The aim of this project was to test hypotheses relating to nucleosome positioning in Arabidopsis to provide a basis for better understanding of epigenetic transcriptional regulation in plants. Prior to this study, virtually no information existed regarding nucleosome positioning in plants. Eukaryote chromosomes consist of chromatin, composed of nucleosomes separated by linker DNA of variable lengths. Nucleosomes consist of 147 bp of DNA wrapped 1.7 times around a histone octamer. Whilst no consensus nucleosome positioning DNA sequence exists, sequence preferences influence positioning, and contribute to the complex epigenetic processes which act to control transcriptional activity. These details of the underlying mechanisms are known to differ between the plant and animal kingdoms. High-throughput sequencing technologies were utilised to generate large datasets of mono- and di-nucleosome sequences from wild-type Arabidopsis. These enabled genome-wide analysis and inference of plant-specific patterns of nucleosome positioning and sequence properties. Further data were generated from a methyltransferase antisense (MET1) which is depleted in methylated CG epigenetic marks. The internal distributions of dinucleotides within Arabidopsis nucleosomes were similar to those observed in non-plant eukaryotes. A unique periodicity in the distribution of linker lengths was detected in Arabidopsis wild type chromatin. In contrast, the MET1 antisense line displayed the expected periodicity, indicating systematic differences in chromatin organisation. There was a significant increase in nucleosome occupancy within exons compared with introns. However, this difference was less marked in the MET1 antisense. Specific patterns of nucleosome phasing were observed around transcription start sites. Linker lengths within rRNA gene clusters associated with nucleolar organiser regions (NORs) differed depending on chromosome of origin, suggesting differences in higher order chromatin structure between the NORs. Comparison of the nucleosome position and DNA methylation within the rRNA gene cluster revealed interesting differences between the two regions, which may reflect interactions affecting chromatin structure and transcriptional regulation.

Corticotrophin releasing hormone (CRH) and CRH receptor (CRH-R) appear to play a number of important roles in human pregnancy. The purpose of the first part of my project was to clone and sequence CRH-R subtypes from human myometrial biopsies. In order to understand the molecular mechanisms that direct the expression of the CRH-R gene, the objective of the second part of my research project was to clone and characterise the promoter region of the human CRH-R2 gene. Our results demonstrated the presence of multiple CRH-R mRNAs in the human myometrium. Six subtypes of the CRH receptor, 1a, 1b, 1c, 2a, 2b, and 2g, were found in the pregnant human myometrium, whereas only three subtypes, 1a, 1b, and 2b were found in the nonpregnant myometrium. Multiple CRH-R mRNAs have been identified in human myometrium with differential expression pattern during pregnancy, which argues for multiple roles for CRH and/or related peptides in myometrial function and suggests distinct functional roles for each receptor during pregnancy. These findings suggest that CRH and its receptor play an important modulatory role in myometrial function. The genomic organisation of human CRH-R2 alternative exons has been determined in the project. The coding region of the gene spans over 18kb. The genomic orders of the alternative exons are CRH-R2b-2g-2a. The 5'-flanking region of the human CRH-R2 gene was cloned by the genomic walking method. Using 5'RACE, the transcription start sites were mapped. The results suggest that there may be a single promoter regulating the expression of all (2a, 2b and 2g) subtypes. The CRH-R2 5'-flanking sequence has many characteristics of the housekeeping gene promoter. Therefore, we speculated that the strong and housekeeping gene-like activity of the CRH-2R gene promoter may contribute to the ubiquitous expression of the CRH-R2 gene. For functional analysis, 5'-flanking sequences up to-1393 bp were fused to the Chloramphenicol acetyltransferase (CAT) gene and tested using a HEK-293 cell line by transfection CAT assays. The results confirmed that the DNA region indicated demonstrated strong basal promoter activity.

The Xenopus pronephros is a simple paired organ; each nephron consists of a single large glomus, one set of tubules and a single duct. Previous work done in our laboratory demonstrates that the tubules and duct are specified at stages 12.5 and 14 respectively and the glomus is specified at stage 12.5. The kidney unit functions to control water balance and dispose of wastes. The simple organisation of the pronephros and the amenability of Xenopus laevis embryos to manipulation make the Xenopus pronephros an attractive system in which to study organogenesis. It has been shown that pronephric tubules can be induced to form in presumptive ectodermal tissue by treatment with RA and activin. Members of this laboratory have used this system in a subtractive hybridisation screen that resulted in the cloning of Xenopus laevis annexin IV (Xanx-4). In this thesis it has been demonstrated that Xanx-4 transcripts are specifically located to the developing pronephric tubules, and the protein to the luminal surface of these tubules. Temporal expression shows zygotic transcription is upregulated at the time of pronephric tubule specification. The temporal and spatial expression pattern of Xanx-4 suggests it may have a role in pronephric tubule development. Overexpression of Xanx-4 yields no apparent phenotype. Depletion of Xanx-4 dsRNA was tested and was shown to specifically reduce levels of Xanx-4 protein in oocytes. Xanx-4 siRNA was also tested for efficacy and was shown to cause a reduced pronephric tubule phenotype. However analysis of the effects of dsRNA and siRNA on mRNA levels have proved inconclusive and cast doubt on the usefulness of dsRNAs in Xenopus laevis. Further studies on RNAi in Xenopus will be required before it can be judged a reliable method for interfering with gene expression. Xanx-4 depletion, using morpholinos produces a shortened, enlarged tubule phenotype. The phenotype observed can be rescued by coinjection of Xanx-4 mRNA. Thus, Xanx-4 can be successfully depleted in embryos using morpholino oligos.

This thesis presents the modelling and analysis of an engineered genetic oscillator in E.coli. Genetic oscillators composed of transcriptional feedback loops are the central components of circadian clocks [16]. Thus understanding small genetic oscillators is key for understanding the complex regulatory networks of circadian clocks. In order to monitor clock function, a new colony based imaging assay was set up, based on luminescent transcriptional reporter constructs, that allows for automated data collection over long time spans and for the screening of clock mutants. Clock runs produced damped oscillatory behaviour after starting the clock by removal of the lac inducer IPTG or by giving a metabolic stimulus by transferring cells onto fresh agar plates. A detailed mathematical model of the clock was constructed, taking into account discrete and stochastic regulatory binding events at the promoter sites. From this model, using the theory of heterogeneous systems [69, 66], deterministic equations were derived and analysed to yield conditions for the occurrence of stable oscillations based on the system's nullclines. To facilitate the modelling, an algorithm was devised and implemented, that allows for automated construction of Markov chain models of gene activity states based on DNA binding events. In sum, the work constitutes the establishment and analysis of an integrated experimental and modelling system, which opens possibilities for further investigation in order to yield insight into the properties of genetic oscillators.

India is rapidly becoming established as a major player in the stem cell sector. However, concerns have been raised about the use of unproven stem cell therapies and the exploitation of parents for cord blood banking. This study aims to explore the nature of stem cell activities, how key stakeholders generate expectations around them and frame the ethical issues they raise, and why the biomedical governance system is unable to regulate these emerging practices. The study involved a survey, documentary analysis and qualitative interviews with key scientists, clinicians, representatives of firms and policymakers. The thesis observes that, unlike international commentaries which largely focus on embryonic stem cell treatments, in India it is adult and cord blood stem cells which are dominant in research and clinical settings. Expectations are configured on the basis that stem cells have the potential to: solve the problem of organ shortage; help patients with ailments; provide affordable health care; and establish India as a global player. The creation of expectations is ethically problematic given the potential health risks and economic exploitation of both native and international patients. However, the ethically contested activities are justified by clinicians on the basis that the Helsinki Declaration allows to use an experimental therapy; there are many 'desperate patients' demanding these treatments; and adult stem cells are safe. To date, the government of India appears to be unable to prevent these activities. Contrary to suggestions in previous literature and by some informants that new legislation is needed to address the problem, this thesis finds that state-led mechanisms for biomedical governance lack the ability to implement existing oversight measures. This implementation gap is partly because other forms of governance are not strong enough and partly because there are high expectations at state level aimed at establishing India as a global player in the stem cell sector.

Research on human genetic variation has shown that the human genome is not a fixed, invariant framework, but that there can be extensive structural variation. This variation includes copy number variation (CNV), which can lead to changes in DNA dosage contributing significantly to variation between individual human genomes and heritable traits. Human beta-defensins are small, secreted antimicrobial peptides encoded by DEFB genes located in a cluster of at least seven genes on 8p23.1. These genes are highly variable in copy number but accurate measurement of multiallelic copy number variants is challenging, particularly for high copy numbers, and has not been intensively studied until recently. A new PRT-based (Paralogue Ratio Test) triplex assay was developed to accurately measure the multiallelic beta-defensin copy number variation. The Triplex assay was demonstrated to be an accurate and powerful method to measure copy number variation in large case-control association studies. This method was used to study the beta-defensin CNV in psoriasis disease, showing that high beta-defensin copy number is associated with susceptibility to psoriasis in Caucasians. Studying population variation of CNV showed that variation in copy number of beta-defensin is not significantly different across human populations. To understand the evolutionary history of beta-defensin CNV in the primate lineage, the study of CNV at this locus was carried out in great apes. Beta-densin genes are copy variable in human and chimpanzee, but not in gorilla, suggesting that variation in copy number of beta-defensin genes may have arisen in the human-chimpanzee lineage after the divergence with gorilla.

Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. Htz1 (H2A.Z), a highly conserved histone H2A variant, is incorporated into the nucleosomes around the promoters of many genes in S. cerevisiae. Htz1 is involved in the maintenance of genome stability, DNA transcription activation, DNA replication and DNA repair. In this study, I employed Saccharomyces cerevisiae, as a model organism. In this thesis, I examined the role of histone Htz1 in the response to UV light at specific regions using a high resolution approach and throughout the entire yeast genome using chromatin-immunoprecipitation coupled to microarrays. htz1Δ and its deposition related mutants are more sensitive to UV and CPD removal was impaired in total DNA from both htz1Δ and swr1Δ strains. Acetylation of Htz1 at K3, 8, 10, 14 does not contribute to UV sensitivity or CPD removal from total DNA. CPD removal experiments at the MFA2 promoter and the HMRa1 coding region have showed that Htz1 has a role in NER and it likely only affects repair at local nucleosomes containing Htz1. My ChIP experiments at MFA2 and HMRa1 indicate that Htz1 enhances the binding of the HAT Gcn5 to Htz1 containing chromatin and this promotes histone H3 hyperacetylation in Htz1 nucleosomes in the MFA2 promoter after UV irradiation. As a result of this optimal level of histone H3 acetylation occur and the binding of Rad14 to damaged DNA is also enhanced at this region. My genome-wide study shows that UV does not influence the localization of Htz1 as it still resides at the pre-UV sites. Htz1 and H3 K9K14 acetylation is found to be associated with each other genome-wide. This is believed to be via the interaction between Htz1 and Gcn5. These results show that there is a positive role of Htz1 in promoting efficient GG-NER.

For the second approach, monoclonal antibodies were raised against rabbit, rat, mouse and human C6. The two most interesting antibodies were raised against human C6 and inhibited complement-mediated haemolysis in a cell-based assay. Both of these antibodies were species specific, excluding the possibility of testing their therapeutic properties in animal models of complement-mediated disease. Instead, an ex vivo model of cardiopulmonary bypass was established and used to test the ability of these antibodies to block soluble C5b-9 formation. Neither antibody inhibited soluble C5b-9 formation, suggesting that they might be interfering with the insertion of C6 into the cell membrane during MAC assembly.

Adult articular cartilage has a limited repair capacity. This leads to an increasing demand for optimised repair techniques. Furthermore, current procedures to regenerate articular cartilage fail to achieve sufficient results. Previous work within our group suggested that combination of functional tissue engineering and gene transfer represents a promising alternative approach. In this thesis, different viral gene transfer methods were investigated and optimised. A clinically relevant three dimensional transduction model was developed. These results were directly implemented in further work aiming to investigate the combined effect of multiaxial mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 on human chondroprogenitor cell chondrogenesis and progression towards hypertrophy. Two cell sources were investigated, namely human mesenchymal stem cells and human articular cartilage progenitor cells. The combined approached enhanced human mesenchymal stem cell chondrogenesis. Yet, it was not possible to completely prevent progression towards hypertrophy. For human articular cartilage progenitor cells, over-expression of bone morphogenetic protein 2 did enhance their chondrogenic differentiation potential. However, mechanical stimulation alone, in the absence of exogenous growth factors, led to stable chondrogenic induction without signs of hypertrophic differentiation. This suggests these cells should be further investigated. Additionally, the potential of Dorsomorphin, as possible agent to block hypertrophic differentiation by inhibition of bone morphogenetic protein signalling, was investigated in a fibrin polyurethane composite system, using human mesenchymal stem cells. As opposed to the pellet culture model, application of Dorsomorphin led to a cytotoxic effect which decreased the general differentiation potential. Finally, the chondrogenic potential of the two cell types was directly compared, using the pellet culture model. Under serum-free conditions, human articular cartilage progenitor cells were not able to undergo chondrogenesis. The reasons for this remain to be elucidated. The combined results of the thesis can help to develop a novel one-step procedure to treat articular cartilage defects.

Activation of complement and its terminal pathway leads to the formation and insertion of the membrane attack complex (MAC) in the membranes of target cells. Complement is activated in tumours as is clear from the presence of complement activation products in cancer tissues. Over-expression of membrane bound complement regulators on tumour cells together with endogenous recovery mechanisms restricts complement activity and results in escape from lytic killing; nevertheless, sublytic MAC deposition is not without consequence. Sublytic MAC assembly on nucleated cells causes cell activation, secretion of extracellular matrix and pro-inflammatory cytokines and may cause protection from apoptosis. Signalling of these events is unclear. The global effects of sublytic MAC were addressed in the murine colon carcinoma cell line (CT26) through the use of microarray technology. Cells were exposed to sublytic complement attack using pooled normal human serum (pNHS) and compared to MAC-inhibited controls generated using pNHS containing the C5 inhibitor OmCI. Total RNA was extracted at 0, 1 and 12 hours post-exposure and subjected to microarray analysis using the Illumina platform. Top statistically significant changes were then identified and a list of genes upregulated at both time points was uploaded to MetaCore and a gene network generated. From this a number of co-regulated genes which converged on the EGFR were highlighted. These were cxcl1, amphiregulin and matrix metalloproteinases (Mmp) 3 and 13. Both the top statistically significant and network derived genes were validated using qPCR. Changes in protein levels were then tested using western blot analyses for Mmp3 and Areg. Inhibition of the MEK/ERK, and to a lesser extent PI3K/Akt, signalling suppressed the gene upregulation that occurred in response to MAC but inhibition of p38 and JNK had no effect, implicating a MEK/ERK- PI3K co-activation. MAC deposition and not C5a/C5aR axis signalling was shown to be responsible for the mmp3 gene upregulation response. Identification of MAC-mediated events and the signalling pathways involved may provide insight into the mechanisms by which complement activation influences tumour growth. In particular the data suggest that sublytic MAC deposition might promote a gene expression response which pushes the cells to a more aggressive phenotype by the upregulation of proliferative, survival, invasion and migratory signals. This in turn will inform strategies that seek to harness complement or complement regulation in tumour immunotherapy.