Programmed Cell death (PCD) is a highly conserved and genetically controlled event
that plays important roles in animal development, homeostasis and disease. Our first
objective was to discover and characterize new genes involved in PCD. Since many PCD
genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands
(SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we
used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene
Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed
transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton
remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to
knownlpredicted genes and were 5-fold differentially expressed prior to cell death.
Next, we investigated the role of differentially expressed genes from SAGE, in cell
death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila
cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment,
and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis
assays to identify the death-related genes and determined their ecdysone dependency and
function in cell death regulation. Our RNAi screen identified six new pro-death related
genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN.
Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox
box proteins may have opposing roles in ecdysone-mediated cell death.
Our final objective was to elucidate the function of CG409], a Drosophila
homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from
SAGE. We created loss-of-function and overexpression mutants of CG4091 to study
gene function in vivo and employed immunoprecipitation and mass-spectrometry assays
to identify proteins interacting with CG409] in vitro. We identified two proteins that are
involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction
partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is
necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect
cellular functions such as autophagy and lipid metabolism/trafficking that require an
intact cytoskeleton. Together, our studies provided new insights into the molecular
mechanisms involved in Drosophila SG cell death.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU.2429/2725 |
| Date | 05 1900 |
| Creators | Chittaranjan, Suganthi |
| Publisher | University of British Columbia |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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