The Rpf/DSF system and the regulation of virulence in the plant pathogen Xanthomonas campestris

The full virulence of Xanthomonas campestris pv. campestris (Xcc) to plants depends upon cell-to-cell signalling mediated by the signal molecule DSF (for diffusible signal factor), that has been characterised as cis-11-methyl-2-dodecenoic acid. DSF-mediated signalling regulates motility, biofilm dynamics and the synthesis of particular virulence determinants. The synthesis and perception of the DSF signal molecule involves products of the rpf (regulation of pathogenicity factors) gene cluster. DSF synthesis is fully dependent on RpfF, which encodes a putative enoyl-CoA hydratase. A two-component system, comprising the complex sensor histidine kinase RpfC and the HD-GYP domain regulator RpfG, is implicated in DSF perception. The HD-GYP domain of RpfG is a phosphodiesterase working on cyclic di-GMP; DSF perception is thereby linked to the turnover of this intracellular second messenger. The full range of regulatory influences of the Rpf/DSF system and of cyclic di-GMP in Xcc has yet to be established. In order to further characterise the Rpf/DSF regulatory network in Xcc, a proteomic approach was used to compare protein expression in the wildtype and defined rpf mutants. This work shows that the Rpf/DSF system regulates a range of biological functions that are associated with virulence and biofilm formation but also reveals new functions mediated by DSF regulation. These functions include antibiotic resistance, detoxification and stress tolerance. Mutational analysis showed that several of these regulated protein functions contribute to virulence in Chinese radish. Interestingly, it was demonstrated that different patterns of protein expression are associated with mutations of rpfF, rpfC and rpfG. This suggests that RpfG and RpfC have broader roles in regulation other than perception and transduction of DSF. Taken together, this analysis indicates the broad and complex regulatory role of Rpf/DSF system and identifies a number of new functions under Rpf/DSF control, which were shown to play a role in virulence.

Impact of the smoke-free workplace legislation on smoking behaviour, risk perception & stigmatisation

The Republic of Ireland became the first European country to implement nationwide smoke-free workplace legislation. Aims: To determine prevalence of smoking among bar workers and estimate the impact of the smoke-free workplace legislation on their smoking behaviour to that of a comparable general population sample. To approximate the influence of tobacco control measures on risk perception of second-hand smoke (SHS) among the general population. To explore the de-normalisation of smoking behaviour and the potential increased stigmatisation of smokers and their smoking. Methods: Prevalence estimates and behavioural changes were examined among a random sample of bar workers before and 1 year after the smoke-free legislation; comparisons made with a general population sub-sample. Changes in risk knowledge related to SHS exposure were based on general population data. Qualitative interviews were conducted among a purposive sample of smokers and non-smokers four years after the implementation of the legislation. Results: Smoking prevalence was extremely high among bar workers. Smoking prevalence dropped in bar workers and significantly among the general population 1 year post ban while cigarette consumption dropped significantly among bar workers. Disparity in knowledge between smokers and non-smoker of risk associated with SHS exposure reduced. Lack of understanding of the risk of ear infections in children posed by SHS exposure was notable. Evidence for advanced de-normalisation of smoking behaviour and intensification of stigma because of the introduction of the legislation was dependent on many factors, quality of smoking facilities played a key role. Conclusions: Ireland’s smoke-free legislation was associated with a drop in prevalence and cigarette consumption. Disparity in knowledge between smokers and non-smokers of the risk posed by SHS exposure reduced however the risk of ear infections in children needs to be effectively disseminated. The proliferation of ‘good’ smoking areas may diminish the potential to reduce smoking behaviour and de-normalise smoking.

The Ford administration, Angola and the perception of Post-Vietnam US credibility, 1974-1976

This thesis is a study of how the Gerald Ford administration struggled to address a perceived loss of US credibility after the collapse of Vietnam, with a focus on the role of Secretary of State Henry Kissinger in the formulation, implementation and subsequent defence of US Angolan policy. By examining the immediate post-Vietnam period, this thesis shows that Vietnam had a significant impact on Kissinger’s actions on Angola, which resulted in an ill conceived covert operation in another third world conflict. In 1974, Africa was a neglected region in Cold War US foreign policy, yet the effects of the Portuguese revolution led to a rapid decolonization of its African territories, of which Angola was to become the focus of superpower competition. After South Vietnam collapsed in April 1975, Kissinger became fixated on restoring the perceived loss of US prestige, Angola provided the first opportunity to address this. Despite objections from his advisors, Kissinger methodically engineered a covert program to assist two anti-Marxist guerrilla groups in Angola. As the crisis escalated, the media discovered the operation and the Congress decided to cease all funding. A period of heated tensions ensued, resulting in Kissinger creating a new African policy to outmanoeuvre his critics publicly, while privately castigating them to foreign leaders. This thesis argues that Kissinger’s dismissal of internal dissent and opposition from the Congress was influenced by what he perceived as bureaucrats being affected by the Vietnam syndrome, and his obsession with restoring US credibility. By looking at the private and public records – as expressed in government meetings and official reports, US newspaper and television coverage and diplomatic cables – this thesis addresses the question of how the lessons of Vietnam failed to influence Kissinger’s actions in Angola, but the lessons of Angola were heavily influential in the construction of a new US-African policy.

Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.