Exploring Genetic Susceptibility: Using a combined systems biology, in vitro and ex vivo approach to understand the pathology of ulcerative colitis

The overall aim of this PhD is to use a multidisciplinary approach to determine the function of Ulcerative Colitis (UC) associated SNPs, to help understand the role of SNPs in the pathogenesis of UC in general and in a patient specific context. UC is a chronic, relapsing inflammatory disease of the large bowel for which the aetiology is thought to be a trifecta of 1) dysregulation of the immune system in response to 2) an environmental trigger in a 3) genetically susceptible host. Genetic susceptibility or susceptibility loci for UC have been identified by Genome Wide Associations Scanning and subsequent fine mapping and deep sequencing. This work intended to further characterise these susceptibility loci at a global level and a patient specific level using both a systems biology approach and experimental validation of the in-silico work. Using publicly available datasets non exonic UC associated SNPs were functionally annotated to regulatory regions within the genome. Exonic SNPs were also analysed looking at impacts in protein linear motifs and splice enhancement motifs. Bioinformatics was used to identify interacting proteins and create a UC-interactome network. This suggested that UC was a disease of fine regulators as opposed to a disease of specific target proteins. Analysis of the UC-interactome identified the focal adhesion complex (FAC) that is involved in regulating wound healing as major component of the network. One member of the FAC, Leupaxin (LPXN), was identified as a potential target for validation. Using CRISPR-Cas9 technology, LPXN overexpressing cell lines and knock out cell lines were created. Wound healing assays and cytokine analysis identified that overexpression of LPXN impaired wound healing and reduced the secretion of MCP-1. In addition, using genotyped colonic biopsies from UC patients and control patients in a polarised in vitro organ culture (pIVOC) system we show that the LPXN risk allele may impact on cytokine production. Finally, UKIBD genetics consortium data was used to access a pilot dataset of 58 patients’ SNP profiles from Immunochip data who were patients at the Norfolk and Norwich University Hospital to create patient-specific UC-interactomes. Analysis of these footprints identified convergent interacting proteins affected by multiple SNPs and novel pathogenic pathways

CHARACTERIZATION OF P53-MEDIATED TRANSCRIPTION DEREGULATION BY THE HEPATITIS B VIRUS X PROTEIN

Ph.DDOCTOR OF PHILOSOPH

Towards ending maternal and infant preventable deaths : omics tools to support vaccine development against Plasmodium falciparum malaria and Streptococcus agalactiae disease

The United Nations sustainable developmental goal 3 “good health and well-being” includes the aim to significantly reduce global maternal mortality and preventable deaths of newborns and children under 5 years of age until the year 2030. Two major contributors to global maternal and infant morbidity and mortality are Plasmodium falciparum severe malaria and Group B Streptococcus (GBS) invasive disease. The central aspect in the WHO strategy towards the elimination of these two diseases is the development of effective malaria and GBS vaccines. In the case of malaria, the immunization with radiation-attenuated P. falciparum sporozoites (PfSPZ) has been shown to convey protective immunity against controlled human malaria infection (CHMI), making this a promising vaccination approach. However, the molecular mechanisms underlying protective anti-malarial immune responses as well as the reasons for the poor immunogenicity of the PfSPZ vaccine in malaria-experienced individuals compared to malaria-naïve volunteers, remain poorly understood. Emerging system analysis approaches, including genome-wide accession of gene expression using RNA-Sequencing (RNA-Seq) provide valuable insight into post-vaccination systemic molecular dynamics and can help to identify immunological correlates of protection. In the case of GBS, multivalent glycoconjugate vaccines, targeting selected GBS capsular polysaccharide types, are currently under clinical trial evaluation. With demonstrated good safety and immunogenicity profiles, the licensure of such vaccines is foreseeable. Large-scale monitoring of vaccine recipients for GBS carriage and assessment of vaccine impact on vaginal colonization, potential serotype replacement and emergence of escape strains will be an important aspect of post-licensure epidemiological studies. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), has emerged as the method of choice for high-throughput microbial species identification in clinical microbiology and has been suggested for strain level typing of bacteria. The overall aims of this thesis therefore included to (i) evaluate the safety and protective efficacy against CHMI of PfSPZ vaccination in Tanzanian volunteers and (ii) elucidate gene expression dynamics in unvaccinated Tanzanian volunteers following CHMI and (iii) to establish a MALDI-TOF MS typing method for GBS for rapid screening of circulating and emerging genotypes. Building on these objectives, the here presented thesis is structured around five manuscripts

Molecular characterisation and functional analysis of eEF1B subunits in mammals

During the elongation of the polypeptide chain in eukaryotic protein synthesis, GTP-bound eukaryotic translation elongation factor 1A recruits the aminoacyl tRNA to the A-site of the ribosome. The GDP-GTP recycling is catalysed by the elongation factor 1B complex (eEF1B) which in higher eukaryotes consists of three different subunits: alpha, delta and gamma. Previous studies on eEF1B focused mainly on biochemical analysis and reports of overexpression in tumours and correlation to decreased survival rate but not a lot is known about is biology. The aim of this PhD is to characterise the eEF1B subunits at the molecular level in view of their potential involvement in tumourigenesis using a variety of bioinformatic and laboratory techniques. All three subunits were found to be ubiquitously expressed at mRNA and protein levels in all mouse tissues analysed. In addition, eEF1Bβ has several transcript variants in mice derived from alternative splicing and multiple isoforms, including a brain and testis specific heavier isoform and a muscle-specific form in addition to other forms. The characteristics of each eEF1B subunit were catalogued by further bioinformatic analysis. eEF1Bα was not detectable at early mouse developmental stages, eEF1Bβ showed stronger expression at pre-natal and early post-natal stages than adult stage whereas eEF1Bγ is ubiquitously expressed at similar levels throughout mouse development. In adult mice and human tissues, eEF1B subunits appeared to be expressed in different cell types and cell sub-populations. Surprisingly, cytoplasmic and some nuclear expression was observed in vivo. This nuclear expression pattern could not be observed in cell lines and it was not related to the cell cycle stage in vitro. The expression of eEF1B subunits did not change during the cell cycle except eEF1Bγ which was highly expressed in S-phase arrested cells. Knockdown by siRNAs of eEF1B subunits leads to decreased proliferation, increased number of cells in G0/G1 phase and increase in apoptosis in HeLa, HCT116, DLD1 and HepG2 cells. In contrast, overexpression in HeLa cells with a V5-tagged constructs lead to increased proliferation, increased number of cells in the G2/M phase and increased viability. Knockdown of eEF1Bα and eEF1Bβ leads to a reduction in eEF1Bγ levels; it is therefore possible that the phenotype shown by the knockdown of each subunit individually might be due to the reduced levels of eEF1Bγ. However, overexpression of each subunit did not affect the protein levels of the other subunits. The presence of multiple forms, the complex expression pattern and distribution of each eEF1B subunit in mouse and human tissues, and the knockdown and overexpression effect on cells suggests that the eEF1B complex might have different quaternary forms throughout development and in different cell types, possibly a more intricate role in translation, potential non-canonical functions any of which may be implicated in the potential role of eEF1B subunits in tumourgenesis