Epigenetic Profiling and Molecular Characterisation of Non-melanoma Skin Cancer

PhDNon-melanoma skin (NMSC) cancer is the most common human malignancy. Cutaneous squamous cell carcinoma (cSCC) and its precursor, actinic keratosis (AK) affect tens of thousands of people each year in the UK. Merkel cell carcinoma is a rare, yet aggressive type of NMSC recently linked with Merkel Cell Polyomavirus (MCPyV). In spite of the clinical burden of NMSC, key molecular regulatory patterns remain largely unknown. The aims of this thesis were to investigate genome-wide genetic, epigenetic and transcriptional changes in AK and cSCC, and assess the prevalence of MCPyV and its effect on methylation in NMSC. Copy-number analysis revealed that AK harbours significantly more genomic aberrations compared to skin, the majority of which occurs on chromosomes 8 and 9. Transcriptional profiling has found 292 and 308 genes as differentially expressed in AK compared to non-sunexposed and sun-exposed skin, respectively, and gene-set enrichment analysis (GSEA) revealed dysregulation of PPAR pathway in this lesion. Expression profiling of cSCC and AK has revealed 346 differentially expressed genes, and GSEA detected dysregulation in several canonical pathways including TGF-β and MAPK pathway. Aberrant methylation in cSCC cell lines occurs in the promoters of many developmental genes. A total of 1085 hyper- and 833 hypomethylated genes were detected in cSCCs, and GSEA revealed dysregulation of critical signalling pathways (WNT, MAPK signalling pathways). Methylation analysis of AK revealed a total of 4194 differentially methylated genes, and implicated FOXF2, PITX2, RUNX1 and SMAD3 transcription factors in this lesions. MiRNA profiling of cSCC and normal skin revealed significant dysregulation of 38 miRNAs including several of viral origin. MCPyV was shown to be common in NMSC, yet MCPyV nor human papillomavirus does not affect cSCC methylation. Taken together, this work provides novel insight into molecular regulation of cSCC oncogenesis, and identifies potential epigenetic targets for functional evaluation in this malignancy.British Skin Foundation and the Barts and the London Charity research grant

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

High Conservation Level of CD8(+) T Cell Immunogenic Regions Within an Unusual H1N2 Human Influenza Variant

Current seasonal influenza vaccines require regular updates due to antigenic drift causing loss of effectiveness and therefore providing little or no protection against novel influenza A subtypes. Next generation vaccines capable of eliciting CD8(+) T cell (CTL) mediated cross-protective immunity may offer a long-term alternative strategy. However, measuring pre- and existing levels of CTL cross-protection in humans is confounded by differences in infection histories across individuals. During 2000-2003, H1N2 viruses circulated persistently in the human population for the first time and we hypothesized that the viral nucleoprotein (NP) contained novel CTL epitopes that may have contributed to the survival of the viruses. This study describes the immunogenic NP peptides of H1N1, H2N2, and H3N2 influenza viruses isolated from humans over the past century, 1918-2003, by comparing this historical dataset to reference NP peptides from H1N2 that circulated in humans during 2000-2003. Observed peptides sequences ranged from highly conserved (15%) to highly variable (12%), with variation unrelated to reported immunodominance. No unique NP peptides which were exclusive to the H1N2 viruses were noted. However, the virus had inherited the NP from a recently emerged H3N2 variant containing novel peptides, which may have assisted its persistence. Any advantage due to this novelty was subsequently lost with emergence of a newer H3N2 variant in 2003. Our approach has potential to provide insight into the population context in which influenza viruses emerge, and may help to inform immunogenic peptide selection for CTL-inducing influenza vaccines. J. Med. Virol. 88:1725-1732, 2016. © 2016 Wiley Periodicals, Inc

Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities

The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus–specific CD8(+) T lymphocytes (CTLs) can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16–57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701(,) B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development

FOXO1 constrains activation and regulates senescence in CD8 T cells

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span

Recovery from severe H7N9 disease is associated with diverse response mechanisms dominated by CD8(+) T cells

The avian origin A/H7N9 influenza virus causes high admission rates (>99%) and mortality (>30%), with ultimately favourable outcomes ranging from rapid recovery to prolonged hospitalization. Using a multicolour assay for monitoring adaptive and innate immunity, here we dissect the kinetic emergence of different effector mechanisms across the spectrum of H7N9 disease and recovery. We find that a diversity of response mechanisms contribute to resolution and survival. Patients discharged within 2-3 weeks have early prominent H7N9-specific CD8(+) T-cell responses, while individuals with prolonged hospital stays have late recruitment of CD8(+)/CD4(+) T cells and antibodies simultaneously (recovery by week 4), augmented even later by prominent NK cell responses (recovery >30 days). In contrast, those who succumbed have minimal influenza-specific immunity and little evidence of T-cell activation. Our study illustrates the importance of robust CD8(+) T-cell memory for protection against severe influenza disease caused by newly emerging influenza A viruses