The human BTG/TOB protein family comprises six members (BTG1, BTG2/PC3/Tis21, BTG3/Ana, BTG4/PC3B, TOB1/Tob, and TOB2) that display anti-proliferative activity in a number of cell types. They are characterised by a conserved N-terminal BTG domain that mediates interactions with the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylases. It was unclear whether the anti-proliferative activity of the BTG/TOB proteins was mediated through interactions with Caf1a (CNOT7) and Caf1b (CNOT8). To address this we further characterised the amino acid residues located along the BTG2 and TOB1 interaction surface with Caf1a (CNOT7)/Caf1b (CNOT8) to identify residues required for the interaction. We then analysed the role of BTG2 and TOB1 in the regulation of cell proliferation, translation and mRNA abundance using a mutant that is no longer able to interact with Caf1a (CNOT7)/Caf1b (CNOT8). We conclude that the anti-proliferative activity of BTG/TOB proteins is mediated through interactions with the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase enzymes. We also demonstrate that recruitment of BTG2 and TOB1 to mRNA leads to reduced protein levels and mRNA degradation. Furthermore, we show that the regulation of mRNA abundance and protein levels is dependent on Caf1a (CNOT7)/Caf1b (CNOT8), but does not appear to require other Ccr4-Not components, including the Ccr4a (CNOT6)/Ccr4b (CNOT6L) deadenylases, or the non-catalytic subunits CNOT1 or CNOT3.

In this thesis I describe an investigation into the function of the protein Icb-1, a homologue of Themis1 in B cells and monocytes. Themis1 is important for T cell positive and negative selections. Yet its function in T cell development is not clear. Although it shows characteristics of an adaptor protein and involvement in TCR-induced signalling, the exact signalling defects in Themis1-/- T cells remain obscure. Icb-1 is similar to Themis1 in sequence, function and binding partners. It has been studied in human tumour and macrophage cell lines, leading to limited conclusions. Its role in B cells has never been published. Given the link with Themis1, it is of great interest to investigate the function of Icb-1. My study has been focused on the comparison between Icb-1 knockout mice with wild-type controls. I characterised the B cell development in Icb-1-/- mice, either naturally born or produced as mixed adult bone marrow chimeras reconstituted from WT and Icb-1-/- donor cells. I examined the possible compensation and redundancy of Themis1 and Icb-1, by characterising Thems1/Icb-1 double knockout mice. The Ig-HEL mouse models were used to examine the change in B cell repertoire due to negative and positive selections. The mice were challenged with SRBCs or NP-CGG to examine the germinal centre response to foreign antigen when Icb-1 is absent. In vitro stimulation of B cells with soluble and membrane-bound antigens was used to investigate early B cell responses in detail and to give insights into the defects found in in vivo challenges. Finally, I examined the BCR-induced phosphorylation of key signalling molecules and Ca2+ flux in splenic B cells. The study revealed largely normal B cell development with subtle selection impairments, but a partially defected B cell immune response to antigens in Icb-1-/- mice. The marginal zone B cell population was enlarged in the absence of Icb-1, while the positive selection of B1 B cells induced by intracellular self-antigen was impaired. The deficient mice showed a reduction in germinal centre B cell generation. The defects are associated with impaired BCR-induced cell signalling to low abundance and/or low avidity antigens. In particular, Ca2+ flux and Erk1/2 phosphorylation were clearly reduced under certain conditions. The results shine a light on the function of protein Icb-1, and also improve our knowledge of Themis1 and the Themis family. They provide a new avenue of investigation into the regulation of BCR signalling, especially in Ca2+ flux induction and Erk1/2 activation. They also provide insight into how differential signalling is controlled within cells during activation and differentiation in response to antigens that vary in terms of affinity, avidity and frequency.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) receptors are heterodimeric complexes composed of the calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP). Association with RAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 or RAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributed throughout the body and play important roles and are implicated in many diseases including migraine, heart failure and sepsis. Recently, CGRP has been shown to promote nitric oxide (NO) production and inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells via ERK activation. CGRP is known to induce iNOS/NO production in thoracic artery smooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, the molecular mechanism of CGRP-induced iNOS/NO production in TA-SMC is unknown. Therefore, in order to determine if CGRP induces iNOS/NO production via ERK activation, I first investigated the exact mechanisms through which CGRP activates ERK1-2 in HEK cells. By using different inhibitors I showed that CGRPinduced ERK activation is mainly activated through two major pathways. I showed for the first time that CGRP induces ERK activation through transactivation of ErbB1 and as expected through the cAMP/PKA pathway. Then, in order to characterise a suitable model to study CGRP-induced iNOS expression, I used primary TA-SMC and I showed that CGRP induces iNOS upregulation, which is reduced when cells are incubated with U0126, a MEK inhibitor. Thus, these results suggest that CGRP induces iNOS expression via ERK activation in TA-SMC. However, further experimentation is required to determine the exact ERK pathway responsible for iNOS induction. Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressing CLR•RAMP2, I investigated the molecular mechanisms regulating the ADM receptor. I first showed that, unlike CLR•RAMP1, even transient stimulation of CLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicating that this ADM receptor does not recycle to the cell-surface. Moreover, I showed that CLR, not RAMP2, is constitutively ubiquitinated, which was further enhanced upon ADM stimulation. In order to elucidate the role of ADM-mediated ubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. I showed that ubiquitination of CLR did not affect ADM-induced trafficking of CLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signalling of CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rate of degradation of the receptor. Together, these results indicate that CLR•RAMP2 does not recycle and is degraded via a molecular mechanism that is accelerated by ADM-induced ubiquitination of CLR.

In recent years a great deal of research has focussed on understanding and exploiting self assembling peptides as they form fibrillar hydrogels for use in a variety of different applications, such as tissue engineering and drug delivery. A particular class of such peptide systems are ionic-complementary peptides, composed of alternating hydrophobic and hydrophilic amino acids. Their simple structure is generally seen to assemble into β sheet rich fibrils, and easy modification of the primary structure is possible to allow the inclusion of recognition motifs tailored for a specific use. This can be done simply via physical mixing. To maximise the potential of such systems it is important to understand the interactions that govern the self-assembly behaviour. Here a variety of different peptides have been studied to elucidate control of peptide conformation and fibril morphology. The ability to easily tune the mechanical strength of the hydrogel has been explored by mixing peptide systems. The peptide FEFEFKFK (FEKII) was seen to assemble into β sheet rich fibrils of ~3 nm in diameter. Control of pH and hence the charge state of the E and K side chains altered sample properties. Gelation at pH 2.8 occurred at a concentration between 20 30 mg ml 1. At pH 4, 5 and 10 where the peptide has a lower net charge gelation was lowered to ~10 mg ml 1. Mechanical properties varied with G' values of 20-1200 Pa as pH was altered. Stronger gels were formed with lower net peptide charge. Hierarchical fibre assembly was observed for positively charged peptides, with fibres forming from lateral association of fibrils. Negatively charged peptides at pH 10 showed no such hierarchical assembly, and lower fibril persistence length. This was related to the change in charge along the fibril structure. At pH 7, where the peptide has no net charge, precipitation occurred. This showed a net charge was required on the peptide to disperse fibrils and prevent aggregation. The work showed the importance of ionic-interactions in determining both network morphology and bulk properties, and also elucidated control of such behaviour. AEAEAKAK (AEKII) was shown to assemble into α helix fibres. Alanine (A) is less hydrophobic than F, and is a known helix former. The role of F and A in assembly was assessed by the design of peptides FEAEFKAK (FAIEKII) and FEFEAKAK (FAIIEKII). Mixing A with F disrupted the peptides' ability to form a β sheet network by lowering the driving force for assembly given by the F residues. Trace amounts of β sheet were observed at low concentration, but at a critical concentration β sheet content increased and gelation occurred. This was found to be pH dependent. FAIEKII formed β-sheet fibrils at a lower concentration than FAIIEKII. While FAIEKII was able to assemble into different fibril structures, FAIIEKII showed no specific aggregation. This not only highlighted the importance of Hydrophobicity as a key driving force to assembly but also how the grouping of these amino acids in the primary sequence can determine the overall assembly characteristics of the peptide. The peptides FEFEFKFKGGFEFEFKFK (FEKII18-1) and FEFEFKFKGGFKFKFEFE (FEKII18-2) were designed to co-assemble with FEKII. Individually both peptides were seen to assemble into β sheet fibrils. FEKII18-1 formed fibrils of 2.3 3.1 nm in size, a result of folding along the chain caused by intra molecular attractive ionic interactions. FEKII18-2 formed larger fibrils of 4.4 5.2 nm from a straightened peptide chain given by the change in charge distribution. When co assembled with FEKII mechanical properties were enhanced, with G' increasing from 40 Pa at 20 mg ml 1 to 2400 Pa, depending on the concentration of FEKII18-1/FEKII18-2 added to the system. This was a result of these peptides providing fibril connections acting as cross links. This work has detailed control over the assembly process via peptide conformation and fibril interactions and the effect this has on overall macroscopic sample properties. This is vital in determining the viability of such systems in various biomedical applications.

No description available.