Recombinant expression and bioinformatic analysis of the Hepatitis B virus X protein

There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer. The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer and has been reported to manipulate several critical cellular pathways. These include the cell cycle, the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of animal models and infectable cell lines together with solubility and stability issues related to the HBx protein have made progress difficult. The reliance on approximate cellular and animal models has yielded many discordant studies that have confounded our interpretations of the role of HBx. There have been no novel approaches attempting to express HBx at a quantity and quality sufficient for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be responsible for these challenges have not been reported. This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus (SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed HBx. This result was despite the positive visualisation of HBx using the same antibodies and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity of HBx. Furthermore these findings supported the decision to develop eukaryotic-based HBx expression vectors as results suggested structural differences between eukaryote and prokaryote expressed protein. HBx was subsequently detected and purified in a soluble and active form using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point from which further work at optimising HBx expression and structural elucidation can occur. Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly regulated and thus experimental systems employing overexpression methodologies may encourage cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding synonymous and non-synonymous substitutions at the overlapping and non-overlapping domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution occurred in the overlapping partner gene. The presence of sites under positive, neutral and negative selection were identified across the length of HBx. The different genotypes showed positive selection indicating selective pressures unique to each, thus offering a contributing explanation for the variable disease severity observed between the subtypes. Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and mammalian cells. These methods, together with an increased understanding of the nature of HBx sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the progression to liver cancer

Biological hydrogen production using an anaerobic fluidised bed bioreactor

Faculty of Science School of Molecular and Cell Biology 9904041r [email protected] production of H2 was monitored using an automated, semi-continuously fed anaerobic fluidised bed bioreactor containing 2 facultatively anaerobic bacteria, Enterobacter cloacae (E. cloacae Ecl) and Citrobacter freundii (C. freundii Cf1). Shake flask tests using Endo formulation with modified C:N:P ratios, showed that a 334:28:5.6 ratio gave the highest attached counts of E. cloacae Ecl and C. freundii Cf1 in both single and binary species biofilms grown on granular activated carbon. Once the reactor had achieved steady state after 30 days using the modified C:N:P ratio, pH, redox potential, temperature, volatile fatty acids and the H2 production rate were monitored. The H2 production rate of 95 mmol H2 / (l x h) compared favourably with previous studies. Bacterial biofilms counts for both E. cloacae Ecl and C. freundii Cf1 remained high around 9.0 log cfu/g granular activated carbon, although biomass overgrowth could not be controlled in the reactor. The efficiency of converting sucrose into H2 was calculated at 20.5%. Therefore use of this technology to power a 5.0kW proton exchange fuel cell for a single rural household is currently not feasible due to the high organic load required. Pooling of wastewater generation capacity, improvement of bacterial strain selection and feed formulation, pH control, gas removal and purification are factors that need to be considered for future improvement of conversion efficiencies. Use of this technology would be most suited for industrial processes generating large volumes of wastewater high in carbohydrates. Alternatively, municipal wastewater treatment facilities could be converted into electricity generating facilities through the combination of this technology and proton exchange membrane fuel cells

Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma

Primary liver cancer is the fifth most common malignancy in the world and is a leading cause of cancer-related mortality. Available treatment for hepatocellular carcinoma (HCC), the commonest primary liver cancer, is rarely curative and there is a need to develop therapy that is more effective. Specific and powerful gene silencing that can be achieved by activating RNA interference (RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy. Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus (HBV) and hepatitis C virus (HCV). Proof of principle studies have demonstrated promising results, and an early clinical trial assessing RNAi-based HBV therapy is currently in progress. Although the data augur well, there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized. Particularly important are the efficient and safe delivery of RNAi effectors to target malignant tissue and the limitation of unintended harmful non-specific effects

Search for Scalar Diphoton Resonances in the Mass Range 65−60065-600 GeV with the ATLAS Detector in pppp Collision Data at s\sqrt{s} = 8 TeVTeV

A search for scalar particles decaying via narrow resonances into two photons in the mass range 65–600 GeV is performed using $20.3\text{}\text{}{\mathrm{fb}}^{-1}$ of $\sqrt{s}=8\text{}\text{}\mathrm{TeV}$ $pp$ collision data collected with the ATLAS detector at the Large Hadron Collider. The recently discovered Higgs boson is treated as a background. No significant evidence for an additional signal is observed. The results are presented as limits at the 95% confidence level on the production cross section of a scalar boson times branching ratio into two photons, in a fiducial volume where the reconstruction efficiency is approximately independent of the event topology. The upper limits set extend over a considerably wider mass range than previous searches