 n’est actuellement en clinique. L’inhibition de cet anti-apoptotique est donc l’un des objectifs du laboratoire. Sachant que Mcl-1 est extrêmement régulée, que l’inhibition de la voie de signalisation PI3K/Akt/mTOR conduit à l’inhibition de cet anti-apoptotique, et que le calcium est capable de moduler cette voie, nous nous sommes demandés si la modulation des flux calciques permettait l’inhibition de Mcl-1 dans nos cellules. Ces travaux de thèse ont pu montrer dans un premier temps que la chélation calcique par le BAPTA-AM permettait d’inhiber Mcl-1 via la voie mTORC1 et de sensibiliser les cellules à l’ABT-737. Dans un second temps, l’étude de l’effet d’un inhibiteur des flux calciques en essais cliniques, le carboxyamidotriazole a permis de mettre en évidence que l’inhibition des canaux calciques capacitifs pouvait entraîner l’inhibition de Mcl-1 de nouveau en inhibant mTORC1 et induire la mort cellulaire en combinaison avec l’ABT-737. Enfin des observations morphologiques ont montré que le CAI induisait un changement morphologique aboutissant à la mort des cellules. Ce type de mort semble être lié à une perturbation du métabolisme des cellules cancéreuses IGROV1-R10 et se rapprocherait d’une mort récemmement décrite dans la littérature : l’autosis. / Ovarian cancer is the leading cause of death from gynecological cancer. There is an urgent need to find new therapeutic strategies due to failure of treatments associated to development of chemoresistance. In this context, the laboratory has shown the cooperation of two anti-apoptotic proteins overexpressed in ovarian cancer, Mcl-1 and Bcl-xL, for preventing apoptotic cell death. ABT-737, a Bcl-xL inhibitor BH3-mimetic, was developed by Abbvie and has a clinically derivative named ABT-263. In contrast, no Mcl-1 inhibitor is currently available in clinic. The inhibition of this anti-apoptotic protein is therefore one of the objectives of the laboratory. Since inhibition of PI3K / Akt / mTOR signaling pathway leads to inhibition of Mcl-1 expression and calcium is able to modulate this pathway, we wondered if the modulation of calcium fluxes allowed the inhibition of Mcl-1 in ovarian cancer cells. First we were able to show that calcium chelation by BAPTA-AM allowed to inhibit Mcl-1 expression via mTORC1 pathway and to sensitize the cells to ABT-737. In a second step, we investigated the effect of an inhibitor of calcium fluxes that is evaluated in clinical studies, carboxyamidotriazole. We show that the inhibition of capacitive calcium channels could lead to Mcl-1 down-expression via inhibition of mTORC1 and promote apoptosis in combination with ABT-737. Finally, we observed that CAI induces a morphological change resulting in cell death. This type of death seems to be related to disruption of metabolism in IGROV1-R10 cancer cells and would be close to a cell death recently described in the literature: autosis.

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular responses including development and proliferation. Calcium (Ca²⁺) is stored in both neutral and acidic stores and its release through gated channels has been implicated in regulating development in Dictyostelium discoideum. This thesis aims to understand the roles of the calcium channel proteins, in particular the two-pore channel proteins (TPCs), found on acidic stores in Ca²⁺ signalling during Dictyostelium development. Bioinformatic analysis indicates conservation of a gene encoding an orthologue of TPC2 in Dictyostelia and, similar to plant TPCs, a Ca²⁺ sensing domain is predicted along with a novel potential calmodulin binding site. To investigate the role of intracellular Ca²⁺ channels, a series of strains was generated, disrupted in one or more of genes encoding the channels TPC2 and mucolipin (TRP-ML), predicted to be located on acidic stores, and IplA, located on neutral stores. All disrupted strains, including one lacking all three channels, are able to complete development. However, strains lacking TPC2 show a pronounced delay in early development, correlating with reduced expression of some early developmental genes. Vesicles derived from tpc2-null cells show normal Ca²⁺ release compared to parental cells but an increased rate of Ca²⁺ uptake. During early development, the pH of acidic vesicles is increased in the absence of TPC2. However development of tpc2-null cells showed increased sensitivity to weak bases in producing fewer aggregates but resistance to sodium chloride and weak bases in later development suggesting a complex role for TPC during development. In vivo cytosolic Ca²⁺ responses were analysed in strains expressing an ultra-sensitive Ca²⁺ indicator YC-Nano 15. Growing tpc2^- and iplA^- cells have lower basal cytosolic Ca²⁺ than parental Ax2 cells. Intercellular Ca²⁺ waves were observed in aggregates from Ax2, mcln^- and tpc2^- cells but were greatly reduced in iplA- aggregates, as was the increase in cytosolic Ca²⁺ in response to extracellular cAMP. In tpc2- aggregates, wave frequencies were reduced and the response to cAMP addition abolished after treatment with caffeine, a known adenylyl cyclase inhibitor in Dictyostelium. This work demonstrates that TPC2 plays a role in the early stages of Dictyostelium development. TPC2 is important for pH regulation in acidic vesicles and cytosolic Ca²⁺ levels, either or both of which could influence development either directly or via changes in early developmental gene expression.