No description available.

This study reports upon the investigation of a number of genetic polymorphisms in indigenous population samples of three regions of the British Isles: the Isle of Man, Cumbria and South West Scotland. Sample selection proved to be important because differences were found in the similarly selected indigenous Manx population - between donors and non-donors. In addition to a study of phenotype distributions and gene frequencies in the three selected populations, a regional analysis of the Manx data, though on a limited level, was effected. Though great difficulties were encountered obtaining indigenous samples, comparisons were performed between the Manx and population samples from selected regions of the mainland of the British Isles as well as certain north-west European populations. Possible explanations of the differences observed between the Manx and surrounding populations were proposed, but it was also suggested that an analysis of the demographic data would be most informative.

The male germline of flowering plants is a simple lineage of two cell types: generative (germ) cell and sperm cells. An asymmetric division of the microspore during pollen (male gametophyte) development produces the germ cell, which goes on to divide to form a pair of sperm that later fuse with egg and central cells at double fertilisation. The production of functional sperm cells depends upon cell cycle progression and cell specification, and three regulatory proteins – the MYB transcription factor DUO1, and the C2H2 zinc finger proteins DAZ1 and DAZ2 - acting in a gene network have been identified in Arabidopsis to be essential for both of these processes. Expression of DAZ1/DAZ2 is triggered by DUO1, and DAZ1/DAZ2 show functional redundancy. Understanding the mechanisms by which DAZ1 and DAZ2 control male germline development was a major aim of this thesis. The first objective was to explore sequence diversity among DAZ1/DAZ2 flowering plant homologues. This study established that the number of zinc finger domains differed between species, and mutating the zinc finger domains of DAZ1 had different effects on DAZ1 function. Functional analysis of DAZ1 EAR motifs showed that these sequences are important for in planta activity. DAZ1 and DAZ2 interact with the transcriptional co-repressor TPL, and a second objective was to investigate the spatiotemporal expression pattern of TPL and its family members in pollen, with male germline expression was observed for TPL and TPR2. The final objective was to identify genes under the regulation of DAZ1/DAZ2. While DAZ1 was predicted to be a transcriptional repressor, through transcriptomics it was revealed that a broad suite of genes were positively regulated by DAZ1, overlapping with targets of DUO1. The findings communicated in this thesis provide new insights into the molecular mechanisms controlling male germline development.

No description available.

Campylobacter jejuni is a significant gastrointestinal pathogen of humans. Many proposed virulence factors in C. jejuni remain poorly characterised, although many studies have already identified chemotaxis as a significant virulence factor. The chemotaxis pathway allows motile bacteria to sense and migrate toward or away from different environmental signals. Transducers like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable this pathogen to respond to changing nutrient concentrations in the intestinal environment through mediating taxis toward or away from chemoeffector stimuli. In spite of recent research to characterise the role of Tlps in chemotaxis in C. jejuni, these proteins are still not fully understood, in particular, the cytoplasmic group C Tlps (Tlp 5, 6, and 8) which have not been extensively investigated. Here, the role of group C Tlps of C. jejuni NCTC 11168 in chemotaxis, and biofilm formation were investigated. Also the localisation of those proteins was determined. The genes encoding Tlp 5, 6 and 8 were successfully mutated and complemented. Inactivation of tlps showed important phenotypes associated with motility, chemotaxis and biofilm formation. The tlp5-, Δtlp6, tlp8- mutants all showed an altered phenotype in the swarm assay. The tlp5- and tlp8- mutants swarmed significantly further than the wild-type motile-variant, whereas the Δtlp6 mutant showed no differences in spreading on plates. No significant growth differences were seen between mutants, complements and wild-type. The modified Hard-Agar Plug (t-HAP) assay and μ-slide chemotaxis assay were developed to improve the determination of ligand specificities of Tlp chemoreceptors. The t-HAP assay showed a promising quantitative result which can be used to compare different concentrations of chemoattractant in a short time. Data derived from the t-HAP assay indicated that mutation of tlp 5, 6 and 8 has a significant effect on the chemotactic response to L-serine, L-aspartate and L-proline compared to wild type. The mutant phenotypes in the μ-slide chemotaxis assay correlated with the result of the t-HAP assay, where both show that C. jejuni has preferential chemotactic patterns, with L-serine more preferred to L-proline, L-glutamate and lastly L-aspartate. The mutation of tlp5 and tlp6 change the chemotaxis patterns to L-serine and L- aspartate compared to wild type, which may be related to the adaptation of the methylation status involved in the response to chemoattracts. The tlp8- mutant showed less chemotactic response to all amino acids tested in this study compared to wild type which may relate to a link with energy taxis. For the first time the localisation of Tlps in C. jejuni using a fluorescent reporter, iLOV, has been examined where the iLOV system was expressed using different promoters. Tlp5 and Tlp8 were shown to be localised at the cell poles. In addition, this is the first report to demonstrate that c-di-GMP-responsive adaption is present in C. jejuni and that there is a link between biofilm dynamics and the level of c-di-GMP. Tlp8 was named CbdA for Campylobacter biofilm dispersion as it was found to be involved in the modulation of c-di-GMP levels to mediate biofilm dispersion. Biofilm dispersion in C. jejuni was also found to be responsive to D-amino acids. The findings in this thesis indicate that transducer like proteins (group C) have an important role not only in chemotaxis but also in biofilm formation, which may provide a basis for understanding the pathogenicity of this pathogen.

Identifying genotypes using genetic material was at first a painstaking laboratory task. In the decades since the first gene was sequenced, techniques have progressed through milestones requiring massive international collaboration. Today’s genotype sequencing facilities use high-throughput technology to sequence entire genomes within days. Despite these technological improvements, and the resultant volume of genetic data, the identification of meaningful genotype-phenotype associations has not been as straightforward as was anticipated in the pre-genome era. The genetic architecture of many common diseases is complex, and heritability often cannot be explained when simple statistical tests are used. This thesis addresses a clinically important problem in statistical genetics - that of predicting disease risk based on genotype information. First, we review progress and current limitations in genetic risk prediction. We then introduce penalised regression. This thesis focusses on ridge regression, a penalised regression approach that has shown promise in risk prediction for high-dimensional data. The choice of the ridge parameter, which controls the amount of penalisation in ridge regression, has not been addressed in the literature with the specific aim of analysing genetic data. We present a method for automatically choosing the ridge parameter based on genome-wide SNP data. Software implementing the method is available to the community. We evaluate the method using simulation studies and a real data example. A ridge regression model does not indicate the strength of association of individual variants with the outcome, a property that is often of interest to geneticists. To this end we extend a previously proposed test of significance in ridge regression models to high-dimensional data and to the logistic model which commonly occurs in the biomedical context. This test is evaluated by comparison to a permutation test, which we view as a benchmark. This test is integrated into the software package mentioned above.

Effective population size (Ne) is an important statistic in conservation science and in the broader topics of evolutionary genetics. Ne is often used to quantify the rate of evolutionary events such as losses in genetic diversity. Estimating and interpreting such quantity can however be challenging. Chapter 2 focuses on the change in allele frequency between two or more time points due to genetic drift. A new likelihood-based estimator N̂_B for contemporary Ne estimation is proposed by adopting a hidden Markov algorithm and continuous approximations. N̂_B is found to be several-fold faster than the existing methods without sacrificing accuracy. It also relaxes the upper bound of Ne to several million and which is currently limited to about 50000 due to computing limitations. Chapter 3 extends N̂_B to handle multialleleic loci through using Dirichlet-multinomial distributions. An R package is also provided and available for download. Chapter 4 explores the signatures of linkage disequilibrium (LD) between a pair of loci induced by genetic drift as a function of recombination rate and historical population sizes. E[r²] can be expressed as the weighted sum of the probability of coalescent at different time points of which information about Ne is contained. This relationship is verified by computer simulation and then applied to historical Ne estimation as illustrated in an example of Anopheles coluzzii population. A new likelihood-based routine Constrained ML is suggested in chapter 5 to estimate haplotype frequencies and r² from genotypes under Hardy-Weinberg Equilibrium. It is shown to be identical to existing EM algorithm under normal conditions but far less sensitive to initial conditions. A new “unbiased” sample size correction is also proposed to estimate r². To summarise, this work pushes the Ne estimation to its current boundary and more importantly provides suitable tools to analyse the ever-growing datasets.

Campylobacter jejuni is a major cause of bacterial food-borne disease, but the available knowledge of the biology and molecular basis of its pathogenesis is limited. Iron is an essential co-factor for the effective intestinal colonisation of C. jejuni. However, iron is not readily available due to its being tightly bound to proteins such as haemoglobin, transferrin and lactoferrin (Lf). Studies in different organisms have reported the contribution of GAPDH to iron acquisition from ferric-Tf and ferric-Lf; iron binding proteins. Therefore, this study aimed to determine the role of C.jejuni NCTC11168 GAPDH in iron uptake from host ferric-lactoferrin complex. Due to essentiality of gapA, use of the Campylobacter complementation pC46 plasmids to inactivate this gene was required. Therefore, gapA mutated strains were successfully created in which the wild type gapA allele was knocked out in a gapA merodiploid strains while the expression of the complemented gapA allele is driven by promoters of different strength. In addition, site-directed mutagenesis was used to create C150S-GAPDH protein which lacks glycolytic activity. The results of gene expression studies in this research show that the mutated gapA strains overexpressed GAPDH. Iron growth assays in MEMα medium supplemented with low concentrations of human ferric-Lf as the sole iron source indicated that GAPDH has an important role in iron uptake from Lf. This role was supported by the inhibition of the growth of C. jejuni incubated with anti-GapA antibody in cultures supplemented with ferric-Lf as the sole iron source. Moreover, GAPDH was identified for the first time as an outer membrane associated protein in C. jejuni. Using different binding assays, it was shown that GAPDH could act as an extracellular receptor for Lf. Both purified wild type GAPDH and non-glycolytic functional C150S-cjGAPDH bound different ferric-Lf family iron binding proteins, with high affinity binding of Lf compared to the other iron glycoproteins. Therefore, the binding function of GAPDH is independent from glycolytic function. In conclusion, this research demonstrates that GAPDH of C. jejuni is important agent in iron uptake from human ferric-Lf, and therefore it is probably required for successful colonisation of C. jejuni.

Accurate replication of the genome during cell division is essential for maintaining genome integrity and suppressing diseases such as cancer. DNA replication forks can stall, collapse and/or break when they encounter obstacles present in DNA. This causes DNA replication stress, which is a major source of genome instability and mutations during carcinogenesis and cancer evolution. A number of studies have investigated how cells respond to replication stress by using various genotoxic agents such as radiation or therapeutic agents. However, these agents cause genome-wide damage; thereby, preventing control over where replication forks collapse or break, and preventing replication stress-associated molecular events from being studied directly. Here, we aimed to overcome these current methodological limitations by developing an innovative FLP-FRT system. The FLP-FRT system is designed to induce the generation of broken DNA replication forks at specific loci in mammalian cells. The system utilises a mutant FLP recombinase that binds to an integrated FLP recognition target site (FRT) marked with a florescent locus labelling system and generates an irreversible protein adduct and DNA single-strand break. Upon DNA replication, the fork encounters the gap leading to fork breakage and the formation of a single-ended DNA double-strand break. Using the ANCHOR labelling tool, the ANCH3-ANCH4 sites flanking the FRT sequence were simultaneously visualized. Our findings suggest that cells with a broken fork display a notable reduction in cell viability when compared to a direct DSB, suggesting they are more cytotoxic, as hypothesized. In conclusion, our study shows that the features of FRT-FLP system offers a widely applicable and powerful tool to directly study replication stress at a very high resolution at the damage site within single cells in vivo. This novel tool permits genetic, biological and microscopic analyses of broken replication fork resolution in mammalian cells, which can shed light on important aspects of cancer cell biology and the development of new therapeutic clinical strategies.

Transposable elements (TEs) are endogenous components of eukaryotic genomes, constituting 45% of human DNA. The human genome project revealed that human endogenous retroviruses (HERVs) constitute about 8% of the sequence. HERVs are derived from sequences integrated into germ cells during retrovirus infections, up to 25 million years ago. However, most copies of HERVs are defective in multiple ways. The HERV-K family is the youngest family and likely has significant biological activity because of its protein coding capacity. HERV-K activity may be involved in a variety of cancers, and in particular may play an important role in human melanoma. In this project, HERV activity in melanoma and different cancer cell lines was investigated. Our results showed the HERV-K pol gene is expressed in melanoma and in breast, prostate and colon cancers. Furthermore, the response to serum starvation conditions is not simply related to increased expression of HERV-K genes, and changes in cellular phenotype under serum starvation are limited to particular melanoma cell lines, rather than being a general phenomenon. HERV-K env protein expression in melanoma cells was compared to normal primary melanocytes - 1% FBS serum starvation can increase expression of this protein in SKMel5 cells that retain an adherent phenotype. Moreover, env protein expression is significantly increased in T47D breast cancer cells under 1% FBS. The analysis of HERV-K LTR methylation state demonstrated that HERV K env and gag proteins in melanoma cells under 10% FBS and 1% FBS conditions decreased. The most interesting finding was the detection of 98 candidate loci as novel proviral insertions where previously these loci were annotated as solo LTRs. This result suggests that proviruses are systematically excluded from assemblies and the census of HERV-K proviruses is much greater than represented in assembled genomes. The Genome-wide amplification of proviral sequences combined with Next Generation Sequences (GAPS –NGS) is established as an effective approach to discover new proviral loci.