A proper innate immune response is essential for multicellular organisms to survive in an environment where they are exposed to hostile microbes. The innate immune system is not only important for protection against foreign pathogens, but also plays a major role in immunological disorders. The NF-kappaB signalling pathway is a major regulator of innate immunity. To understand how the innate immune transcriptional program is activated, the signalling cascades regulating NF-kappaB needs to be characterised. Our aim is to determine how ubiquitylation regulates the activity of mediators of NF-kappaB activation. As NF-kappaB signalling is very complex in mammals, we use Drosophila as a model to elucidate the principal concepts of ubiquitin signalling in innate immunity. Ubiquitylation seems to be an evolutionary conserved requirement for innate immunity, as the E3-ubiquitin ligase activity of IAPs is required for activation of NF-kappaB in both flies and mammals. In addition to being E3-ubiquitin ligases, the IAPs are established caspase regulators. Our results suggest that the caspase drICE acts as a negative regulator of NF-kappaB signalling. Hence, we aim at finding out if drICE regulates signalling by restraining the E3 ligase activity of the Drosophila IAP, DIAP2. Also M1-linked linear ubiquitin chains seems to be important for NF-kappaB activation in mammalian cells. No ligase able to induce linear ubiquitylation has been described in Drosophila. However, we have found a new gene (CG11321), which encodes for a putative linear ubiquitin ligase. Our preliminary data suggests that CG11321 is required for mounting an immune response against Gram-negative bacteria. Characterising this new gene may give us insight into the general principles of linear ubiquitylation in NF-kappaB signalling. For efficient NF-kappaB signalling to proceed, signalling molecules need to be gathered to close enough proximity to interact. Our results indicate that the ubiquitin-binding scaffolding protein p62 indeed is required for activation of NF-kappaB in flies. Hence, we will elucidate if p62 can sequester ubiquitylated proteins to NF-kappaB-activating signalling platforms. In addition, to these approaches to study ubiquitylation in NF-kappaB signalling, we will screen for protein complexes that are associated with ubiquitin upon NF-kappaB activation. The identity of interacting proteins found may provide important clues to the role of ubiquitylation in NF-kappaB signalling.