Understanding two inhibitors of NF-κB: A20 and IκBβ

While prompt activation of NF-κB is essential for optimal immune response, it is equally important to terminate the response to avoid tissue damage and perhaps even death resulting from organ failure. This thesis describes two inhibitors of NF-κB, A20 and IκBβ. A20 is an essential inhibitor of NF-κB mediated inflammation as mice lacking A20 die from multi-organ inflammation and cachexia. Multiple biochemical approaches have suggested that A20 functions as a deubiquitinase by disassembling K63-linked regulatory ubiquitin chains from upstream adapter molecules like RIP1. To determine the contribution of the deubiquitinase role of A20 in downregulating NF-κB, we generated and characterized a knock-in mouse lacking the deubiquitinase activity of A20. However, we find that these mice display normal NF-κB activation and show no signs of inflammation. Our results suggest that the deubiquitinase activity of A20 is dispensable for downregulating NF-κB. The second part of this thesis unravels a new biological pathway mediated by IκBβ. Unlike IκBα, which functions solely as an inhibitor of NF-κB, IκBβ can both inhibit and activate NF-κB depending on the physiological context. We hypothesized that this may be because IκBβ (unlike IκBα ) exists in two forms, a constitutively phosphorylated form and an unphosphorylated form. Prior work from our group has demonstrated that hypophosphorylated IκBβ complexes with p65:cRel and mediates the expression of certain inflammatory genes like TNFα . We report here that Glycogen Synthase Kinase 3β (GSK-3β ) interacts with and phosphorylates IκBβ at Serine-346. This phosphorylation masks the NLS of p65 in the phospho-IκBβ:p65:cRel complex, thereby sequestering the complex in the cytoplasm and mediating the anti-inflammatory role of IκBβ. We discovered a peptide that can inhibit this phosphorylation by abrogating the interaction between GSK-3β and IκBβ. Mice succumb to a sublethal dose of LPS when injected with this peptide because of increased production of TNFα (but not IL-6); thereby demonstrating the inflammatory role of unphosphorylated IκBβ in upregulating specific genes like TNFα. We propose a signaling model by which phosphorylation by GSK-3β can regulate the functions of IκBβ in response to LPS

Design of Peptide-Based Prodrug Chemistry and Its Application to Glucagon-like Peptide I

Peptide-based drugs are highly effective medicines with relatively short duration of action and of variable therapeutic index. Glucagon-like peptide 1 is a hormone that offers promise in the treatment of Type II diabetes. However, the biggest problem in the therapeutic use of GLP-1 is its extremely short half-life in plasma (~2 min). A prodrug of GLP-1 should extend and improve the pharmacodynamics of this peptide hormone. We have designed prodrugs that slowly convert to the parent drug at physiological conditions of 37C and pH 7.2 driven by their inherent chemical instability without the need of any enzymatic cleavage. We observed that amide prodrugs could not convert to the active peptides under physiological conditions. Consequently, we decided to synthesize peptide drugs which had a hydroxy-terminal extension instead of a Nterminal amine. Ester prodrugs were prepared using these hydroxy-peptides as the scaffold. We explored the diketopiperazine and diketomorpholine (DKP and DMP) strategy for the chemical flexibility that it offers to develop prodrugs with variable time actions. The esters proved to be more labile than the corresponding amides and the dynamic range in rate of cleavage ranged from an hour to almost half a week. We found that the rate of cleavage depends on the structure and stereochemistry of the dipeptide pro-moiety and also on the strength of the nucleophile. The careful selection of appropriate functionality that balances chemical, biological and immunological features under physiological conditions has also been reported

Proliferation and fragmentation: Transactions costs and the value of aid

The problem of the proliferation of the number of aid donors and aid channels continues to worsen. It is widely and plausibly believed that this significantly; reduces the value of aid by increasing direct and indirect transactions costs. We contribute to the existing literature by: (a) categorising the apparent adverse effects of proliferation; (b) producing a reliable and fair indicator of the relative degree to which the main bilateral donors proliferate or concentrate their aid; (c) giving some explanation of why some donors proliferate more than others; (d) constructing a reliable measure of the extent to to which recipients suffer from the problem of fragmentation in the sources of their aid; and (e) demonstrating that the worst proliferators among the aid donors are especially likely); to be suppliers of aid to recipients suffering most from fragmentation. There are significant implications for aid policy

Spatial Interactions in Hedonic Pricing Models:The Urban Housing Market of Aveiro, Portugal

Spatial heterogeneity, spatial dependence and spatial scale constitute key features of spatial analysis of housing markets. However, the common practice of modelling spatial dependence as being generated by spatial interactions through a known spatial weights matrix is often not satisfactory. While existing estimators of spatial weights matrices are based on repeat sales or panel data, this paper takes this approach to a cross-section setting. Specifically, based on an a priori definition of housing submarkets and the assumption of a multifactor model, we develop maximum likelihood methodology to estimate hedonic models that facilitate understanding of both spatial heterogeneity and spatial interactions. The methodology, based on statistical orthogonal factor analysis, is applied to the urban housing market of Aveiro, Portugal at two different spatial scales