Long terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as alternative promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported that a member of a specific class ofLTR elements (THE I B) in Hodgkin's Lymphoma (HL) acted as a promoter for the growth factor receptor gene CSF 1 R and that this gene is required for HL cell survival. However, to which extent and how such elements participate in shaping the unique gene expression program of HL is unknown. To address this question we mapped the genome-wide activation ofLTRs in HL using a novel targeted next generation sequencing approach (RACE-Seq). Integration of such data with global gene expression as well as chromatin profiling data from HL and non-HL cell lines discovered a unique pattern of LTR activation impacting on gene expression, including a number of genes associated with the HL phenotype. We also show that global LTR activation is induced by activation of inflammatory signaling pathways. Together these results demonstrate that LTR activation presents an additional layer of gene expression deregulation in HL and highlight the potential for the impact of genome-wide L TR activation in other inflammatory diseases.

Heat shock protein 70 (HSPA/HSP70) gene expression is induced by a wide range of cellular stress conditions. This study investigated HSPA/HSP70 expression in human cell lines exposed to hypoxic conditions, in cancerous and non-cancerous brain tissue specimens from 18 patients (gliomas and normal conditions), and in post mortem rat brain samples exposed to heat shock. Three human glioma cell lines were chosen for this study, each representing various types of glioma: (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. In addition, 18 clinical brain tissue samples were also examined. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in glioblastoma tissue were 1.8 and 8.8 fold higher respectively than in lower grade glioma and control tissues, which is suggestive of a grade related transcription profile. Similar patterns of grade related expression were also observed in corresponding cell lines. The percentage of cells showing positive for HSPA protein in normal cell lines increased from 0 to 33% immediately after exposure to hypoxia, and gradually declined to 11% 24 h after treatment. However, the effects of hypoxia were marginal in glioma cells, due to the already elevated levels of HSPA. Although hypoxia induced HSPA expression in normal cells, it did not achieve the same level of induction in cancer cells, suggesting that there are other factors which contribute to the induction of HSPA. These results suggest that HSPA is induced in cancer cells, not only by hypoxia, but also by other factors. In addition, this study indicated for the first time that HSPA expression in glioma cells may possibly be grade related, and thus may have value as a prognostic marker. However a greater sample size is needed to validate such findings. This study showed that HSPA is expressed at low levels in normal brain tissue, but was more highly expressed in brain tissue subjected to mild heat shock. The levels of HSPA transcripts in heat shocked post mortem brain tissue showed a marked increase in HSPA expression. GAPDH was used as a control gene for these studies, and exhibited a consistent level of expression in normal and tumourous cell lines and tissue samples under normal and hypoxic conditions, and also in post mortem tissues exposed to heat shock. For Homo sapiens GAPDH, the average transcript numbers for normal and tumourous cell lines and brain tissue samples were approximately 145,000 copies per sample. For Rattus norvegicus GAPDH, levels were higher than for human samples, at an average of 268,300 copies per sample. The consistency of these results confirms that GAPDH was a suitable candidate gene for the purpose of this study. Early in the post-mortem period, HSPA is expressed more highly in tissues subjected to single and multiple heat shocks compared to controls. However, later post-mortem intervals of between 3 - 24 h demonstrated inconsistent and irregular results, with no predictive or reproducible patterns. Therefore, although there is demonstrable de novo expression of HSPA in post mortem brain tissue in response to heat shock, it is difficult to predict the full parameters of this induction, probably as a result of other forms of cellular stress affecting these tissues under our experimental methodology. These initial studies indicate that the use of HSPA with the methodologies employed here are not suitable as an accurate indicator of post-mortem interval.

In the absence of any vaccine, prophylactic drug and effective vector control, the fight against human African trypanosomiais (HAT) is based on the the combination of active case-finding and consequent drug treatment of identified positive cases. Unfortunately, low sensitivity and specificity of current diagnostic techniques often result in misdiagnosis, leaving infected patients without cure or exposing them to inappropriate chemotherapy protocols, which use dangerous and expensive drugs. The development of more efficient, simple, cheap and field-robust diagnostic tests is, therefore, urgently needed. In the field, direct observation by light microscopy of trypanosomes in human fluids (blood, lymph node aspirate, cerebrospinal fluid) is considered the ideal way of confirming HAT infection. However, in practice this approach is problematic, especially for the Gambian form of the disease, where patients may present with very low parasitaemia. Detection limits of parasitological techniques can be improved by adding a preliminary step of sample concentration, although this further increases the laboriousness of HAT diagnostic algorithm. Recent advances in fluorescence microscopy could be exploited to facilitate trypanosome detection. The introduction and implementation of fluorescence microscopy in HAT endemic countries would offer the advantages of an increased overall sensitivity of microscopical examination and a more rapid screening of the specimen. In contrast to traditional, expensive and fragile fluorescence microscopes, new LED-illuminated instruments are relatively cheap, very efficient and portable, lending themselves to utilisation in poorly equipped rural settings. In order to design a new diagnostic tool that exploits LED technology, however, selective and reliable fluorescent markers to label trypanosomes in human fluids are needed. The development of new tools to assist in the diagnosis of African trypanosomiasis by use of LED fluorescence microscopy was the overall objective of this project. The work was mainly focused on testing various fluorescent compounds for their ability to selectively stain trypanosomes. Fluorophores were otained from commercial and academic sources, or else directly synthesised during the project. An important requirement evaluated was the compounds’ compatibility with the currently available SMR LED Cytoscience fluorescence microscope, developed and kindly provided by our collaborator Prof. D. Jones (Philipps University, Marburg). The utility of a UV LED-driven microscope in performing the arsenical drug resistance test was also assessed. This assay, developed in our laboratory to detect trypanosome strains resistant to arsenical and diamidine compounds, could represent a useful tool for chemotherapeutic decision making in the field, where resistance to arsenical drugs is a rising problem.

During the past few decades a new approach has emerged for the treatment of human disease. In that short period, the concepts and techniques of gene therapy have progressed from being entirely fanciful to experimental clinical application. A major stumbling block for gene therapy is the inefficiency of gene transfer and the transient nature of therapeutic gene expression. Attempts to deliver therapeutic genes using replication-defective adenoviruses have been hampered by a strong host immune response to the vector, leading to clearance of transduced cells and loss of transgene expression. Current adenovirus vectors have an additional disadvantage of being able to carry only approximately 10 kb of exogenous DNA. The work here describes attempts to improve upon existing adenoviral vectors by addressing these limitations In order to reduce the host immune response to the vector, additional deletions in the residual viral coding regions are required to prevent expression of immunogenic proteins. Deletion of the major late transcription unit (MLTU), which encodes virtually all of the viral structural proteins, would achieve this and would also increase the transgene carrying capacity of the vector. In order to create such a vector, a cell line would be required to complement the growth of the deleted vector by providing late gene functions in trans. The work presented here describes the successful cloning and subsequent analysis of the Ad5 MLTU with expression driven by the major late promoter (MLP). The expression plasmids constructed express one or more late proteins from each late gene segment in a transient assay. The plasmids also carry the EBNA-I and oriP sequences from Epstein-Barr virus which allow the plasmids to be stably maintained in eukaryotic cells. Stable cell lines were constructed using these plasmids but no late protein expression from the MLTU could be detected. Attempts to activate expression from the major late promoter by providing viral transactivating factors in trans also proved to be ineffective. To address these problems, an alternative inducible promoter was chosen. The sheep metallothionein Ia promoter was cloned upstream of the MLTU and this construct was then cloned into an episomal expression vector. This plasmid was also used successfully to regulate the expression of late proteins during transient transfection studies. However, a stable cell line constructed using the same plasmid did not show any expression of late proteins. The reasons for the inability of any cell line constructed to express late proteins are still undetermined. Possible reasons discussed are plasmid rearrangement, promoter down-regulation and possible blocks to post-transcriptional processing and translation of the complex MLTU transcript. Suggested future studies include testing of these possibilities in order to gain further insight into the regulation of expression from the MLTU construct, ultimately leading to the construction of a cell line capable of complementing the growth of adenovirus vectors with late gene deficiencies.

Gene therapy is a novel approach to the treatment of human disease that is in its very early stages of development. Its purpose is to add to and/or alter the pattern of gene expression in cells so as to achieve a therapeutic benefit and is being developed for application to such diverse conditions as simple inherited diseases, cancer, AIDS, and cardiovascular disease. Poor uptake of DNA by cells in vivo is a significant technical barrier for gene therapy. Viruses have evolved to carry their nucleic acid contents into cells very efficiently and are thus considered as potential vectors for gene therapy, provided their pathogenicity and other adverse features can be overcome. Adenovirus is a virus type, which is a prime candidate for development as a vector for efficient transfer of genes into human tissues. In recent years, there have been many studies in order to develop replication deficient adenoviral vectors as gene therapy vectors. Current disadvantages of these vectors include their limited capacity to accommodate exogenous DNA and elucidation of a host immune response against these vectors. Currently they have a capacity for about 9 kilobasepairs of DNA, which is not sufficient for some therapeutic purposes. The work described in this thesis aimed to provide a way to produce highly deleted adenoviral vectors containing deletions in their L1 52/55-kD, L1 52/55-kD and IVa2, or L1 52/55-kD and IX coding regions. These genes encode proteins, which contribute to virus particles, and their assembly and some of which have been reported to increase expression of the viral late (structural protein) genes. Together with the deletions available in currently used vectors, these new deletions would create vectors with an increased capacity to accommodate exogenous DNA. Furthermore, these deletions were expected to make the vectors more replication-deficient and less immunogenic by decreasing the expression of residual viral genes. Initially, cell lines expressing adenovirus L1 52/55-kD, L1 52/55-kD and IVa2, or L1 52/55-kD and IX proteins were constructed for use as complementing cell lines. These were designed to supply in trans the relevant proteins that would be missing during attempts to construct recombinant viruses with deletion mutations in these gene(s). 293-L1 cells were proven in their ability to complement missing L1 52/55-kD protein function using existing adenovirus L1 52/55-kD point mutants ts369 and H5pmSOO1. However, attempts to isolate an L1 52/55-kD coding region deleted recombinant adenovirus using this cell line were unsuccessful, possibly due to the nature of sequences missing in the deleted L1 52/55-kD coding region which might have currently undefined cis acting regulatory functions. L1 52/55-kD protein is known to form a complex with IVa2 and the latter protein has been reported to activate the major late promoter. The effects of L1 52/55-kD protein on the adenovirus major late promoter transactivation were therefore investigated by transient expression experiments carried out by transfection of COS cells with a major late promoter-dependant reporter gene (CAT) and expression vectors for L1 52/55-kD and/or IVa2. These experiments did not reveal any role for the L1 52/55-kD protein in the activation of adenovirus major late promoter.

Contamination of dry surfaces with infectious pathogens can have a significant role in infection spread, particularly if the pathogen is resistant to environmental stressors and the infectious dose is low. The use of antimicrobial surfaces in high risk clinical and community environments could help to reduce cross contamination. Although copper alloys have been known to have medicinal properties for centuries it is only relatively recently that laboratory studies have demonstrated that alloys containing over 60% copper have antimicrobial properties. This study encompasses work done 2008-2013 which has continued to investigate this premise, looking at efficacy of copper and copper alloys to kill newly emerging pathogens which are proving to be a significant risk to global healthcare and also determining the mechanism of pathogen destruction on copper surfaces. Stainless steel which is ubiquitous, partly because of resistance to corrosion, was used as a control surface throughout. Initial work demonstrated that clinical isolates of vancomycin- resistant enterococci were rapidly killed on copper alloy surfaces within a few minutes to 2 hours dependant on the copper content of alloy, size of inoculum or aqueous content of the contamination (mimicking either wet droplet or dry fingertip touch contamination of fomites). In contrast, enterococci persisted on stainless steel for several months. Following increasing concerns about the emergence of infections caused by Gram-negative pathogenic bacteria these studies identified a rapid kill on copper alloys but not stainless steel of food-borne pathogens Escherichia coli O157 and Salmonella, and also multidrug-resistant E. coli and Klebsiella pneumoniae containing the β-lactamase genes bla CTX-M-15 and bla NDM-1, respectively (which are responsible for a wide range of community and hospital acquired infections worldwide with diminishing effective therapies). Further studies identified that release of Cu(I) and Cu(II) ionic species was requisite for copper surface antibacterial toxicity but significant differences in killing mechanism was observed between Gram-positive and Gram-negative bacteria related to their structural dissimilarities. Exposure to copper alloys inhibited respiration in all bacteria tested. Bacterial genomic and plasmid DNA was rapidly destroyed in Gram-positive cells but the cell membrane was not compromised immediately; however in Gram-negative cells the inner cell membrane was immediately depolarised on contact with copper alloys but the DNA breakdown occured more slowly. The outer membrane of Gram-negative bacteria remained intact upon initial contact with copper surfaces. Reactive oxygen species (ROS) are also generated so that in effect the bacteria ‘commit metabolic suicide’ on copper surfaces: hydroxyl radicals generated by Gramnegative bacteria suggested a role for a Fenton reaction although the importance varied between species. In enterococci short term production of superoxide was the principle ROS. The nucleic acid destruction observed in all bacteria tested could prevent the horizontal transfer of antibiotic resistance or virulence genes and allay concerns about the possibility of developing resistance to copper. This was supported when it was determined that transfer of β-lactamase genes from E. coli ST131 and K. pneumoniae to recipient E. coli did occur on stainless steel but not on copper dry surfaces and that transfer was immediate in the former. The incidence of carbapenemase gene bla NDM-1 transfer increased with time on stainless steel, highlighting concerns that persistence of viable cells not only poses an infection risk but also that contamination of the environment with intact DNA also increases the risk of gene transfer. These results support the use of copper alloys as biocidal surfaces to kill pathogenic bacteria and prevent horizontal gene transfer (HGT). The final investigation determined that copper alloys were efficacious in inactivating murine norovirus, a close surrogate for human virus, and exposure to copper surfaces destroyed the RNA genome. Copper ions were still responsible directly or indirectly for the inactivation but ROS were not part of the toxicity mechanism. All the results suggest that copper alloys are effective at destroying a diverse range of pathogenic microorganisms although the mechanisms may be different and multi-faceted. Exposure to copper alloy dry surfaces also prevented the horizontal transfer of genes conferring drug resistance and virulence which has been responsible for the continuing evolution of some of the world’s most dangerous pathogens. The results support the use of copper alloys as constantly killing surfaces in healthcare and community environments in conjunction with regular and efficient cleaning and decontamination regimes using non-chelating reagents that could inhibit the copper ion activity. Recent hospital trials now support this thesis.

Leishmania spp. are protozoan parasites that infect humans and other vertebrates to cause a spectrum of disease, ranging from cutaneous ulceration to visceral dissemination dependent on the species. Leishmaniasis is prevalent across the developing world and is a major global health issue, yet difficulties in the efficacy and administration route of current anti-leishmanial treatments means the existing drug repertoire is inadequate. To address this, further research and development measures are necessary to identify Leishmania proteins representing useful targets for drug inhibition. Essential genes encode proteins that are necessary for parasite survival and therefore represent suitable drug targets, but the study of such genes is limited by the absence of a conditional deletion system. A family of proteins which has previously been shown to regulate crucial aspects of Leishmania biology are the protein kinases. Protein kinases have been validated in mammalian systems as drug targets in cancer therapy, therefore they represent a promising avenue for research into anti-leishmanial drugs. The cdc-related kinases CRK3 has been studied in particular depth in Leishmania, and current reverse genetic techniques have implicated expression of CRK3 as essential to promastigote survival. CRK3 regulates the cell cycle as demonstrated by treatment of cdc2 inhibitors, but a lack of a system to regulate expression prevents more specific phenotypic dissection of the role of CRK3. In addition the validation of CRK3 as a drug target has been limited by an absence of a conditional genetic system to ablate the gene in mammalian infective amastigotes. To regulate CRK3 expression in a conditional manner to assess its function in the cell cycle of promastigotes and validate it as essential for amastigotes, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) for use in L. mexicana. Cre recombinase mediates the excision of DNA sequences flanked by 34bp loxP sites (‘floxed’). diCre is encoded as two separate subunits each linked to rapamycin binding domains (FRB and FKBP12); therefore recombinase activity is induced by rapamycin treatment which causes dimerisation of the subunits. Our method involves replacing both CRK3 alleles with a ‘floxed’ CRK3 open reading frame and the diCre coding sequence through promastigote transfection and homologous recombination. Induction of diCre through rapamycin treatment of promastigotes results in highly efficient deletion of CRK3 and a distinct growth arrest phenotype corresponding to a block in G2/M. Induced loss of CRK3 can be complemented by expression of a CRK3 transgene but not by expression of an inactive site (T178E) CRK3 mutant, showing that protein kinase activity is crucial for CRK3 function. Significantly, inducible deletion of CRK3 in stationary phase promastigotes prevents the establishment of murine infection, thereby demonstrating an essential role in the amastigote cell cycle to further validate CRK3 as a drug target. Promisingly, inducible deletion is functional in lesion-derived amastigotes and will enable direct phenotypic assessment following essential gene loss in this life cycle stage. To establish a basis for future in vivo application of diCre in Leishmania, a murine infection model was developed with which to track bioluminescent parasite burden by in vivo imaging and assess innate immune cell recruitment to the site of infection by flow cytometry analysis. The combination of functional gene regulation in amastigotes and measures of parasite burden and immune response will yield a powerful tool for the further study of Leishmania genes encoding suitable drug targets. The application of the diCre technique to Leishmania would be greatly benefitted by targeting genes where there is evidence of a regulatory role of orthologous genes in model organisms. The utilisation of genome or protein family-wide RNAi screens in Trypanosoma brucei has identified a number of protein kinases which regulate the differentiation of the parasite between life cycle stages. The repressor of differentiation (RDK1) protein regulates bloodstream form to procyclic form differentiation in T. brucei, and the identification of a protein in L. mexicana with high sequence identity suggested a potentially analogous role in preventing Leishmania from undergoing amastigote to promastigote differentiation in vivo. To assess this, a cell line was generated deficient in RDK1 but no effect on differentiation was identified, as parasites were able to maintain murine infection and differentiate between life cycle stages. This study represents an important addition to the reverse genetic toolkit to study aspects of cell cycle regulation in vitro, and further assess essential genes as drug targets by deletion in amastigotes. The application of the diCre conditional deletion method will enhance the discovery and evaluation of suitable drug targets in Leishmania by phenotypic analysis.

Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is the commonest cause of hospital acquired infection in the UK. The organism initiates infection through spore formation and attachment, germination in the gut and then the production of two potent cytotoxins; toxins A and B. While the contribution of toxins A and B to infection is beyond dispute the relative importance of each toxin is a subject of debate. Thus diagnostic assays capable of rapidly detecting the presence of both toxins are needed. To develop such an assay we first characterised the structure of C. difficile spores to better understand their role in pathogenicity and adherence to organic and inorganic surfaces. Following attachment the spore germinates and the resulting vegetative bacteria express toxins. To facilitate the development of an assay capable of detecting both toxins, we employed a bioinformatics based approach which identified highly conserved nucleotide sequences within regions of each toxin which we hypothesised were under strict selective pressure. The specificity of the probes identified was confirmed using a panel of 58 clinical C. difficile isolates, related Clostridium isolates, non-related species and human gut metagenomic DNA samples. Selected probes were incorporated into a metal enhanced fluorescent assay platform and their ability to detect the organism in various organic backgrounds was determined. We were able to detect as few as 10 bacteria in 500 μl of human faecal material within 40 seconds, suggesting that this approach has the potential to be developed into a commercial assay. To support the development of this assay we sought to develop an insect infection model using the worm Manduca sexta. Our inability to initiate infection, inspite of the fact that bioinformatic analysis revealed the presence of genes with homology to known insect virulence factors, suggests that C. difficile may have potential evolutionary association to invertebrates.

Feline immunodeficiency virus (FIV) is an important veterinary pathogen with comparative significance because of its similarities to its human counterpart HIV. Since FIV is the only non-primate lentivirus which induces AIDS-like symptoms in its natural host, it serves as a valuable animal model for both prophylactic and therapeutic studies of HIV. It is accepted that the induction of neutralising antibodies (NAbs) is a key element in the control of lentiviral infection, since T-cell based vaccines alone failed to prevent infection in most experimental animal model systems. In this project a robust and reproducible in vitro neutralisation assay was developed and optimised, permitting the assessment of the NAb response in naturally infected cats and with the potential to evaluate candidate vaccines. It was demonstrated that, in general, primary FIV strains in the UK belong to subtype A, and therefore the development of a regional, subtype A-specific, FIV vaccine could be considered for use in the UK. The identification of a neutralisation resistant isolate of FIV led to the finding that a linear neutralisation determinant was located within the V5 region of Env and mutations in this region may lead to immune evasion in vivo. In addition, a second neutralisation determinant was identified in the C3/V4 region of Env. Finally, it was observed that a small proportion of naturally infected cats generated NAbs against FIV. Of these, only a very small proportion of the cats had antibodies with the potential to cross neutralise strains within the same subtype as the homologous isolate. Nonetheless, a plasma sample from a single cat was identified that neutralised all strains tested, including strains from different subtypes and geographical regions. It is likely that studies of the homologous isolate that induced the broad NAb response may be capable of inducing a similar broad response in vaccinated cats. Such a finding would have important implications for the design of potential novel lentiviral immunogens.

Recent advances in genome sequencing are improving our better understanding of genetic variation. However, the investigation of the genotype-phenotype relationship is still challenging, especially for the interpretation of the myriad of discovered genetic variants that weakly relate to disease. Recently, researchers have confirmed that disease causing genetic variants typically occur at functional sites, such as protein-protein or protein-ligand interaction sites. Giving this observation, several bioinformatics tools have been developed. This thesis first details VarMod (Variant Modeller), an algorithm that predicts whether nonsynonymous single nucleotide variants (nsSNVs) affect protein function. The recent Ebola virus outbreak in West Africa demonstrated the potential for the virus to cause edipdemics and highlighted our limited understanding of Ebola virus biology. The second part of this thesis focuses on the investigation of the molecular determinants of Ebolavirus pathogenicity. In two related analyses knowledge of differing pathogenicity of Ebolavirus species is used. Firstly, comparison of the sequences of Reston viruses (the only Ebolavirus species that is not pathogenic in humans) with the four pathogenic Ebolavirus species, enabled the identification of Specificity Determining Positions (SDPs) that are differentially conserved between these two groups. These SDPs were further investigated using analysis of protein structure and identified variation in the Ebola virus VP24 as likely to have a role in determining species-specific pathogenicity. The second approach investigated rodent-adapted Ebola virus. Ebola virus is not pathogenic in rodents but it can be passaged to induce pathogenicity. Analysis of the mutations identified in four adaption studies identified that very few mutations are required for adaptation to a new species and once again the VP24 is likely to have a central role. Subsequent molecular dynamics simulations compared the interaction of Ebola and Reston virus VP24 with human karyopherin alpha5. The analysis suggests that Reston virus VP24 has weaker binding with karyopherins and we propose that this change in binding may reduce the ability of Reston VP24 to inhibit human interferon signaling.