Phenotypic screening is a powerful technique for discovering small organic molecules with the ability to effect a desired change in biological systems. Yet, because of the diversity of ways that a small molecule can alter a biological system, phenotypic screens provide little to no insight into how a hit molecule elicits such a change. Understanding a molecule’s mechanism of action—which proteins it engages, where it localizes, and its reactivity—is critical to fully developing a hit molecule into a research tool or a therapeutic. In this dissertation, strategic, hypothesis-driven molecular design is used as a cornerstone technique to understand the mechanism of action of molecules that regulate cell death. In the first part, we examine the structural requirements for ferroptotic death induction by the 1,2-dioxolane FINO2. Next, we create a panel of ferroptosis inhibiting molecules that are targeted to specific organelles and used as imaging agents in order to examine the contribution of different organelles to ferroptosis. I then apply molecular design in a target-based context to discover which molecular features of LOC14 promote association with its receptor protein. Finally, I discuss a computational approach to developing a ligand that inhibits protein-protein interactions mediated by the small GTPase Rheb.

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"July 2004" / Explanatory notes on back page. / Bibliography: leaves 131 -181. / vii, 181 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2004

Correct development of multicellular organisms requires the programmed removal of supernumerary, redundant, or damaged cells, a process achieved by apoptosis. Apoptosis, or Programmed Cell Death (PCD) is executed by caspases, a highly conserved family of cysteine proteases. The removal of redundant larval tissues during metamorphosis is controlled by the steroid hormone ecdysone. Ecdysone signalling is mediated by the nuclear receptor heterodimer EcR/Usp, which in turn transcriptionally activates a host of transcription factors which then go on to regulate genes essential for PCD, like caspases. The apical caspase dronc is upregulated in the larval midgut and salivary glands prior to their destruction and is dependent on the BRC and E93 transcription factors. To further understand the role of dronc in development, a dronc mutant fly was generated and a transgenic promoter-reporter strategy was employed to investigate dronc regulation. Larval organs from dronc mutants lack dying cells and, when irradiated, fail to show a radiation-induced PCD response. The midguts from dronc mutants undergo apoptosis and have high caspase activity. These data indicate that a droncindependent caspase activation pathway is active in the midgut. Salivary glands from dronc mutants failed to be removed and have reduced caspase activity. Consequently it is clear that the role of DRONC differs significantly between the midgut and salivary glands. The employment of a transgenic dronc promoter-LacZ reporter system identified promoter regions essential for the correct temporal and spatial expression of dronc. A region of the dronc promoter between 1.1kb and 2.8kb has elements essential for LacZ expression in salivary glands. This region was also dependent on the BR-C and E93 transcription factors for salivary gland expression. A functional ecdysone receptor binding element (EcRBE) was identified in the dronc proximal promoter. The EcR-B1 isoform directly binds this EcRBE and is necessary for correct dronc expression in the larval salivary glands. This work revealed some novel findings regarding the role of DRONC in development and the availability of a specific dronc mutant now makes it possible to explore some of the recently published non- poptotic roles of dronc. This work aids in understanding how nuclear hormones control transcription and shows dronc to be an ideal model gene to explore these molecular and genetic processes. / Thesis (Ph.D.)--Department of Medicine, 2004.

Thesis (Ph.D.)--McMaster University, 2004. / Supervisor: David W. Andrews. Includes bibliographical references (leaves 134-148).

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (March 1, 2007) Vita. Includes bibliographical references.

Optic nerve section (ONS) is an experimental model for damage of the optic nerve associated with diseases such as glaucoma and optic neuritis. Damage to the optic nerve causes loss of retinal ganglion cells that are attached, once the cells are damaged, they are not typically replaced. Recently, Fischer and Reh (2003) found that Müller glia have the potential to adopt phenotypes and functional capabilities similar to those of retinal progenitors, a potential source of retinal regeneration. In the chick, when the specific retinal cells are targeted for damage by chemotoxins, there is widespread apoptosis but also mitotically active cells that label with retinal progenitor markers. Fischer and Reh (2002) also discovered that the combination of growth factors FGF2 and insulin is capable of stimulating the regenerative response of the Müller glia to retinal progenitor cells in chick eyes. This study was conducted to analyse damage to the ganglion cells by optic nerve section in chicks to determine the effect of age on the cell death timeline, the proliferative qualities of the retina and to see if injections of growth factors had the ability to increase the proliferation. Histological methods were used to analyse cellular changes and ultrasound to monitor eye growth. Apoptotic activity preceded retinal thinning and ganglion cell loss, indicating that ONS-related cell death is mediated at least in part by apoptotic mechanisms and age did not affect the time course, although, age did affect the eye growth changes, which may be attributed to the plasticity of the younger eyes. ONS damage elicited proliferative activity in the retina as did growth factor injections alone. The combination of ONS damage and growth factor injections increased the proliferative activity and the overall total number of cells in the ganglion cell layer. These findings can potentially lead to the development of therapeutic strategies for the preservation or restoration of retinal cells in diseased eyes.

The amnioserosa (AS) is an epithelium that plays major roles in two crucial morphogenetic processes during Drosophila embryogenesis: Germ Band Retraction (GBR) and Dorsal Closure (DC). The AS is extraembryonic and as such, it does not contribute to the mature embryo but is eliminated during development by programmed cell death. In this thesis, a comprehensive investigation of the timing and characteristics of the AS death and degeneration is performed. It is demonstrated that AS elimination occurs in two phases: “cell extrusion” during DC, embryonic stages 12 to 14, and “tissue dissociation” following DC, embryonic stages 15 to 16. Ten percent of AS cells are eliminated during phase one while the remaining ninety percent are removed during phase two. It is found that both cell extrusion and tissue dissociation are absent in apoptotic defective backgrounds, as well as in genetic backgrounds associated with increased class I phosphoinositide 3-kinase (PI3K) activity, a key regulator of autophagy. It is also found that extrusion is enhanced two-fold in embryos expressing the pro-apoptotic reaper gene product, and that tissue dissociation also accelerates in this background. Interestingly, our observations suggest that the activation of caspase cascade is not complete until AS cells have lost apical contacts with neighboring cells. Shortly after the loss of apical contact, an apoptotic morphology including membrane blebbing, cell fragmentation, and macrophage engulfment is readily observed. Measurements of the rate of DC demonstrate that this process is protracted in backgrounds lacking extrusion, leading to the conclusion that extrusion contributes towards generating adequate AS tension required for normal DC rates. Overall, our data suggest that phase one extrusion and phase two dissociation are manifestations of the same cellular event and that both are caspase dependent. It is also demonstrated that autophagy is a key component of AS death that acts upstream of apoptosis. Strikingly, our results lead to the suggestion that autophagy may function to trigger apoptosis during the programmed elimination of this extra-embryonic tissue.

The amnioserosa (AS) is an epithelium that plays major roles in two crucial morphogenetic processes during Drosophila embryogenesis: Germ Band Retraction (GBR) and Dorsal Closure (DC). The AS is extraembryonic and as such, it does not contribute to the mature embryo but is eliminated during development by programmed cell death. In this thesis, a comprehensive investigation of the timing and characteristics of the AS death and degeneration is performed. It is demonstrated that AS elimination occurs in two phases: “cell extrusion” during DC, embryonic stages 12 to 14, and “tissue dissociation” following DC, embryonic stages 15 to 16. Ten percent of AS cells are eliminated during phase one while the remaining ninety percent are removed during phase two. It is found that both cell extrusion and tissue dissociation are absent in apoptotic defective backgrounds, as well as in genetic backgrounds associated with increased class I phosphoinositide 3-kinase (PI3K) activity, a key regulator of autophagy. It is also found that extrusion is enhanced two-fold in embryos expressing the pro-apoptotic reaper gene product, and that tissue dissociation also accelerates in this background. Interestingly, our observations suggest that the activation of caspase cascade is not complete until AS cells have lost apical contacts with neighboring cells. Shortly after the loss of apical contact, an apoptotic morphology including membrane blebbing, cell fragmentation, and macrophage engulfment is readily observed. Measurements of the rate of DC demonstrate that this process is protracted in backgrounds lacking extrusion, leading to the conclusion that extrusion contributes towards generating adequate AS tension required for normal DC rates. Overall, our data suggest that phase one extrusion and phase two dissociation are manifestations of the same cellular event and that both are caspase dependent. It is also demonstrated that autophagy is a key component of AS death that acts upstream of apoptosis. Strikingly, our results lead to the suggestion that autophagy may function to trigger apoptosis during the programmed elimination of this extra-embryonic tissue.

The amnioserosa (AS) is an extraembryonic tissue that undergoes programmed cell death (PCD) during the normal course of Drosophila embryogenesis. AS degeneration involves morphological evidence of autophagy as well as caspase activation, but the relationship between these two processes is not well defined. While the bulk of the AS tissue dies at the conclusion of the morphogenetic process of dorsal closure (DC), approximately 10% of AS cells are actively extruded from the epithelium during DC. Using live imaging confocal microscopy and various fluorescent protein sensors, I have been able to observe caspase activation as well as autophagy upregulation in the context of epithelial extrusion events as well as overall AS degeneration. The data show that epithelial extrusion events are caspase-dependent but are also associated with localized onset of autophagy. Furthermore, extensive characterization of loss of function mutants of the key Drosophila regulator Atg1 kinase indicates that autophagy is not required for the normal degeneration of AS, contrary to earlier studies. This thesis also introduces new relationships between caspase activation and autophagic cell death. In addition, new data suggest that the InR/TOR and EGFR/Ras/MAPK signaling pathways interact with the pro-apoptotic protein Head involution defective (Hid) and Atg1 kinase to regulate the progression of programmed cell death in the AS.