Developing an analytic platform to define the heterogeneity of cell death program
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USyd Access
Type
ThesisThesis type
Doctor of PhilosophyAuthor/s
Coleman, MikaelaAbstract
Programmed cell death is critical to maintain cellular homeostasis, resolve infection, and defend against cancer. The biochemical program by which cells die can be predictive of disease outcome and success of drug treatment in infection and cancer. Once thought to be largely distinct, ...
See moreProgrammed cell death is critical to maintain cellular homeostasis, resolve infection, and defend against cancer. The biochemical program by which cells die can be predictive of disease outcome and success of drug treatment in infection and cancer. Once thought to be largely distinct, a mounting number of studies have drawn attention to the high degree of molecular redundancy and interaction between the three major death programs – apoptosis, pyroptosis, and the various forms of necrosis. Currently, no method exists to investigate the concurrence of these three major death programs, making attempts to study the overlap and interactions in cell death machinery across distinct death programs challenging. To address these challenges, an ASC-Azurite-expressing THP-1 cell line and ASC foci detection algorithm was developed to enable the identification of pyroptosis using an imaging flow-cytometry (IFC)-based approach. This novel ASC foci detection algorithm identified both cell-associated and cell-free ASC foci and formed the basis of an IFC-based cell death assay to detect and quantify pyroptosis, early and late apoptosis, and necrosis simultaneously. This approach also readily supported kinetic analysis to trace how interactions evolve as death programs progress from initiation to execution phases. Applying this tool to identify new FDA approved drugs suitable for repurposing as chemotherapeutics, 4 drugs with previously unknown or poorly characterised cytotoxicity in human monocytic cancer cells were identified as candidates for repurposing. The robust analytic capability of this new approach offers an important tool in the intricate study of cell death and will contribute to the development of knowledge about infectious disease pathogenesis, cancer drug discovery, and host-directed therapies.
See less
See moreProgrammed cell death is critical to maintain cellular homeostasis, resolve infection, and defend against cancer. The biochemical program by which cells die can be predictive of disease outcome and success of drug treatment in infection and cancer. Once thought to be largely distinct, a mounting number of studies have drawn attention to the high degree of molecular redundancy and interaction between the three major death programs – apoptosis, pyroptosis, and the various forms of necrosis. Currently, no method exists to investigate the concurrence of these three major death programs, making attempts to study the overlap and interactions in cell death machinery across distinct death programs challenging. To address these challenges, an ASC-Azurite-expressing THP-1 cell line and ASC foci detection algorithm was developed to enable the identification of pyroptosis using an imaging flow-cytometry (IFC)-based approach. This novel ASC foci detection algorithm identified both cell-associated and cell-free ASC foci and formed the basis of an IFC-based cell death assay to detect and quantify pyroptosis, early and late apoptosis, and necrosis simultaneously. This approach also readily supported kinetic analysis to trace how interactions evolve as death programs progress from initiation to execution phases. Applying this tool to identify new FDA approved drugs suitable for repurposing as chemotherapeutics, 4 drugs with previously unknown or poorly characterised cytotoxicity in human monocytic cancer cells were identified as candidates for repurposing. The robust analytic capability of this new approach offers an important tool in the intricate study of cell death and will contribute to the development of knowledge about infectious disease pathogenesis, cancer drug discovery, and host-directed therapies.
See less
Date
2021Rights statement
The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.Faculty/School
Faculty of Medicine and Health, Central Clinical SchoolAwarding institution
The University of SydneyShare