Trypanosoma brucei is an evolutionarily divergent eukaryotic parasite of mammals in sub-Saharan Africa and is transmitted by the tsetse fly vector. To evade the mammalian immune response, T. brucei utilises antigenic variation, which involves switches in the Variant Surface Glycoprotein (VSG) expressed on the cell surface. Such reactions can occur at very high rates (~10-3 switches/cell/generation) and occur primarily by the recombination of VSG genes, selected from an enormous silent archive, into specialised expression sites. It has been previously shown that such VSG switching is a form of homologous recombination, as mutation of RAD51 and a related gene, RAD51-3, impairs the process. BRCA2 has emerged as a significant regulatory factor during RAD51-catalysed recombination. In humans, BRCA2 contains eight BRC repeats, six of which have been shown to bind RAD51. Similar repeats are present in BRCA2 from other organisms, though normally in smaller numbers. This thesis describes a T. brucei BRCA2 homologue that appears exceptional in that it contains up to 12 BRC repeats. Furthermore, the sequence degeneracy that is observed between the BRC repeats in most organisms is absent in T. brucei, with all but the C-terminal proximal repeat being identical. It was hypothesised that this unusual BRCA2 organisation is due to the high levels of RAD51-directed recombination needed during antigenic variation. To examine the function of the putative T. brucei BRCA2 homologue, mutants were generated and found to display impaired growth, sensitivity to induced DNA damage, impairment in the ability to form sub-nuclear RAD51 foci, a reduced ability to recombine DNA constructs into their genome and a reduction in frequency of VSG switching, all of which are consistent with roles for BRCA2 in DNA repair and recombination. Furthermore, genome instability in the mutants was observed through the loss of silent VSG gene copies and substantial reductions in the size of the mega-base chromosomes. Interestingly, other chromosome classes (the so-called mini- and intermediate-chromosomes) appear not to be susceptible to such instability. A potentially novel function for BRCA2 was identified through DNA content analysis of the T. brucei BRCA2 mutants. Mutation of BRCA2 was shown to result in an accumulation of cells with aberrant DNA content that is most readily explained by an increased number of cells that undergo cytokinesis without having completed nuclear division, phenotypes that are not observed in other T. brucei recombination mutants, such as RAD51. This result suggests that BRCA2 has a role in the regulation of cell division, with mutation causing impaired replication of T. brucei nuclear DNA, but without a cell cycle stall, leading to the accumulation of chromosomal aberrations. In order to investigate the potential role of T. brucei BRCA2 in DNA replication and the unusual BRC repeat organisation phenotypes further, various truncations of BRCA2 were expressed in a mutant background. Cell lines expressing BRCA2 with only 1 BRC repeat displayed reduced efficiency in recombination, DNA repair and RAD51 foci formation, indicating that the large BRC repeat expansion in T. brucei BRCA2 plays a critical role in the proteins function. Expression of a BRCA2 variant encompassing only the region of the protein, C-terminal to the BRC repeats appeared able to function, at least partially, in regulating cell cycle progression. Moreover, this DNA replication role appears not to be provided by conserved DNA binding motifs present within the C terminus of BRCA2 since a fusion of T. brucei BRCA2 and the parasites homologue of the replication protein A 70 kDa subunit was impaired in cell division, but was proficient in repair of DNA damage. Taken together, these data infer that T. brucei BRCA2 possesses a function that is distinct from BRCA2’s role as a regulator of RAD51, and acts in DNA replication or cell division. In addition to the above research on BRCA2, I sought to examine the factors that interact with RAD51 in T. brucei. This work demonstrated that it is possible to add an epitope tag for tandem affinity purification (TAP) to the N-terminus of RAD51 in both the bloodstream and procyclic stages of T. brucei without disrupting its function. Preliminary data suggest that TAP is potentially a feasible way of examining RAD51 interacting factors.

Injury to the peripheral nervous system (PNS) stimulates a finely regulated regenerative response that generally leads to some recovery of function. In contrast, the response to injury in the adult mammalian central nervous system (CNS) is abortive and adult CNS neurons do not normally regenerate. We used a microarray approach to identify putative regeneration-associated changes in gene expression in the L4 dorsal root ganglion (DRG) in rat models of PNS and CNS injury. Our models included crush injury to both branches of the bifurcating axon of sensory neurons with cell bodies in the DRG (DRGNs). Injury to the peripheral branch at the level of the spinal nerve (SN) results in axonal regeneration and reinnervation. Crush injury of the central branch in the dorsal root (DR) results in active regeneration up to the point of CNS entry at the DR entry zone (DREZ) and subsequent arrest of further growth, while transection injury within the CNS at the level of the dorsal columns (DC) results in abortive and unsuccessful regeneration attempts. These DRGN injury models therefore allowed us to compare the gene expression programmes elicited during active, arrested and abortive regeneration. Following a pilot microarray experiment to optimize experimental parameters and tract tracing and electrophysiological experiments to confirm time points for harvest of DRGs after DR and SN injury, respectively, male Sprague-Dawley rats underwent an L4 SN crush, an L4 DR crush or a bilateral DC transection at the L3/L4 spinal segment boundary. L4 DRGs were collected at 2 weeks (active regeneration) and 6 weeks (arrested regeneration) after DR crush. DRGs were harvested at 6 weeks after SN crush and 2 weeks after DC transection. DRGs harvested from naïve rats served as a control group. Microarray analysis (Affymetrix Rat genome 230 2.0 array) identified several hundred genes showing differential expression (5% FDR) in comparisons of regenerating with non-regenerating conditions. Selected genes were chosen for validation by qRT-PCR. These genes could represent putative regeneration-associated genes and may suggest novel therapeutic interventions to encourage regeneration of the spinal cord following injury. Additionally, we have identified genes upregulated in the DR active regeneration state relative to DR arrested state, which have relevance to root avulsion injury and may provide insight into the mechanisms that prevent regeneration of DR axons through the DREZ to re-enter the spinal cord. We also present evidence that a transcriptional programme consistent with regeneration is mounted within the DRG following DC transection. This lends support to the idea that CNS neurons have intrinsic regenerative capability and that manipulations of the CNS environment may be sufficient to permit regeneration of CNS axons.

How genetic mutations such as Single Nucleotide Polymorphisms (SNPs) affect the risk of contracting a specific disease is still an open question for numerous different medical conditions. Two problems related to SNPs analysis are (i) the selection of computational techniques to discover possible single and multiple SNP associations; and (ii) the size of the latest datasets, which may contain millions of SNPs. In order to find associations between SNPs and diseases, two popular techniques are investigated and enhanced. Firstly, the ‘Transmission Disequilibrium Test’ for familybased analysis is considered. The fixed length of haplotypes provided by this approach represents a possible limit to the quality of the obtained results. For this reason, an adaptation is proposed to select the minimum number of SNPs that are responsible for disease predisposition. Secondly, decision tree algorithms for case-control analysis in situations of unrelated individuals are considered. The application of a single tool may lead to limited analysis of the genetic association to a specific condition. Thus, a novel consensus approach is proposed exploiting the strengths of three different algorithms, ADTree, C4.5 and Id3. Results obtained suggest the new approach achieves improved performance. The recent explosive growth in size of current SNPs databases has highlighted limitations in current techniques. An example is ‘Linkage Disequilibrium’ which identifies redundancy in multiple SNPs. Despite the high accuracies obtained by this method, it exhibits poor scalability for large datasets, which severely impacts on its performance. Therefore, a new fast scalable tool based on ‘Linkage Disequilibrium’ is developed to reduce the size through the measurement and elimination of redundancy between SNPs included in the initial dataset. Experimental evidence validates the potentially improved performance of the new method.

Here the use of the in vitro spheroid conformation model, is carried out in such a way as to make use of confocal laser scanning microscopy on vital tissues. Its use in the observation of the invasion of stromal tissues by cancer tissues and the progression of this invasion dynamically was investigated. The employment of simple routine laboratory techniques and the adaptation of this method for the dynamic investigation of human tissues and cancers during the invasion process was paramount. The spheroids for the confrontations were produced from re-aggregates of cells grown in monolayer culture stained with vital fluorescent dyes CMFDA and CMTMR.. The confrontation method seemed appropriate for the examination of invasiveness as it enables the observation of the invasive process on a three dimensional structure which emulates the situation in vivo. The use of the clinically significant parameter INVASLOG is described here and its limits have been elucidated. It has been demonstrated that the use of the INVASLOG enables an objective estimation of the invasive process in vital cultures. Autologous clones of mouse melanoma K-1735, of varying invasive behaviour in vivo and human melanomas A375 and A2058 can be distinguished between in their behaviour. The investigation of the effect of retinoic acid has also demonstrated and the usefulness of this method and the invaslog parameter in investigating invasion. Its use in the investigation of and its application to, diagnosis and prognosis of cancer should be investigated particularly where specific therapies for specific cancers can be tested and the treatment regime adapted for each patient individually.

Telomeres cap the end of eukaryotic chromosomes and prevent the natural end of a chromosome from being recognised as a double-stranded DNA break. Dysfunctional telomeres may trigger replicative senescence, or fuse with other telomeres or with non-telomeric DNA breaks. The length of a telomere plays a key role in telomere function. Relatively little is known about how telomeric chromatin influences telomere length and function. A number of studies in mammalian cells have identified a handful of chromatin remodelling proteins and the chromatin marks they deposit in telomere length regulation. However such examples at human telomeres are scarce. The primary aim of this thesis was to investigate whether the chromatin structure of a telomere is a determinant of its length in human cells. Two approaches were taken to address this issue: Firstly, the chromatin structure of telomeres of differing lengths were directly analysed by measuring enrichment of histone modifications known to be prominent at telomeres in other model organisms. Secondly, selected chromatin remodelling proteins were studied to determine whether they play a role in telomeric chromatin structure and telomere length. Single Telomere Length Analysis (STELA) provides a high resolution method to measure telomere length distributions at individual chromosome ends. STELA assays were previously designed for the 2p, 9p, 11q, 12q, 16p, 17p and 18q telomeres. An allele‐specific STELA assay has also been designed for the XpYp chromosome end. In this study novel telomere and telomeric allele‐specific qPCR assays were developed for the same chromosome ends. These qPCR assays, when used in conjunction with ChIP provide a tool for analysing telomeric chromatin structure at individual chromosome ends. Applying this ChIP‐qPCR approach alongside STELA allows any correlations between telomeric chromatin structure and telomere length to be identified. This approach suggested differences in telomeric chromatin structure between telomeres of different lengths in telomerase‐positive HT1080 fibrosarcoma cells. Shorter HT1080 telomeres were less abundant in H3 and TRF1 and also had lower levels of H4K20me3 and, to a lesser extent, H3K4me3 compared to longer telomeres. Differences in chromatin structure were not observed between telomeres of different lengths in telomerase negative MRC5 fibroblasts. Changes in chromatin structure were observed at individual telomeres/telomere alleles were observed between actively proliferating cells and in cells undergoing senescence. Telomeric enrichment of H3 and TRF1 as well as the histone methylation marks H3K4me3, H3K9me3 and H4K20me3 were reduced in senescent cells. The degree of chromatin structural change as the cells entered senescence differed between chromosome ends. This highlights the benefits of using the telomere-specific ChIP-qPCR approach over the more traditional ChIP-dot blot assays which would not be able to differentiate between the chromatin structure of different chromosome ends. To identify roles for chromatin remodelling proteins in telomere length maintenance siRNA mediated knockdown of selected chromatin remodelers was performed in a clonal population of HT1080 cells followed by STELA analysis. RNAi-depletion of the histone methyltransferase (HMTase) III EHMT2 resulted in an increase in very short 17p telomeres whereas loss of another HMTase, DOT1L caused a divergence in the 17p telomere length distribution suggesting the presence of two subpopulations of cells each with differing telomere length distributions. Subtle changes in mean telomere length was observed after siRNA mediated knockdown of the HMTases MLL and EZH2, the histone deacetylases (HDACs) HDAC1 and SIRT6, the ATP dependent chromatin remodelling complex subunit BAF155 and the H3.3 histone chaperone DAXX. However due to certain limitations of the RNAi screen the validity of these observations is questionable and more work would have to be performed to confirm whether these chromatin remodelers have an effect on telomere length. Finally, dramatic telomere shortening was observed in a keratinocyte holoclone population after siRNA mediated knockdown of DAXX at number of chromosome ends. Prolonged depletion of DAXX also caused an increase in telomere‐to‐telomere fusions. A similarly dramatic loss in telomere length was seen in these cells after knockdown of EHMT2.

The present study investigated the possible involvement of nine candidate genes in NF1 tumours by assessing loss of heterozygosity, promoter hypermethylation, and expression in NF1-related tumours. The CDKN2A/p16INK4a , RB1, TP53</italic> and MGMT genes have previously been found to be altered in NF1 tumours, and this was confirmed in this study. However, new candidate genes were also found to be involved in NF1 MPNSTs and rare malignancies (RARB, MLH1 and RASSF1A).

Four genes, connected to either the inflammatory or cardiovascular system or both, were investigated for association with late onset AD in up to 180 late onset cases and 180 age- and sex-matched controls. The I allele of DCP1 has previously been reported to be associated with AD although no associations was detected in this study. Six other polymorphisms within the gene were also studied but yielded no positive genotypic, allelic or haplotype associations. The other genes studied, TACR2, a peptide receptor that maps to a region of suggestive linkage on chromosome 10, ECE1, a potent vasoconstrictor which also maps to region of suggestive linage on chromosome 1 and PI12, a serine protease inhibitor that can form amyloidogenic fragments, were screened for polymorphisms. Fifteen polymorphisms were discovered (five coding) with only one two-marker haplotype in ECE1 (p= 0.001) and a polymorphism upstream of TACR2 (p = 0.05) showing statistically significant association. Neither association retained statistical significance after adjusting for multiple testing.

Colorectal cancer is the 2nd most common cause of death by cancer in the UK, but it is treatable if diagnosed early. In order to increase the likelihood of early diagnosis, more must be understood about the early stages of colorectal tumourigenesis. It is known that intestinal stem cells (ISCs) are the cells of origin of colorectal tumourigenesis, and that an expansion of undifferentiated cell types, akin to ISCs, is one of the earliest events in mouse models of tumourigenesis. This indicates the importance of the relationship between the ISC compartment and tumourigenesis. In order to understand how changes in the ISC compartment may be contributing to tumourigenesis, the ability to accurately quantify this compartment is essential. Currently, analysis of the ISC compartment relies on the analysis of gene expression levels of ISC markers. However, there is a great deal of controversy surrounding the majority of these markers and there is no evidence that alterations in expression levels of these markers results in a functional change in the ISC compartment. Here I present a novel method for assessing the ISC compartment based on a functional capacity of ISCs; the ability to form intestinal organoids in culture. This new method uses organoid formation efficiency as a readout of changes in the ISC compartment, and can be used in conjunction with traditional methods of ISC marker expression to understand the relationship between expression of ISC markers and ISC functionality. I have used this method to further analyse the intestinal phenotype of a range of mouse models of colorectal cancer based on gene deletion of Apc, Cited1, Apc2, Pten and Pml. These experiments have shown that organoid formation efficiency can be a useful method for assessing the ISC compartment, although changes within this compartment may not be accurately predictive of tumourigenesis.

Alzheimer's disease is a neurodegenerative disorder characterised by progressive memory impairment, a decline in language function and a variety of behavioural symptoms. The majority of AD cases have an age at onset above 65 years and exhibit no clear pattern of inheritance. The only known genetic risk factor this late onset AD, LOAD, is the [Special character omitted]4 allele of the apolipoprotein E gene on chromosome 19. There is significant evidence of linkage to LOAD on chromosome 10q21-23. Therefore, nine candidate genes that map to this region were examined as susceptibility loci for the disease. In addition, a purely functional candidate, ADAM12, was examined on the basis of its homology to &alpha;-secretase enzymes, 58,752 basepairs of genomic sequence were screened by DHPLC, covering all exons, intron-exon boundaries and putative promoter regions, and 89 polymorphisms were detected. An additional 25 SNPs were identified from a SNP database. A two-stage strategy was employed. Variants were initially tested for association with AD by genotyping them in a small case-control sample, either in DNA pools or individually. Polymorphism showing a significant&nbsp; difference between cases and controls were carried forward to the second stage and individually genotyped in a larger sample. A number of SNPs initially gave a positive result but after individual genotyping in a larger sample only 2 SNPs in the ADAM12 gene showed evidence for association with LOAD. As a large number of polymorphisms were examined, this may be a false positive finding, but this gene certainly warrants further investigation.

In cell populations there is a great deal of heterogeneity. Cells can be seen to react different to the same stimulus despite being the same cell type and under the same conditions. Single cell analysis of a population is, therefore, crucial to understanding the nature of these differences. Using different technologies to study a single cell can help a researcher gather more data on the nature of these differences. The first part of this study looked at the development of a microwell array as a technique to allow correlative microscopy of non-adherent cell types. Laser ablation was used to generate microwells in both glass and the silicone polymer, polydimethylsiloxane (PDMS). Different methods were tested to increase quality of the microwells with the use of a sacrificial layer proving successful. Microwells were determined to be suitable for both live cell imaging and scanning electron microscopy imaging with correlative microscopy of non-adherent cells demonstrated. A new microwell design to allow the tracking of a single well through different imaging steps including sectioning was devised. Surface modification was required to make PDMS more cytophilic and different methods were investigated in the next part of this thesis. More cytocompatible surfaces were produced for PDMS using silanisation of the surface to produce amine moieties proved the most successful. In the final part of the thesis the cell penetrating peptide (CPP) octaargine was investigated along with the pro-apoptotic peptides PAD and the Bcl-2 converter peptide. The blebbing effects of the two peptides were analysed using non-adherent cells in microwells and fixed cells on a normal surface. Apoptosis as means of cell death by these peptides was disputed and the necrotic blebbing caused by theses peptides investigated.