Co-infection with HIV in patients with chronic HCV infection is a common occurrence, which accelerates disease progression and the rate of liver fibrosis. Current literature report conflicting results on the effect of HIV co-infection on HCV at the genetic level. This thesis sets out to further explore the genetic variation of HCV in this group of patients, and use new techniques which may impact upon the diagnosis and management of HCV. In resource-limited settings such as sub-Saharan Africa, there is a paucity of data concerning rates of active HCV infection in patients with HIV infection. The limited infrastructure and financial restrictions are contributing factors in this. Consequently, cheaper and novel diagnostic procedures are required to promote testing in these important cohorts. A real-time PCR assay for use with pooled plasma and dried plasma spot specimens was developed to screen a large cohort of HIV-infected individuals from Ghana and assess its suitability for screening in such a setting. The prevalence of active HCV infection in this cohort was similar to previous estimates from blood donors but was much lower than estimates from serological tests, highlighting the potential risks in relying on these tests alone in this region. The diversity of strains found within this population agreed well with previous studies. As the yield of HCV infections was low in Ghana, further studies were completed with the UK and European cohort. A deep sequencing approach was utilised to two effects. The first focussed on a specific notable polymorphism – Q80K in the NS3 gene – to determine whether deep sequencing would benefit the clinic in the detection of this polymorphism at low frequencies, which are below the threshold of detection by traditional population sequencing. The second use of deep sequencing aimed to determine the impact of HIV co-infection on the presence of resistance associated mutations occurring at baseline, studying the largest cohort to date. The Q80K polymorphism was not observed in any sample as a minority variant, suggesting that the current clinical procedures for pre-treatment screening are suitable and provide sufficient sensitivity. Overall, resistance mutations were relatively common among patients and it was observed that, generally, there were no differences in the prevalences of resistance mutations between HCV mono-infected and HIV/HCV co-infected patients. This finding is in agreement with previous small-scale studies. This work has direct clinical implications on the use of deep sequencing for screening patients for the presence of resistance mutations. Furthermore, it suggests that co-infected individuals are not at risk of further complications for the treatment of HCV.

The pneumococcus remains a pathogen of global importance due to its ability to circumvent antibiotic and vaccine pressures. For resource poor countries such as Malawi, the threat posed by this pathogen is perhaps the strongest, as access to timely and effective healthcare is limited. Coupled with this, high rates of HIV mean that pneumococcal disease is particularly prevalent in Malawi. The recent rollout of vaccination, anti-retroviral therapy to combat HIV, and increased availability to antimicrobials is helping to control this pathogen. However, recent genetic analyses have already demonstrated the potential for the pneumococcus to circumvent such measures, through vaccine-escape, and antibiotic resistance. The pneumococcus inhabits the nasopharynx where it is thought to recombine with other closely related mitis group species. Such recombination events are thought to have allowed this pathogen to first acquire beta-lactam resistance- a class of antibiotics favoured for the treatment of pneumococcal diseases. Survival within this niche is nonetheless challenging, as it is subject to high levels of attack by charged oxygen particles, termed oxidative stress. Oxidative stress results not only from attack by the host’s immune system, but also from the pneumococci’s metabolism. Such attack has been found to cause a wide variety of genetic lesions in the pneumococcus, and consequently the repair of such damage is likely to be essential for survival. Despite this, the pneumococcus lacks many of the normal repair machineries found in bacteria such as E. coli, and as such some have suggested a role for recombination in this repair process. Detection of genetic damage within collections of clinical pneumococcal isolates in silico indicated deletional damage occurred frequently, and genome-wide. This implied that the damage detected was not a consequence of replication errors, which would be expected to occur clustered close to the origin of replication. Deletion repair was identified as an essential mechanism in order to subjugate the fitness costs associated with such damage in situ. Recombination with siblings (self-repair) appeared to be the dominant force in this process. Attempts to model genetic damage in vitro were however limited, with exogenously delivered hydrogen peroxide appearing to poorly reflect the natural condition. It may therefore be important to consider pneumococci subject to abnormal levels of oxidative stress in clinical settings to better characterise this repair process. Recombination has additionally played a major role in the ability for pneumococci to respond to clinical interventions. The ability for pneumococci, and other mitis group species to circulate globally was found to play an important role in the acquisition of beta-lactam resistance in the Malawian pneumococcal population. Continued epidemiological surveillance of this pathogen will therefore be important in order to assess how this pathogen responds to the current clinical interventions, such as PCV13 vaccination. Although recombination within the mitis group was identified, the interactions between pneumococci and other members of this group remain poorly characterised. Such events however appear infrequent, likely restricted to a subset of the population, such as in infants. Finally, to better elucidate the pathways by which beta-lactam resistance arises clinically; a genome wide approach was taken to identifying resistance associated genes. In addition to indicating novel resistance mechanisms, a role for interspecies recombination within these sites was identified.

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In the HIV-1 infected cell, a specific recognition event has to take place between the structural polyprotein precursor known as gag, and the virion genomic RNA. This interaction is essential for ensuring that the assembling virus packages a genome. The interaction is specific as cellular RNAs and spliced genomic RNAs are largely excluded from the virus particle. Several published reports have suggested that the p55 gag protein specifically interacts with RNA sequences in the 5' untranslated region of the virion genome. This thesis describes in-vitro investigations designed to further understand this interaction with the aim of determining a minimal binding site for the HIV-1 gag protein on the genomic RNA. Using a deletion type approach the interaction was analysed by filter binding techniques with a GST-p55 gag fusion protein and in-vitro transcribed RNA. This interaction was then subsequently investigated by ribonuclease footprinting. These methodologies identified regions of RNA rich in secondary structure, between the 5' splice donor and up to and including the gag start codon, which interact with GST-p55 in solution. The location of the gag-RNA interaction in infected cells had not been established. I investigated this using subcellular fractionation techniques, UV photo affinity cross-linking of RNA-protein interactions, immunoprecipitation and RT-PCR. These experiments show that gag-RNA interactions do not occur in the nuclei of the infected cell. This interaction probably occurs at some location in the soluble cytoplasm, as gag-RNA complexes could be identified in the soluble cytoplasmic fractions after fractionation on sucrose gradients which separate out cytosol and membrane fractions. Preliminary experiments suggest that gag interacts with HIV-1 genomic RNA sequences in the cytoplasm and not subgenomic HIV-1 RNAs, as required for virion morphogenesis.

Foetal islets are functionally immature but retain their capacity for proliferation if harvested and cultured in an appropriate manner. Graft function was shown to depend largely on the gestational age and conditions of organ culture prior to transplantation. The required period of organ culture for optimal graft function was investigated for foetal mouse pancreas of different gestational ages. The growth of the graft in situ also depended on the diabetic state of the host, and chronic hyperglycaemia appeared to impair graft function. Subsequent studies using NOD recipient mice as a model for IDDM showed that recurrent autoimmune disease was seen in foetal islet isografts but rapid rejection of allografts and foetal pig xenografts also occurred. The striking differences seen between the allo-, and xenograft response was the presence of many eosinophils that dominated the infiltrate at the xenograft site. However, HAR was not a problem in this discordant xenograft and Gal(1-3)Gal expression, the major epitope for xenoreactive Ab, was not present on differentiated cells but was detectable on ductal cells. A brief treatment with a specific anti-CD4 MAb (GK1.5) had a profound effect in the survival of xenografts in NOD mice. There were consistent differences in xenograft survival and in the number of circulating T and B cells in other strains of mice, e.g. CBA, BALB/c, C57BL/6 compared to NOD mice. Prolongation of xenograft survival for up to 12 weeks was achieved with the use of peri-transplant and weekly treatment with anti-CD4 or anti-CD3 MAbs especially when the graft has been "immunomodulated" by using 90% O2 in organ culture. Using this protocol foetal pig xenografts maturing under the kidney capsule of spontaneously diabetic NOD mice reversed hyperglycaemia and appeared also to secrete growth factor(s) that induced regeneration of cells in the host pancreas.

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