A non-classical LysR-type transcriptional regulator PA2206 is required for an effective oxidative stress response in Pseudomonas aeruginosa

LysR-type transcriptional regulators (LTTRs) are emerging as key circuit components in regulating microbial stress responses and are implicated in modulating oxidative stress in the human opportunistic pathogen Pseudomonas aeruginosa. The oxidative stress response encapsulates several strategies to overcome the deleterious effects of reactive oxygen species. However, many of the regulatory components and associated molecular mechanisms underpinning this key adaptive response remain to be characterised. Comparative analysis of publically available transcriptomic datasets led to the identification of a novel LTTR, PA2206, whose expression was altered in response to a range of host signals in addition to oxidative stress. PA2206 was found to be required for tolerance to H2O2 in vitro and lethality in vivo in the Zebrafish embryo model of infection. Transcriptomic analysis in the presence of H2O2 showed that PA2206 altered the expression of 58 genes, including a large repertoire of oxidative stress and iron responsive genes, independent of the master regulator of oxidative stress, OxyR. Contrary to the classic mechanism of LysR regulation, PA2206 did not autoregulate its own expression and did not influence expression of adjacent or divergently transcribed genes. The PA2214-15 operon was identified as a direct target of PA2206 with truncated promoter fragments revealing binding to the 5'-ATTGCCTGGGGTTAT-3' LysR box adjacent to the predicted -35 region. PA2206 also interacted with the pvdS promoter suggesting a global dimension to the PA2206 regulon, and suggests PA2206 is an important regulatory component of P. aeruginosa adaptation during oxidative stress.

Realisable climate change narratives in public discourse: an East/West comparative analysis of environmental, social, and corporate governance (ESG) as a response to global climate change

This thesis looks at how non-experts develop an opinion on climate change, and how those opinions could be changed by public discourse. I use Hubert Dreyfus’ account of skill acquisition to distinguish between experts and non-experts. I then use a combination of Walter Fisher’s narrative paradigm and the hermeneutics of Paul Ricœur to explore how non-experts form opinions, and how public narratives can provide a point of critique. In order to develop robust narratives, they must be financially realistic. I therefore consider the burgeoning field of environmental, social, and corporate governance (ESG) analysis as a way of informing realistic public narratives. I identify a potential problem with this approach: the Western assumptions of ESG analysis might make for public narratives that are not convincing to a non-Western audience. I then demonstrate how elements of the Chinese tradition, the Confucian, Neo-Confucian, and Daoist schools, as presented by David Hall and Roger Ames, can provide alternative assumptions to ESG analysis so that the public narratives will be more culturally adaptable. This research contributes to the discipline by bringing disparate traditions together in a unique way, into a practical project with a view towards applications. I conclude by considering avenues for further research.

Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

Immunostimulatory effects of different aspects of aquaculture on the host response in the edible sea urchin, paracentrotus lividus

Aquaculture is a fast-growing industry contributing to global food security and sustainable aquaculture, which may reduce pressures on capture fisheries. The overall objective of this thesis was to look at the immunostimulatory effects of different aspects of aquaculture on the host response of the edible sea urchin, Paracentrotus lividus, which are a prized delicacy (roe) in many Asian and Mediterranean countries. In Chapter 1, the importance of understanding the biology, ecology, and physiology of P. lividus, as well as the current status in the culture of this organism for mass production and introducing the thesis objectives for following chapters is discussed. As the research commenced, the difficulties of identifying individuals for repeat sampling became clear; therefore, Chapter 2 was a tagging experiment that indicated PIT tagging was a successful way of identifying individual sea urchins over time with a high tag retention rate. However, it was also found that repeat sampling via syringe to measure host response of an individual caused stress which masked results and thus animals would be sampled and sacrificed going forward. Additionally, from personal observations and discussion with peers, it was suggested to look at the effect that diet has on sea urchin immune function and the parameters I measured which led to Chapter 3. In this chapter, both Laminaria digitata and Mytilus edulis were shown to influence measured immune parameters of differential cell counts, nitric oxide production, and lysozyme activity. Therefore, trials commencing after Trial 5 in Chapter 4, were modified to include starvation in order to remove any effect of diet. Another important aspect of culturing any organism is the study of their immune function and its response to several immunostimulatory agents (Chapter 4). Zymosan A was shown to be an effective immunostimulatory agent in P. lividus. Further work on handled/stored animals (Chapter 5) showed Zymosan A reduced the measured levels of some immune parameters measured relative to the control, which may reduce the amount of stress in the animals. In Chapter 6, animals were infected with Vibrio anguillarum and, although V. anguillarum, impacted immune parameters of P. lividus, it did not cause mortality as predicted. Lastly, throughout this thesis work, it was noted that the immune parameters measured produced different values at different times of the year (Chapter 7); therefore, using collated baseline (control) data, results were compiled to observe seasonal effects. It was determined that both seasonality and sourcing sites influenced immune parameter measurements taken at different times throughout the year. In conclusion, this thesis work fits into the framework of development of aquaculture practices that affect immune function of the host and future research focusing on the edible sea urchin, P. lividus.