An investigation into the role of Bcl-3 in toll-like receptor signalling

Through the recognition of potentially harmful stimuli, Toll-like receptors (TLRs) initiate the innate immune response and induce the expression of hundreds of immune and pro-inflammatory genes. TLRs are critical in mounting a defence against invading pathogens however, strict control of TLR signalling is vital to prevent host damage from excessive or prolonged immune activation. In this thesis the role of the IκB protein Bcl (B-cell lymphoma)-3 in the regulation of TLR signalling is investigated. Bcl3-/- mice and cells are hyper responsive to TLR stimulation and are defective in LPS tolerance. Bcl-3 interacts with and blocks the ubiquitination of homodimers of the NF-κB subunit, p50. Through stabilisation of inhibitory p50 homodimers, Bcl-3 negatively regulates NF-κB dependent inflammatory gene transcription following TLR activation. Firstly, we investigated the nature of the interaction between Bcl-3 and p50 and using peptide array technology. Key amino acids required for the formation of the p50:Bcl-3 immunosuppressor complex were identified. Furthermore, we demonstrate for the first time that interaction between Bcl-3 and p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3. Using the data generated from peptide array analysis we then generated cell permeable peptides designed to mimic Bcl-3 function and stabilise p50 homodimers. These Bcl-3 derived peptides are potent inhibitors of NF-κB dependent transcription activity in vitro and provide a solid basis for the development of novel gene-specific approaches in the treatment of inflammatory diseases. Secondly, we demonstrate that Bcl-3 mediated regulation of TLR signalling is not limited to NF-κB and identify the MAK3K Tumour Progression Locus (Tpl)-2 as a new binding partner of Bcl-3. Our data establishes role for Bcl-3 as a negative regulator of the MAPK-ERK pathway.

Variations in total and differential milk somatic cell counts and plasmin levels and their role in proteolysis and quality of milk and cheese

Increased plasmin and plasminogen levels and elevated somatic cell counts (SCC) and polymorphonuclear leucocyte levels (PMN) were evident in late lactation milk. Compositional changes in these milks were associated with increased SCC. The quality of late lactation milks was related to nutritional status of herds, with milks from herds on a high plane of nutrition having composition and clotting properties similar to, or superior to, early-mid lactation milks. Nutritionally-deficient cows had elevated numbers of polymorphonuclear leucocytes (PMNs) in their milk, elevated plasmin levels and increased overall proteolytic activity. The dominant effect of plasmin on proteolysis in milks of low SCC was established. When present in elevated numbers, somatic cells and PMNs in particular had a more significant influence on the proteolysis of both raw and pasteurised milks than plasmin. PMN protease action on the caseins showed proteolysis products of two specific enzymes, cathepsin B and elastase, which were also shown in high SCC milk. Crude extracts of somatic cells had a high specificity on αs1-casein. Cheeses made from late lactation milks had increased breakdown of αs1-casein, suggestive of the action of somatic cell proteinases, which may be linked to textural defects in cheese. Late lactation cheeses also showed decreased production of small peptides and amino acids, the reason for which is unknown. Plasmin, which is elevated in activity in late lactation milk, accelerated the ripening of Gouda-type cheese, but was not associated with defects of texture or flavour. The retention of somatic cell enzymes in cheese curd was confirmed, and a potential role in production of bitter peptides identified. Cheeses made from milks containing high levels of PMNs had accelerated αs1-casein breakdown relative to cheeses made from low PMN milk of the same total SCC, consistent with the demonstrated action of PMN proteinases. The two types of cheese were determined significantly different by blind triangle testing.