The Role of Slp-76 Phosphotyrosines in TCR Signal Transduction and T Cell Differentiation

The cytosolic adapter protein src homology 2(SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) lacks enzymatic activity but nucleates a multi-molecular signaling complex that links early T cell receptor (TCR)-induced phosphorylation events into multiple downstream signaling pathways. The N-terminus of SLP-76 contains three tyrosines at residues 112,128 and 145 that are phosphorylated following TCR ligation and, although the mechanisms are not entirely clear, they are required for optimal TCR signal transduction. TCR signals are required for T cell proliferation, cytokine production, and effector and memory differentiation. The experiments described in this dissertation have first tested the biochemical mechanisms by which the SLP-76 tyrosines transmit signals and second tested how alterations in the TCR signals transmitted through SLP-76 tyrosines influence T cell differentiation and effector function. Experiments were performed using two genomic knock-in (KI) mice that express tyrosine to phenylalanine mutations at residue 145 (Y145F) or 112 and 128 together (Y112/128F). Using biochemistry-, flow cytometry- and microscopy-based approaches we show that mutations in the tyrosines of SLP-76 result in graded defects in TCR-induced signals and function depending on the tyrosine(s) affected. Surprisingly, localization of SH2 domain containing effector proteins to mutant SLP-76-nucleated signaling complexes was not lost and therefore could not account for the observed signaling defects. Infection of SLP-76 KI mice with lymphocytic choriomeningitis virus (LCMV) resulted in normal CD8 expansion but graded enhancement of memory differentiation consistent with a model in which weaker TCR signals preferentially promote memory versus effector differentiation. Furthermore CD8+ effector and memory KI T cells failed to produce appropriate cytokine upon antigen restimulation. Similarly, in vitro polarized KI Th17 and Th2 cells failed to produce IL17a and IL4, respectively, following TCR restimulation. Taken together our data show that SLP-76 tyrosines are essential for optimal TCR signal transduction and, moreover, TCR signals sufficient to promote T cell differentiation are different than those required to elicit inflammatory cytokine production

Calibrating mars orbiter laser altimeter pulse widths at mars science laboratory candidate landing sites

Accurate estimates of surface roughness allow quantitative comparisons between planetary terrains. These comparisons enable us to improve our understanding of commonly occurring surface processes, and develop a more complete analysis of candidate landing and roving sites. A (secondary) science goal of the Mars Orbiter Laser Altimeter was to map surface roughness within the laser footprint using the backscatter pulse-widths of individual pulses, at finer scales than can be derived from the elevation profiles. On arrival at the surface, these pulses are thought to have diverged to between 70 and 170 m, corresponding to surface roughness estimates at 35 and 70 m baselines respectively; however, the true baseline and relationship remains unknown. This work compares the Mars Orbiter Laser Altimeter pulse-widths to surface roughness estimates at various baselines from high-resolution digital terrain models at the final four candidate landing sites of Mars Science Laboratory. The objective was to determine the true baseline at which surface roughness can be estimated, and the relationship between surface roughness and the pulse-widths, to improve the reliability of current global surface roughness estimates from pulse-width maps. The results seem to indicate that pulse-widths from individual shots are an unreliable indicator of surface roughness, and instead, the pulse-widths should be downsampled to indicate regional roughness, with the Slope-Corrected pulse-width dataset performing best. Where Rough Patches are spatially large compared to the footprint of the pulse, pulse-widths can be used as an indicator of surface roughness at baselines of 150 to 300 m; where these patches are spatially small, as observed at Mawrth Vallis, pulse-widths show no correlation to surface roughness. This suggests that a more complex relationship exists, with varying correlations observed, which appear dependent on the distribution of roughness across the sites

Mechanism and function of Vav1 localisation in TCR signalling

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux

LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells

The serine/threonine kinase LKB1 has a conserved role in Drosophila and nematodes to co-ordinate cell metabolism. During T lymphocyte development in the thymus, progenitors need to synchronize increased metabolism with the onset of proliferation and differentiation to ensure that they can meet the energy requirements for development. The present study explores the role of LKB1 in this process and shows that loss of LKB1 prevents thymocyte differentiation and the production of peripheral T lymphocytes. We find that LKB1 is required for several key metabolic processes in T-cell progenitors. For example, LKB1 controls expression of CD98, a key subunit of the l-system aa transporter and is also required for the pre-TCR to induce and sustain the regulated phosphorylation of the ribosomal S6 subunit, a key regulator of protein synthesis. In the absence of LKB1 TCR-β-selected thymocytes failed to proliferate and did not survive. LBK1 was also required for survival and proliferation of peripheral T cells. These data thus reveal a conserved and essential role for LKB1 in the proliferative responses of both thymocytes and mature T cells

Cotton Rats and House Sparrows as Hosts for North and South American Strains of Eastern Equine Encephalitis Virus

TOC summary: Wild rodents and wild birds can serve as amplification hosts

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1

Innovation in tourism: Re-conceptualising and measuring the absorptive capacity of the hotel sector

Recent reviews of research on innovation in tourism have highlighted a number of weaknesses in the literature. Among these is the limited theorising and empirical investigation of innovative practices by tourism organisations. This paper responds to these concerns by examining one important dimension of innovation within commercial tourism organisations, namely their ability to acquire, assimilate and utilise external knowledge (absorptive capacity) for competitive advantage. The topic is pertinent because there is evidence to suggest that tourism organisations are particularly dependent on external sources of knowledge when compared with businesses in other sectors. Following a discussion of the conceptual antecedents of absorptive capacity and its dimensions, a validated instrument for its measurement is developed and used to measure the absorptive capacity of the British hotel sector. The results suggest that current conceptions of absorptive capacity have limitations when applied to tourism enterprises. Absorptive capacity is re-conceptualised to overcome these deficiencies. The research and policy implications of the findings are discussed. © 2014 Elsevier Ltd

The constitution of risk communication in advanced liberal societies

This article aims to bring to the fore some of the underlying rationales that inform common conceptions of the constitution of risk communication in academic and policy communities. ‘Normative’, ‘instrumental’ and ‘substantive’ imperatives typically employed in the utilisation of risk communication are first outlined. In light of these considerations a theoretical scheme is subsequently devised leading to the articulation of four fundamental ‘idealised’ models of risk communication termed the ‘risk message’ model, the ‘risk dialogue’ model, the ‘risk field’ model and the ‘risk government’ model respectively. It is contended that the diverse conceptual foundations underlying the orientation of each model suggest a further need for a more contextualised view of risk communication that takes account not only of the strengths and limitations of different formulations and functions of risk communication, but also the underlying knowledge/power dynamics that underlie its constitution. In particular it is hoped that the reflexive theoretical understanding presented here will help to bring some much needed conceptual clarity to academic and policy discourses about the use and utility of risk communication in advanced liberal societies