Histones are basic nuclear proteins that bind to genomic DNA. There are five subclasses: core histones H2A, H2B, H3 and H4, and linker histone H1. Release of extracellular histones has been shown in a range of critical illness animal models and human patients including sepsis and acute pancreatitis. Extracellular histones act as damage-associated molecular pattern molecules (DAMPs) on parenchymal epithelial and endothelial cells, cardiomyocytes, immune cells (neutrophils, monocytes, macrophages, dendritic cells and lymphocytes) and platelets to activate Toll-like receptors (TLRs; TLR2 and TLR4) and/or NLR Family Pyrin Domain Containing 3 (NLPR3) inflammasome as well as induce calcium influx, proinflammatory cytokine production, thrombin generation and cell death. Anti-histone strategies such as anti-histone antibodies or pharmacological neutralisers alleviate histone-induced toxicity. To progress the translational relevance of histones in circulation, this thesis explores the development of xMAP technology based assay for measuring relevant toxic histones and also the significance of histone determination in animal models and patients with acute pancreatitis. Truncated histones were generated by recombinant DNA technology and the purity was 75.6-95.1%, except for histone H2A C-terminal (35.5%). Anti-histone single chain variable fragment (ahscFv) was also generated (purity > 95%) and shown to bind to histone subclasses by Western blot and IAsys resonant biosensor. Fluorescein isothiocyanate (FITC)-full length and FITC-truncated histones (H1.1 C-, H2A N-, H3.1 N-terminal) bound to the cell membrane and induced calcium influx in EA.hy926 cells, while other truncated histones did not. ahscFv significantly prevented histone-induced cell death. To measure all toxic histones in one assay, Luminex xMAP multiplex technology was developed. Standard curves of both singleplex and multiplex assays were developed and used to measure the levels of circulating histones in plasma of patients with severe trauma, sepsis and pancreatitis. However, the recovery ratio in spiked plasma was low and the assay failed to detect histones in patient plasma which were detectable by Western blot. Release of circulating histones were investigated in mice with acute pancreatitis induced by either 4 or 12 injection of caerulein (50 µg/kg/h; CER-AP) or by infusion of pancreatic duct with sodium taurocholate (3.5%, 1 ml/kg; NaTC-AP). Four and 12 injections of caerulein resulted in oedematous and necrotising CER-AP, respectively, with marked systemic inflammation and multiple organ injury observed in the necrotising CER-AP. NaTC-AP caused more diffuse pancreatic necrosis, systemic inflammation and multiple organ injury. Circulating histones, as measured by Western blots, were elevated early with further increases in necrotising CER-AP (peak > 100 µg/ml) and NaTC-AP (peak > 140 µg/ml) as disease progressed but not in oedematous CER-AP which were comparable to saline controls. The levels of circulating histones were significantly associated with pancreatic necrosis and multiple organ injury parameters. Circulating histones were then measured from healthy volunteers and a consecutive cohort of acute pancreatitis patients admitted to Royal Liverpool University Hospital (RLUH) within 48 h of disease onset. The predictive values of circulating histones for persistent organ failure (POF), major infection and mortality were compared with biochemical markers and clinical scores. A total of 236 patients (mild 156, moderate 57, severe 23 as per Revised Atlanta Classification) and 47 heathy volunteers were included. The median histone level in severe acute pancreatitis was 18.8 µg/ml (interquartile range: 5.9-33.8), significantly higher than mild 1.1 µg/ml (0.6-2.1) or moderate 1.3 µg/ml (0.5-2.8) category which was comparable with heathy volunteers 1.0 µg/ml (0.5-1.6). The area under the receiver-operating characteristic (AUC) curve of histones for predicting POF and mortality was 0.92 (95% confidence interval [CI]: 0.85-0.99) and 0.96 (0.92-1.00) respectively, which was as or more accurate than tested biomarkers or clinical scores. For infected pancreatic necrosis and/or sepsis, the AUC of histones was 0.78 (0.62-0.94). Histones did not predict or correlate with local pancreatic complications and transient organ failure, but negatively correlated with leucocyte cell viability (r = -0.511, P < 0.01). A study of consecutive acute pancreatitis patients with primary admission to RLUH (n = 260, blood sampling < 24 h) or referred (n = 52) from other hospitals and healthy controls (n = 47) were recruited. Referred patients had POF > 48 h (blood sampling < 24 h of admission to ICU of the RLUH then daily for one week) within 3 weeks of disease onset. Histones, cytokines and routine biochemical markers were measured. Multivariable analyses determined associations between circulating histone levels and variables. There were 235 patients in Group 1 (no POF), 25 in Group 2 (POF < 24 h) and 52 in Group 3 (POF > 48 h). Circulating histones were significantly correlated with tested proinflammatory cytokines, clinical severity scores and individual organ injury parameters. Circulating histones were significantly more elevated in Group 3 compared to Group 2 but both were higher than in Group 1 or healthy volunteers. Multivariable analyses revealed that it was POF, but not pancreatic necrosis or other variables that most significantly associated with elevated circulating histones (odds ratio: 98.1, 95% CI: 14.4-669.0, P < 0.001).

This thesis is concerned with the development and application of AMPLE, a software pipeline for macromolecular crystallographic Molecular Replacement, to different classes of proteins. The ability of AMPLE to solve protein structures via Molecular Replacement was first explored with two new classes of proteins: coiled-coils and transmembrane helical proteins. The results were very positive, with AMPLE solving 75 of 94 (80%) of the coiled-coil and 10 of 15 (67%) of the transmembrane protein test cases. In both cases the performance of AMPLE was benchmarked against a library of ideal helices. The performance of idea helices was found to be surprisingly good (solving 44 of the coiled-coil and 7 of the transmembrane test cases), but the performance of AMPLE was significantly better. AMPLE's truncation and ensembling pipeline was then applied to the solution of protein structures using very distant homologs, and compared with the performance of the current state-of-the-art in automated Molecular Replacement in MRBUMP. The AMPLE pipeline was able to solve structures that could be be solved using MRBUMP, showing how AMPLE is able to find the evolutionarily conserved structural core from homologs that cannot be accessed using existing protocols. Work was also carried out to optimise AMPLE's cluster and truncate procedure. This has resulted in a significant improvement on AMPLE's ability to solve the structures in a difficult set of test cases (solving 11 of 18 test cases compared with 6 for the original protocol), despite only a modest increase in processing time. As part of this work, AMPLE has been extended from a prototype piece of software consisting of a collection of independent scripts, to a coherent, modularised program incorporating a range of software best practice.

Environmental change will have significant impacts on microbial ecosystems. Microorganisms dominate most biogeochemical pathways, and environmental perturbations may alter these functions. Such functions include nutrient cycling, pollution abatement and greenhouse gas emission, and it is paramount that the impact of environmental change on ecosystems is understood. High throughput DNA sequencing provides a window into complex microbial communities and their functional potential, thus allowing us to empirically study how such communities respond to predicted future environments. There are, however, caveats and challenges associated with such technologies, particularly with converting billions of sequencing base calls into species and function counts. This thesis firstly quantifies the performances of sequence annotation tools and parameters using a simulated metagenome. It is found that tools differ in performance, and that parameter selection can significantly reduce annotation accuracy e.g. One Codex correctly annotated many sequences at the genus level, whereas MG-RAST RefSeq produced many false positive annotations. The results provide a guideline to quantitatively inform researchers about the impacts of certain choices on annotation performance, and show that some published studies may be drawing incorrect conclusions. This thesis also investigates the impacts of increased flooding frequency and duration on soil microbial ecosystems, in line with predicted climate change. Increased frequency has significant impacts on biodiversity, community composition and potential function. SkyLine, a novel, continuous gas flux measuring system, was used to record CO2 and CH4 fluxes. Increased flooding duration significantly reduced CH4 oxidation and increased CO2 assimilation, with the combined global warming potential of these gasses reduced.

The unfolded protein response (UPR) is a highly conserved mechanism by which all eukaryotes respond to endoplasmic reticulum (ER) stress. In higher eukaryotes this response is mediated by three ER transmembrane stress sensors: activating transcription factor 6 (ATF6/), PKR-like ER kinase (PERK) and inositol requiring 1 (IRE1/). IRE1 is the most highly conserved of the three ER stress sensors and is also the only sensor to mediated UPR signalling via two different enzymatic domains. It is currently believed that during prolonged ER stress, the RNase domain of IRE1α provides cytoprotection via XBP1 splicing, whilst the kinase domain initiates proapoptotic JNK signalling via interaction with the adaptor protein TRAF2. However, characterising how these domains contribute to cell fate decisions is complicated by the fact that traditional models use ER stress mimetic drugs, which activate all three branches of the UPR and thus make it difficult to attribute downstream events to individual effectors. Therefore, the aim of the research presented in this thesis was to produce a model that allowed isolated activation of IRE1α in order to determine the contribution of its kinase and RNase activities to proapoptotic JNK signalling, without input from other upstream effectors. Using the Fv2E-IRE1α system, the data presented in this thesis provides novel insight into the mechanism by which IRE1α instigates proapoptotic JNK signalling by suggesting that a functional kinase domain is not required for IRE1α to interact with TRAF2 and that endoribonuclease function may be required for downstream JNK activation in humans. Furthermore, evidence is also provided to suggest that, whilst kinase activity is not required for interaction with TRAF2, it is required for downstream JNK activation. This gives rise to the possibility that, contrary to current knowledge, the IRE1α kinase domain has the capacity to phosphorylate proteins other than IRE1α.

Plant NBLRR proteins are immune receptors named for their characteristic domains. Their mode of action is currently undetermined. The potato NBLRR protein Rx1 has been shown to possess a DNA binding activity in vitro. This thesis presents evidence that Rx1 binds DNA in response to its cognate elicitor CP106 in fixed N. benthamiana leaf material using a novel FRET-FLIM assay. The Rx1 CC and NBARC domains were both shown to possess this DNA binding activity. A nucleocytoplasmic distribution of Rx1 was shown to be required for DNA binding. Potential regulators of Rx1 DNA binding activity were identified using a yeast 2-hybrid screen against the CC domain of Rx1 and their effects on Rx1 DNA binding and Rx1 mediated immunity characterised. The transcription factor NbGLK1 was identified and characterised as a promoter of Rx1 DNA binding using FRET-FLIM and a promotor of Rx1 mediated extreme resistance to PVX. However, NbGLK1 was not found to affect Rx1 mediated HR. The protein NbMLHP was also identified in the yeast 2-hybrid screen. This protein was not found to impact Rx1 DNA binding in FRET-FLIM assays. It was, however, identified as a suppressor of Rx1 mediated extreme resistance to PVX (but not HR), and Rx1 did inhibit NbMLHP DNA binding.

Protein palmitoylation is an attachment of palmitate which is one of the most common post-translational modifications (PTMs) of proteins. It is important in various cellular events, and dysregulation has been observed in many diseases including cancer and neurological/neuropsychiatric disorders. There is increased interest in the development of novel therapeutic agents targeting palmitoyl acyltransferases (PATs); however, progress in understanding palmitoylated proteins/PATs has been slow due to the nature of modification and the lack of reliable assays until the recent development of the bioorthogonal ligation technique. Within the rich array of palmitoylated proteins, the main focus of this thesis was on Hedgehog (Hh) proteins which are irreversibly palmitoylated unlike the majority of reversibly palmitoilated proteins. Hh proteins act as morphogens in embryonic development and play a crucial role in tissue homeostasis in adult organisms. Mutations in various Hh signal transduction effectors and aberrant Hh signalling results in developmental malformation and tumourigenesis in adults. Hh proteins are dual lipidated (N-terminal cysteine by palmitate and C terminal glycine by cholesterol) in order to produce mature, fully-functional Hh signalling ligands. Irreversible N-palmitoylation of Hh proteins is catalysed by a member of membrane bound O-acyltransferases (MBOATs) called Hedgehog acyltransferase (HHAT). Efforts have been made to find pharmacologic agents to control the Hh signalling pathway; however, inhibition of downstream Hh signal components has led to the emergence of drug resistance, thus there being an urgent need to circumvent such issues. RU-SKI candidate HHAT inhibitors have opened new avenues to control Hh signalling by inhibiting palmitoylation of Hh ligands, hence attenuating the activation of the Hh pathway. This thesis describes the utilisation of the bio-orthogonal ligation technique to profile palmitoylated proteins, particularly the most abundant Hh homologue, Sonic Hedgehog (Shh) protein, using a palmitate mimic, YnPalm, in cell-based assays. After profiling Shh palmitoylation in depth, detailed characterisation of RU-SKI inhibitors has been performed to understand the effect of Shh secretion and cell signal transduction upon HHAT inhibition. Known PAT inhibitors were derived from chemical reporters in order to elucidate the target proteins by chemical proteomics approach. In short, RU-SKI inhibitors are highly selective against HHAT in terms of profiling their effects in global palmitoylation; however, their toxic effects towards signal reporter cell lines have interrogated the accurate read out in cell signalling activity. From the PAT inhibitor characterisation work, it has been reiterated that there are no specific PAT inhibitors available to date.

As the physical boundary separating the cell interior from the outside world, the plasma membrane is arguably one of the most important cellular organelles. Along with the proteins embedded within it, the plasma membrane plays a part in a number of vital roles including intercellular communication, cell motility and homeostasis. Despite this, comparatively little is known about the exact structure of the plasma membrane. The plasma membrane is a lipid bilayer that is widely believed to undergo transient phase separation into domains of different viscosities, which may be responsible for certain membrane properties. By using molecular rotors (organic fluorophores whose fluorescence properties depend on the viscosity of their surrounding environment) in conjunction with fluorescence lifetime imaging microscopy (FLIM), it is possible to generate a viscosity map of a heterogeneous biological system. In this thesis, we use molecular rotors to investigate the viscoelastic properties and phase behaviour of a range of lipid bilayer systems. We begin by using molecular rotors based on a boron-dipyrrin (BODIPY) core to investigate membrane viscosity within model systems, finding that different lipid phases display different viscosities, and visualising lipid phase separation within giant vesicles. We go on to use BODIPY rotors to image viscosity within the plasma membranes of live Escherichia coli bacteria at different temperatures, finding that they display a much higher degree of membrane ordering than previously observed in eukaryotic cells. Next we explore synthetic alternatives to the conventionally used BODIPY rotors in order to address their shortcomings, before using molecular rotors based on a thiophene moiety to determine the viscosity of the plasma membranes of live human cell lines. Our results from these studies suggest that phase-separation may indeed occur within the plasma membrane.

The work presented in this thesis is aimed to develop and evaluate methodologies for noninvasive measurements of metabolism using fluorescence microscopy. The use of 3D cell cultures in biomedical research is increasing and these require appropriate tools and techniques to provide quantitative readouts for image-based studies. Fluorescence lifetime imaging microscopy (FLIM) can provide robust readouts in complex optical samples and here I have investigated its application to map changes in the response of genetically expressed biosensors utilising Förster resonance energy transfer (FRET) in spheroids. In particular, I adapted a FRET biosensor for the activity of a key metabolic enzyme, AMP activated protein kinase (AMPK), by substituting the donor fluorescent protein ECFP for mTurquoise2, in order to improve its performance in FLIM-based assays. I developed spheroid cultures expressing FRET biosensors and studied these using quantitative FRET readouts. To take account of possible influences of the microenvironment of 3D culture on the fluorescence lifetime measurements, I generated spheroids expressing simple fluorescent proteins and expressing an inactive mutation of the FRET biosensor. I evaluated the new AMPK FRET biosensor, demonstrating improved performance for fluorescence lifetime readouts, and compared dose responses for a direct activator of AMPK with the biosensor expressed in “2D” monolayer cultures and in spheroids, consistently observing a uniform response. In contrast, the dose response of an indirect activator of AMPK in spheroids presented a spatially varying AMPK activation. I further explored the application of FLIM to map the readout of a genetically expressed FRET biosensor for glucose and again observed a spatially varying response in spheroids. I then explored cell specific AMPK activities using FRET biosensors in prostate cancer cells and bone marrow stromal cells with a spheroid system of tumour stromal interactions. I also used biosensors for ATP and glucose concentration in a similar manner and undertook measurements of oxygen consumption rates using a metabolic flux analyser. I observed changes in metabolism that indicate the prostate cancer cells were metabolically benefitting from the interaction with bone marrow cells.

Maintaining cellular homeostasis involves a repertoire of intricate systems being able to respond to internal changes and environmental stimuli. Co-ordinating the process of post-transcriptional gene regulation is a number of ribonucleases, including polynucleotide phosphorylase (PNPase). PNPase controls steady-state transcript levels and thus regulates the production of various proteins, including enzymes involved in central metabolism. A feedback mechanism between central metabolism and RNA turnover has been previously suggested for the bacterium Escherichia coli. The Krebs cycle metabolite citrate was observed to modulate the activity of E. coli PNPase in vitro and in vivo. To discover whether such interactions are conserved across evolution, PNPase homologs from bacteria, eukarya and archaea were studied. Notably, citrate co-crystallises within the active site of Homo sapiens PNPase, suggesting that the citrate-PNPase communicative link may be conserved in eukaryotes. In the current study, a combination of bioinformatics and in silico molecular docking approaches, show that citrate is predicted to bind PNPase and related exoribonucleolytic proteins, from diverse bacterial species, eukaryotic organelles and archaea. Furthermore, in vitro results suggested that PNPase, from another bacterial species Synechocystis sp, may also be susceptible to inhibition/attenuation by citrate, and that this attenuation may therefore be commonplace amongst prokaryotes. Moreover, both eukaryotic PNPase from human mitochondria and the archaeal exosome complex from Sulfolobus solfataricus, is similarly inhibited/attenuated by citrate. The recurring interaction between citrate and PNPase homologs across all three domains, may represent an ancient and evolutionarily conserved mechanism of regulating RNA turnover. Using the same in silico and in vitro approaches, the tricarboxylic acid (TCA) metabolites acetyl-CoA and succinyl-CoA were also shown to affect hPNPase and EcPNPase 3’-5’ phosphorolytic activity. Results indicated that the nucleotide component of CoA in these metabolites, may bind and occlude the active site in a similar way to citrate. Accordingly, other nucleotide-based metabolites were investigated; phosphate-rich nucleotides and signalling molecules (GTP, ppppG, ppGpp) were predicted to bind to the active site of hPNPase. The results from gel-based assays then demonstrated that GTP, ppppG and ppGpp could affect the activity of both hPNPase and EcPNPase. It was also observed that the activity of hPNPase was more affected by these metabolites than EcPNPase and this was supported by previous research that suggested that PNPase homologs, across evolutionarily diverse organisms, have different phosphate preferences. Whether other PNPases can similarly interact with phosphate-rich nucleotides needs to be investigated. Likewise, the in vivo effects and physiological relevance of these metabolite-PNPase interactions remain to be discovered. In summary, this study demonstrates that a metabolite-PNPase regulatory mechanism has the capacity to be conserved amongst all three domains of life and proposes that metabolite-mediated, post-transcriptional mechanisms are widespread. A system where central metabolism can influence RNA stability in a feedback loop, provides another tier of added complexity to the current hierarchal process governing the cellular flow of information. This mechanism potentially facilitates the fine-tuned response that is required to modify cellular functioning for adaptation and or survival. A greater understanding of the intricate network of interactions, occurring in cells, is invaluable for developing novel medical and biotechnological applications.

The relative spatial organisation of protein complexes involved in the light reactions of photosynthesis in plants, (photosystem II (PSII), cytochrome b6f (cytb6f), photosystem I (PSI), and ATP synthase) remains unknown, but is crucial as it determines the diffusive path of mobile electron carriers plastoquinone (PQ) and plastocyanin (PC). Protocols were developed for purifying grana and stromal lamellae membranes for atomic force microscopy (AFM) in model plants spinach (Spinacia oleracea) and Arabidopsis thaliana. The development of methods to purify stromal lamellae from spinach for AFM allowed for the nanoscale imaging of PSI and ATP synthase for the first time. The 3.2 nm, stroma-protruding subunits of PSI were clearly visible in the AFM images, facilitating its identification. Contrary to the consensus that PSI in plants is monomeric, 25% of PSI were found to exist as dimers, which may facilitate diffusion of protein complexes within the stromal lamellae. ATP synthase was present and intact within the purified stromal lamellae membranes and showed the same height and width as purified Bos taurus ATP synthase incorporated into a DOPC lipid bilayer. The implications of the observed organisation of PSII, cytb6f, and PSI for the rate of electron transport were investigated using Monte Carlo simulations. AFM data revealed that long-range ( > 100 nm) diffusion distances are required of the thylakoid luminal protein plastocyanin (PC). The simulations revealed that the diffusion of PC is significantly restricted due the crowded nature of the thylakoid lumen and was affected by the size of the grana/stromal lamellae interface. It was proposed that the size of the interface is, therefore, the result of a compromise between a large interface, which maximises the rate of PC transport, and a small interface which minimises the excitation energy spillover from PSII to PSI.