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Chikungunya virus (CHIKV) is a pathogenic, single-stranded, positive-sense RNA virus transmitted to humans by Aedes spp. mosquitoes. After decades of low-level endemic circulation, CHIKV has re-emerged to establish local transmission on five continents, infecting upwards of 3,000,000 people. There are no currently available vaccines or direct-acting anti-viral therapeutic agents. A greater understanding of the CHIKV replication cycle is essential, as much of what is known about the replication cycle is assumed from studies of related but divergent viruses, which have provided conflicting reports. Preliminary work carried out by A. Tuplin (University of Leeds) suggested a highly ordered structured region at the 5′ end of the CHIKV genome, spanning ~300 nt including the 5′ untranslated region (UTR) and the 5′ coding sequence of nsp1. The aim of this project was to determine the phenotypic importance of secondary structure in this region for the CHIKV lifecycle in human and mosquito cells at multiple stages of viral replication and to investigate the sequence and structure requirements for functional interactions. This study represents the first investigation of functional elements within the 5′ UTR and adjacent nsP1-coding region in CHIKV. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, the wild-type secondary structure of the 5′ 300 nt of the CHIKV genome was found to be essential for genome replication in human- and Ae. albopictus-derived cells. Six RNA stem-loops were determined to individually enhance CHIKV genome replication - including novel structures analysed for the first time in this study. Comparative analysis in human and mosquito-derived cell lines revealed that the novel stem-loop SL47 in the 5′ UTR functions in a host-independent manner while stem-loops in nsp1 function in a host-dependent manner. Stem-loops were demonstrated to function within the positive-strand genomic RNA, via predominantly structure-dependent mechanisms. Furthermore, single-host passaging studies suggested strong selection pressure to regenerate secondary structures and highlighted potential differences in translational recoding between host species. Finally, the potential for tertiary structure formation was explored. In addition to furthering knowledge of fundamental aspects of the molecular virology of this important human pathogen, this study will inform rational design of a genetically stable attenuated vaccine.

Despite the importance of calcifications in early detection of breast cancer, and their proposed association with tumour growth, remarkably little detail is known about their chemical composition, or how this relates to pathology. One reason for this gap is the difficulty of systematically and precisely locating calcifications for analysis, particularly in sections taken from diagnostic archives. Two simple methods were developed which can achieve this in sections cut from wax embedded breast tissue. These are based on micro-CT and x-ray fluoroscopy mapping, and were used to locate calcifications for further study. The elemental composition of calcifications in histological sections was measured using energy-dispersive x-ray spectroscopy in an environmental scanning electron microscope. Variations in Ca:P ratio could in principle be detected non-invasively by dual energy absorptiometry, as demonstrated in a proof of principle experiment. However, the Ca:P ratio was found to lie in a narrow range similar to bone, with no significant difference between benign and malignant. In contrast, a substantial and significant difference in Na:Ca ratio was found between benign and malignant specimens. This has potential for revealing malignant changes in the vicinity of a core needle biopsy. The phase composition and crystallographic parameters within calcifications was measured using synchrotron x-ray diffraction. This is the first time crystallite size and lattice parameters have been measured in breast calcifications, and it was found that these both parallel closely the changes in these parameters with age observed in foetal bone. It was also discovered that these calcifications contain a small proportion of magnesium whitlockite, and that this proportion increases from benign, to carcinoma in-situ, to invasive cancer. When combined with other recent evidence on the effect of magnesium on hydroxyapatite precipitation, this suggests a mechanism explaining observations that carbonate levels within breast calcifications are lower in malignant specimens.

Early detection of Barrett’s oesophagus and associated neoplasia is key to preventing progression to oesophageal adenocarcinoma. Improving surveillance and introducing population screening for Barrett’s are major goals of current research: this project aimed to apply emerging techniques in vibrational spectroscopy to these problems. Fourier transform infrared (FTIR) mapping was used to develop an automated histology tool for detection of Barrett’s and Barrett’s neoplasia in tissue biopsies. 45 FTIR maps were measured from 22 tissue samples from 19 patients. Principal component analysis (PCA) fed linear discriminant analysis (LDA) was used to build classification models based on spectral differences, tested using leave one sample out cross validation (LOSOCV). Classification of normal squamous samples versus ‘abnormal’ samples (any stage of Barrett’s) was performed with 100% sensitivity and specificity. Using a 3-group model to differentiate normal squamous, non-dysplastic Barrett’s and neoplastic Barrett’s (dysplasia or adenocarcinoma), neoplastic Barrett’s was identified with 95.6% sensitivity and 86.4% specificity. Non-endoscopic cell collection devices have recently been developed for population screening for Barrett’s oesophagus. A further aim of this project was to evaluate FTIR for classification of oesophageal cells. Cytology brushings were collected at endoscopy, cytospun onto slides and FTIR maps measured. Cytology review and contemporaneous histology was used to inform a training dataset containing 141 cells from 17 patients. A classification model was constructed using PCA-fed LDA. Applying this training model to the entire dataset of 115 FTIR maps from 66 patients, whole samples were classified with sensitivity and specificity respectively as follows: normal squamous 79.0% and 77.0%, non-dysplastic Barrett’s 31.3% and 100%, and neoplastic Barrett’s 83.3% and 54.2%. Raman spectroscopy was also evaluated as a tool for tissue diagnosis, but several strands of enquiry were limited by instrument problems. FTIR mapping could be used as an accurate, automated tool for processing biopsies in Barrett’s surveillance. Analysis of oesophageal cell samples can be performed using FTIR with reasonable sensitivity for Barrett’s neoplasia, though poor specificity with the current technique.