Paleovirology is the study of viruses over evolutionary timescales. Contemporary paleovirological analyses often rely on sequence data, derived from organism genome assemblies. These sequences are the germline inherited remnants of past viral infection, in the form of endogenous viral elements and the host immune genes that are evolving to combat viruses. Their study has found that viruses have exerted profound influences on host evolution, and highlighted the conflicts between viruses and host immunity. As genome sequencing technology cheapens, the accumulation of genome data increases, furthering the potential for paleovirological insights. However, data on ERVs, EVEs and antiviral gene evolution, are often not captured by automated annotation pipelines. As such, there is scope for investigations and tools that investigate the burgeoning bulk of genome data for virus and and antiviral gene sequence data in the search of paleovirological insight.

The aim of this thesis was to further our knowledge about the contribution of silent alleles on megabase chromosomes to the late stages of trypanosome infection and test the hypothesis that this contribution takes shape in a hierarchy of expression due to differences between alleles in terms both of flanking regions and coding sequence. This was achieved through a combination of bioinformatics and molecular studies. The initial approach was to undertake an extensive manual curation of the available VSG archive; this endeavour resulted in establishment of a fertile collaboration with the Trypanosoma brucei genome sequencing project, and in creation, with the aid of P. Ward and S. Menon, of a dedicated web-based tool to handle and query curated VSG genes. Out of an updated estimate of ~1600 VSG genes, 940 (between half and three quarters) were annotated and shown to be arranged in subtelomeric arrays and to be largely present as pseudogenes (~90%). By considering separately the hypervariable N-terminal domain (three types, A, B and C) and the more conserved C-terminal domain (types 1 to 4, with two additional types identified in this study), it appeared that most of the degeneracy lay in the C-terminal domain. This suggested that N-terminal domains (one third of them being intact) would be utilised by a process of segmental gene conversion yielding hybrid genes, by recombination with functional C-terminal ends resident at the expression site. Under the assumption that “order” within the genome (the presence of patterns within the VSG archive) helps inform “order” in VSG expression (a hierarchy based on different activation probabilities), it was somewhat surprising to detect little evidence of clear substructuring within the archive: no “classes” of VSGs could be identified, based on coding sequence and flanking sequence features. In keeping with the observed high level of divergence within the VSG archive, clear orthologue groups (here defined as alleles sharing >60% amino acid sequence identity) were found not to include more than three to four members and to be scattered at random across the arrays. Putative functional genes could not be separated into groups based on expected differences in activation probabilities, such as a different number of upstream 70-bp repeats, shown to be involved in copying silent alleles to the expression site.

Troponin C is a regulatory protein of the myofilament which binds to calcium to trigger the process of contraction. This protein exists in two isoforms, skeletal and cardiac, which are spatially and temporally regulated. Work in this project builds the primary stage of a long-term project, for using the gene transfer method to overexpress the skeletal isoform of Troponin C in cardiomyocytes. The long-term aim is to achieve complete or partial substitution of the native cardiac isoform and study the effects on contractile force produced, in normal and ischemic cardiomyocytes, both in vitro and in vivo. This project has involved designing, constructing and analyzing expression of adenoviral gene transfer vectors overexpressing the sTnC isoform. Several adenoviral vectors were generated with the wild type sTnC gene under the control of muscle-specific promoters. To facilitate analysis of protein expression and its subcellular localization, the sTnC protein was tagged with epitope tags and adenovirus generated, with this gene under the control of constitutive (CMV) and cardiac-specific (HCA) promoters. Epitope-tagged adenoviruses were expressed in vitro using mouse fibroblast (NIH3T3) cells and analyzed by western blot analysis, showing successful constitutive expression. Recombinant adenoviruses containing epitope-tagged-sTnC under the control of the human cardiac actin promoter showed cardiac-specific expression in cultured cardiomyocytes, in situ, using immunocytochemistry. The constitutively-expressing sTnC adenoviral vector showed successful expression in cardiomyocytes in culture, using northern blot analysis. A range of adenoviral vectors have been successfully generated, and constitutive and tissue-specific expression has been established for some of these vectors. Successes attained in this project have established the initial requirements to achieve the long-term goal to alter calcium sensitivity of myofilaments, by overexpression of sTnC isoform in cardiomyocytes, both in vitro and in vivo.

Hodgkin lymphoma (HL) is one of the most common haematological malignancies in the Western world. Although HL responds favourably to cytotoxic therapy in the majority of cases, late side-effects such as secondary malignancies and cardiovascular disease are becoming of great concern, particularly in younger patients. The current challenges are to maintain treatment efficacy whilst reducing side-effects and to develop biomarkers to predict response to treatment(s). Protein degradation pathways in HL cells have remained largely unstudied in this malignancy and represent an opportunity for more targeted therapy. This thesis describes the activities of protein degradation pathways in HL-derived cell lines and their sensitivities to inhibition. The results suggest that the use of proteasome and HDAC6 inhibitors, alone or in combination, may be of clinical benefit in the treatment of HL in the future. The presence of p62 was used to monitor protein handling stress; however, its diverse expression patterns in HL-derived cell lines and in paraffin-embedded HL biopsy material preclude its use as an informative biomarker in HL. p62 was found to traffic between the cytoplasm and nucleus in HL-derived cell lines and its association with a DNA damage marker in both cellular compartments implicate it as a chaperone for the cytoplasmic degradation of nuclear proteins. A previously unreported nuclear expression of the lysosomal enzyme cathepsin B in HL-derived cell lines was identified, and this may have implications for the aberrant transcriptional profile of these cells given the regulatory activities of other members of this family of cysteine proteases. The non-lymphoid origin of the HD-MyZ cell line, a putative HL-derived cell line, was corroborated by phenotypic differences in protein handling pathways in comparison to accepted HL-derived cell lines. The activities and insensitivity to inhibition of protein handling pathways in this cell line suggest that it will be an interesting model for studying alternative protein degradation pathways. The presence of a number of small cell populations with cancer stem cell (CSC)-like characteristics was confirmed in HL-derived cell lines and, although these populations require further characterisation, this has implications for HL patients who suffer relapse.

Hodgkin lymphoma (HL) is a malignant lymphoma that is diagnosed mostly in young adults, and is the second most common malignancy to affect this age group. This disease is subdivided into two entities with different aetiologies: classical HL (cHL) (~95% of cases) and nodular lymphocyte-predominant HL. In Europe, ~82% of young adults with cHL are non-Epstein-Barr virus associated and epidemiological studies have suggested that a common infectious agent may play a key role in the aetiology of these cases. The molecular biology of HL is not well understood, primarily due to the low number of Hodgkin and Reed-Sternberg (HRS) cells present within these tumours. However, recently developed techniques for the selection and micromanipulation of single HRS cells from tumours, and the development of molecular cytogenetic techniques (i.e. array-comparative genomic hybridisation (CGH)) are overcoming these difficulties. To investigate a potential candidate virus, DNA samples from cHL biopsies were screened for the measles virus (MV) and polyomaviruses (PyV), using immunohistochemistry and highly sensitive PCR assays. Chromosomal imbalances in six well-established cHL-derived cell lines and a cHL case were analysed by array-CGH. To obtain sufficient DNA for array-CGH from the cHL case, single HRS cells were isolated using laser microdissection. DNA was extracted then amplified by degenerate oligonucleotide primer polymerase chain reaction. MV and PyV genomes were not detected within cHL biopsies. Recurrent chromosomal imbalances were confirmed within the cHL-derived cell lines and cHL case, in addition to several novel imbalances. This is the first time that a cHL case has been analysed by array-CGH.

Parasite life history theory predicts that lifetime reproductive success evolves through differential allocation of energy to life history traits constrained by trade-offs. These life history traits govern the characteristics of parasites such as their virulence, transmission and infection phenotypes, so understanding their evolution is a key concern for infectious disease prediction and management. This thesis uses the powerful tool of experimental evolution to gain a fuller understanding of the factors and constraints involved in parasite life history evolution, using bacteriophage Φ2 as a model. I found that the evolution of life history in this phage is sensitive to spatial structure, UV-C exposure and coparasitism with plasmids, and evolution can be mediated by co-evolution with the host. The high levels of variance I observed here suggest that evolution of parasite life history is more complex than a single trajectory towards a predicted optimum, and likely involves some degree of epistasis or pleiotropy with genes elsewhere on the genome. There was some degree of independent evolution of individual life-history traits, indicating that simple direct trade-offs were not in operation. I demonstrated that co-evolution with the host provided additional mutational input, resulting in a greater degree of evolution in co-evolved populations than those evolved to a static host. Furthermore, I note that co-parasitism with phage and plasmid may provide the necessary conditions for plasmid persistence under fluctuating selection for plasmid-encoded traits, and that the efficacy and suitability of phage as therapeutic agents against plasmid-encoded antibiotic resistance is complicated. No direct link between mutation and phenotype could be elucidated in this study, suggesting that evolution in life history is either governed by genes not examined in this thesis, or involves epistasis and pleiotropy with genes elsewhere on the genome. I concluded that it is important to consider the specific ecology of the focal parasite, its host and any co-occuring symbionts in order to make informed predictions of life history evolution, and general predictions may not be achievable.

The under-reporting of cases of infectious diseases is a substantial impediment to the control and management of infectious diseases in both epidemic and endemic contexts. Information about infectious disease dynamics can be recovered from sequence data using time-varying coalescent approaches, and phylodynamic models have been developed in order to reconstruct demographic changes of the numbers of infected hosts through time. In this study I have demonstrated the general concordance between empirically observed epidemiological incidence data and viral demography inferred through analysis of foot-and-mouth disease virus VP1 coding sequences belonging to the CATHAY topotype over large temporal and spatial scales. However a more precise and robust relationship between the effective population size (N_e) of a virus population and the number of infected hosts (or 'host units') (N) has proven elusive. The detailed epidemiological data from the exhaustively-sampled UK 2001 foot-and-mouth (FMD) epidemic combined with extensive amounts of whole genome sequence data from viral isolates from infected premises presents an excellent opportunity to study this relationship in more detail. Using a combination of real and simulated data from the outbreak I explored the relationship between N_e, as estimated through a Bayesian skyline analysis, and the empirical number of infected cases. I investigated the nature of this scaling defining prevalence according to different possible timings of FMD disease progression, and attempting to account for complex variability in the population structure. I demonstrated that the variability in the number of secondary cases per primary infection R_t and the population structure greatly impact on effective scaling of N_e. I further explored how the demographic signal carried by sequence data becomes imprecise and weaker when reducing the number of samples are described, including how the extent of the size and structure of the sampled dataset impact on the accuracy of a reconstructed viral demography at any level of the transmission process. Methods drawn from phylodynamic inference combine powerful epidemiological and population genetic tools which can provide valuable insights into the dynamics of viral disease. However, the strict and sensitive dependency of the majority of these models on their assumptions makes estimates very fragile when these assumptions are violated. It is therefore essential that for these methods to be applied as reliable tools supporting control programs, more focused theoretical research is undertaken to model the epidemiological dynamics of infected populations using sequence data.

It is well established that adenoviruses degrade components of the cellular DNA damage response, such as p53, DNA ligase IV and Mre11, in order to avoid detection from the host cell and thus, promote viral replication. Here we show TAB182, a protein of previously unknown function, is degraded following adenovirus serotype 5 and 12 infection. Similarly to other DNA damage proteins, together with the cellular Cullin 5 (during Ad5 infection) and Cullin 2 (during Ad12 infection). Interestingly, siRNA-mediated knockdown of TAB182 appears to be beneficial for adenovirus infection, as denoted by an increased expression of the adenoviral E1A protein and Cyclin E during adenovirus infection. Together with other studies, we confirm that TAB182 interacts with the large, multi-subunit CNOT complex. This complex has no defined function in mammalian cells, but is known to play a role in gene regulation in yeast. Interestingly, components of the CNOT complex are also degraded during adenovirus infection, whether adenovirus degrades TAB182 as well as CNOT for the same advantage is currently unknown. Cells deficient in TAB182 are hypersensitive to agents that induce DNA replication stress and also exhibit abnormal replication dynamics following release from hydroxyurea-induced fork stalling. In particular, they display increased fork restart and elevated new origin firing following release from hydroxyurea treatment, suggesting that TAB182 prevents fork recovery and suppresses new origin firing following replication stress. Depletion of some components of the CNOT complex is able to rescue the phenotypes observed in TAB182 deficient cells, suggesting that TAB182 and the CNOT complex may act in concert at the replication fork. TAB182 deficient cells display less DNA gaps and breaks but increased levels of 53BP1 bodies in G1 and micronuclei, which are markers of genome instability, following replication stress. Whether TAB182 acts directly at the replication fork, or in conjunction with other proteins known to be involved in replication restart such as helicases, nucleases, or chromatin remodelling complexes, remains to be elucidated.

The role of the E4 protein in the human papillomavirus (HPV) life cycle is an enigma even though it has varied effects on cell behaviour and organisation in overexpression studies. Full-length E4 proteins are derived from E1^E4 spliced RNA transcripts and E1^E4 proteins from diverse HPV types interact with serine-arginine (SR)-specific protein kinase SRPK1, that regulates diverse cellular functions including RNA splicing. This thesis has sought to address the hypothesis that E1^E4 alters SRPK1 activity and influences SRPK1 functions in the HPV life cycle. This study has uncovered the novel finding that E1^E4 protein of HPV1, but not HPV5, 16 and 18, is a potent inhibitor of SRPK1 activity in vitro and in vivo and inhibition is dependent upon E1^E4 binding to SRPK1. Whilst HPV1 E1^E4 inhibits SRPK1 phosphorylation of cellular (ASF/SF2, SRp20, SC35, 9G8 and SRp75) and viral (HPV E2) SR protein substrates, it has only weak effects on SR protein cellular localisation and on cellular and viral RNA splicing in minigene systems. Addition of the small molecule inhibitor of SRPK, SRPIN340 to organotypic raft cultures of HPV18 genome-containing keratinocytes enhances the morphological features of HPV viral replication suggesting that the HPV may modulate SRPK activity to facilitate the virus life cycle.