Thesis advisor: Marc A. T. Muskavitch / Notch signaling is an evolutionarily conserved developmental pathway regulated by two classes of transmembrane proteins: the Notch receptors and the Delta/Serrate/LAG-2 (DSL) ligands. Notch and DSL ligands mediate cell-cell communication that results in a downstream signaling cascade that affects many aspects of metazoan development. Additional regulatory mechanisms that affect Notch signaling are being discovered continuously, and recent findings highlight the importance of endocytosis, ubiquitylation and subcellular trafficking as essential requirements for proper signaling. In order to obtain further insights into the regulation of Notch signaling, I took a two-fold approach, combining genetic and molecular techniques in <italic>Drosophila</italic>. First, I took part in a large-scale transposon-based screen in the developing <italic>Drosophila</italic> eye to identify additional genes involved in the pathway. We screened 10,447 transposon lines from the Exelixis collection for modifiers of cell fate alterations caused by overexpression of Delta, and we identified 170 distinct modifier lines that may affect up to 274 genes. I further analyzed a previously uncharacterized gene, which we have named <italic>Amun</italic>, and showed that it localizes to the nucleus and contains a putative DNA glycosylase domain. Further analyses of Amun reveal that altered levels of Amun function interfere with cell fate specification during eye and sensory organ development. Second, to investigate structural requirements for ubiquitylation of Delta, I analyzed four individual lysine residues in the Delta intracellular domain, and assessed their necessity for Delta signaling activity. I find that a conserved residue, DeltaK742, is essential for Notch signaling in the <italic>Drosophila</italic> imaginal wing disc and is apparently required for ubiquitylation of Delta by the E3 ubiquitin ligase, Mind bomb1 (one of two E3 ubiquitin ligases required for Delta signaling activity). Taken together, the findings from this thesis research contribute to the advancement of our understanding of different aspects of Notch signaling and Notch-mediated development. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.