Quantification of gamma-secretase activity in an endogenous context reveals biphasic GSI-mediated Notch/APP selectivity switch and the novel detection of potential proteolytic cleavage fragments of the Notch Intracellular Domain

Gamma-secretase is a promiscuous intra-membrane protease implicated in the proteolytic processing of two notable substrates: amyloid-precursor-protein (APP), in which gamma-secretase will irreversibly cleave to produce Amyloid-beta (Aβ) in Alzheimer’s disease; and the Notch receptor, where gamma-secretase is essential for liberating the Notch intracellular domain (NICD) to activate Notch-mediated transcriptional regulation in the nucleus. Gamma-secretase inhibitors such as DAPT and Avagacestat have been tested as therapies to prevent the formation of amyloid plaques, however, off-target interference with the Notch signalling pathway leading to Notch-related malignant side effects in clinical trials makes these pharmaceuticals unsuitable. The high-throughput search for selective drugs that block the production of Aβ but don’t interfere with the Notch signalling pathway has been hindered by a lack of reliability in detecting Notch signal inhibition in pre-clinical, cell-based assays with ectopic substrate expression. Therefore, the development of a highly-sensitive, high-throughput cell-based assay to quantify the level of proteolytic processing of APP and Notch by gamma-secretase is a promising addition to the gamma-secretase inhibitor drug discovery pipeline. This thesis presents the combination of immunofluorescence staining, western blotting, and the bromo-deoxyuridine (BrdU) cell proliferation assay as three orthogonal methods to sensitively quantify the gamma-secretase cleavage of Notch and APP in SH-SY5Y human neuroblastoma cells, which rely on active Notch signalling to maintain proliferation. Using our assay, we found that the selectivity for gamma-secretase to cleave APP versus Notch was dependent on the time the GSI was replenished before harvesting, which may directly reflect a GSI concentration-dependent selective potency. Using this high-throughput cell-based assay, cleavage of APP and Notch activation by gamma-secretase was sensitively quantified; while a novel profile of cell-type specific proteolytic fragments of the Notch ICD have been identified that may have biological implications in normal development and the pathological proliferation and metastasis of some cancers

The drone question: Legality, ethics, and the need to recognize transnational armed conflict

This work focuses on the legality and ethics of targeted killings via drones conducted by the United States. The first section of this work looks at the use of drone strikes by the U.S. government as they fall outside of the traditional notion of a zone of armed conflict, that being one that can be defined geographically and temporally, and explores whether these strikes could be considered legal under international humanitarian law and the international law of self-defense. This work assumes that an armed conflict exists between the United States and the non-state armed actors that have been targeted by these drone strikes to explore whether the strikes could be considered legal under those two international legal regimes. The second section looks at these strikes from an ethics perspective. It explores if the ethical questions raised by drones are indeed novel, if drone strikes conducted by the United States satisfy the principles provided under Just War Theory, the impact drones have on the application of military ethics, and the ethical concerns raised by fully-autonomous weapon systems. This work argues that drones can be a legal and ethical weapon system, but the failure of international law to recognize a new, hybrid category of armed conflict has clouded the debate surrounding drones. The adoption of transnational armed conflict as a new type of conflict classification is presented as a solution to the problem posed by drones and contemporary conflict dynamics to better define and explain conflicts involving a state and a non-state armed actor that crosses and/or maintains bases of operations across international borders