Malaria, a parasitic disease caused by five species of the protozoan parasite Plasmodium, still kills an estimated 0.6 million people each year, almost all in the third world African countries. With renewed emphasis on global eradication of malaria, genome-based discovery of novel anti-transmission candidates has been identified as one of the priority research areas for the immediate future. The aim of this study was to exploit the benefits of a combination of classical forward/reverse genetics approaches, flow cytometry and high throughput whole genome sequencing to examine the molecular basis of gametocytogenesis in the rodent malaria parasite, P. berghei. Plasmodium is known to spontaneously generate gametocyte non-producing (GNP) mutants if asexually maintained for a long time. Using a sex-specific fluorescently labelled P. berghei parental line, ten parallel isogenic lineages were asexually maintained in mice by repeated mechanical passage for a year. Three, out of the potential ten lines, developed the GNP phenotype at the end of the study. The three GNP and their isogenic parental lines were sequenced using the Illumina platform and the sequence variations analysed. It was discovered that one single gene, a transcription factor with an AP2 domain (PBANKA_143750), was uniquely mutated in all the three de novo GNP mutants and also in the two pre-existing GNP mutants. The gene, called AP2-G, was thus implicated in regulating a switch associated with commitment to gametocytogenesis. Further conclusive evidence was generated using targeted AP2-G knockout studies (producing the GNP phenotype) and complementation studies in the AP2-G mutants (restoring the WT phenotype). AP2-G was also shown to recognize and bind to a conserved DNA motif in the selected gene promoters in a sequence-specific manner. Inhibition of this interaction by a synthetic customized polyamide compound, ISS-15, was also demonstrated in vitro. Collectively, the work done in the thesis (together with simultaneous independent evidence of involvement of the P. falciparum orthologue of AP2-G in gametocytogenesis) established AP2-G as the critical regulator of the commitment to gametocytogenesis in the form of a molecular switch.

Regenerative medicine and tissue engineering are being revolutionised by developments in the field of stem cell science. Mesenchymal Stem Cells (MSCs) are emerging as a desirable tool in regenerative medicine and cell therapy due to their wide ranging differentiation potential, large expansion capacity, and their lack of immune rejection following transplantation. Early in vivo studies have demonstrated therapeutic effects of hMSCs; however to clinically exploit the potential of hMSCs, the adherent cell type must be expanded to therapeutically relevant lot sizes (109 to 1012 cells). Hence now there is a need to develop protocols for stable, controlled in vitro expansion, isolation and preservation of a homogenous population of functionally viable cells. Specifically a practical, clinically safe and scalable system which adheres to current GMP guidelines is required to develop reproducible and cost effective therapeutic products. Here we describe the design, manufacture and characterisation of biofunctionalised hydrogel microcarriers containing ECM derived adhesion peptides and a range of compressive moduli for adipose derived stem cell expansion. Microfluidic devices were employed to produce monodisperse spherical particles which were polymerised in situ. In addition, these microcarriers have tunable characteristics which make them a particularly useful tool for the systematic investigation of cellular responses. Microcarriers modified to contain fibronectin and laminin derived peptides supported ADSC attachment and growth in a concentration dependent manner. ADSCs cultured on peptide modified microcarriers were capable of differentiating into osteocytes, chondrocytes and adipocytes, indicating cells cultured on microcarriers maintained multipotency. Substrate compressibility was found to effect ADSC differentiation, corroborating previous literature reports. Bioreactor culture demonstrated successful ADSC expansion with fold increases in cell number far higher than have previously been reported in the literature. High cell seeding densities produced large quantities of viable cells. However, decreasing initial cell seeding density, increased the total fold expansion and reduced cell doubling rates.

I have analysed the regulation and function of the Him gene to gain new insights into Drosophila heart development and its controlling factors. My results show that Him is important during the early specification of pericardial cells and cardioblasts. Loss of Him leads to a reduced number in both of these cell types by the end of embryogenesis. Over-expression of Him throughout the heart results in supernumerary pericardial cells. Him is expressed in all embryonic pericardial cells from embryonic stage 12 to approximately stage 15. I have identified an enhancer fragment that reproduces this expression pattern. Phylogenetic footprinting revealed three highly conserved regions within this sequence. I undertook an extensive mutational analysis of this enhancer to identify regulatory elements within it. I identified Tinman as a direct activator of Him expression. My data indicate that Him is activated in a widespread area of the dorsal mesoderm and the amnioserosa and is actively limited to the pericardial cells. A 5 bp mutation within the enhancer sequence allows for expression within the cardioblasts. Both heart cell types develop from the dorsal mesoderm and some share immediate progenitors. By stage 13, Him is pericardial cell specific and Mef2 is cardioblast specific. This is essential for normal heart development. If Him is not excluded from the cardioblasts, expression of the muscle-cell specific differentiation gene myosin is disrupted, similar to what has been described for Mef2 null mutants. If Mef2 is expressed in pericardial cells, the larval development of the pericardial cells is severely disturbed. A possible explanation for these data is that Him is part of a genetic program that prevents the premature differentiation of heart cells and its down-regulation permits the pericardial cells to undergo their correct development.

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

Transcriptional regulator protein family members Activator Protein-1 (AP1) bind to their target site TGAC/GTCA during the normal cell cycle. Their over-expression is linked to the initiation of cancer. Regulating cFos and cJun interactions with AP1 binding sites is a potential cancer therapy strategy. How the proteins find their target sites and whether non-specific DNA binding occurs will be investigated. The Protein Fragment Complementation Assay (PCA) derived inhibitor FosW is also capable of interfering with its target cJun. To study these proteins, DNA tightropes were created where single strands of λ, pUC19, pUCap1 and target-free λ (TFλ) DNA were suspended above the surface of a glass coverslip on 5 μm high pedestals. Oblique Angle Fluorescence (OAF) microscopy was used to image Quantum dot (Qdot) conjugated proteins in vitro. The protein combinations cFos:cFos, cJun:cJun, cFos:cJun, FosW and FosW+cJun (Mason et al. 2006, Worrall and Mason 2011) were studied interacting with the different DNA substrates and within the AP1 family. 71 ± 3.1% cJun:cJun, 53 ± 6.1% cFos:cJun heterodimers diffused 3-Dimensionally and 1-Dimensionally along λ DNA, indicating this is a crucial part of their search mechanism. Surprisingly, cFos is capable of dimerising, a previously unseen observation. 45 ± 3.7% of these cFos:cFos homodimers also diffused 3-Dimensionally and 1-Dimensionally. Diffusion decreased when the proteins interacted with pUC19 and pUCap1 and cJun only showed 3 ± 1.5% movement on TFλ, an unexpected observation. The interaction between FosW and cJun:cJun indicated clear interference with cJun dimerization. 55 ± 11.0% FosW and 39 ± 11.0% FosW+cJun diffused 3-Dimensionally and 1-Dimensionally. This was observed to occur directly on DNA and clarifies the mechanism of competitive inhibition and partner exchange in the AP1 family. This insight may significantly impact our understanding on how these proteins regulate transcription and help define new mechanisms of inhibition.

iv Abstract Low Copy Number Quantification of DNA Utilising Loop - mediated Amplification (LAMP) with Biolumines cent Assay in Real - Time (BART) Reporter Real time quantitative PCR is the benchmark technology of molecular diagnostics in a wide range of fields including forensic science, clinical diagnosis and the detection of genetically modified (GM) c rops. T here is a requirement for rapid, cheap and simple portable quantitative and specific diagnostics . Quantitative PCR is limited by a number of factors in this regard: t he complex hardware is often expensive and largely laboratory limited. Bioluminescent Assay in Re al Time (BART) is a nucleic acid amplification detection system that converts inorganic pyrophosphate (PPi) , a by - product of DNA synthesis , into light output. The pyrophosphate is converted into ATP which is utilised by a thermostable luciferase to convert luciferin to oxyluciferin with the emission of light. The development of isothermal amplification techniques that use the strand displacement properties of certain DNA polymerases enables the BART detection to be utilised in simple and cheap hardware at a single temperature. Loop - mediated amplification (LAMP) is an isothermal amplification method which is highly specific to the DNA target sequence and produ ces high concentrations of PPi.

Over the past few decades stem cells have been extensively investigated due to their potentially invaluable therapeutic use. Embryonic stem cells (ESCs) have wide-ranging therapeutic applications in tissue repair and regeneration due to their pluripotent properties and their ability to self-renew indefinitely. However, ethical concerns surround their use and hence alternatives are sought. Adult stem cells (ASCs) have been isolated from various adult tissues including the oral mucosa lamina propria (OMLP). This study aims to isolate ASCs from the OMLP, reprogram these cells to induced pluripotent stem cells (iPSCs) and determine the potential for both to differentiate into functional neurons due to the limited regeneration of neurons in the central nervous system. Such investigations into strategies for the treatment of neural damage are invaluable and timely due to current limitations in the availability of human-derived cells for potential autologous or allogeneic tissue repair. OMLP-PCs represent an ideal cell source for use in regenerative medicine given their ease of isolation, proliferative potential, multipotent properties and immunosuppressive activities. Work in this Thesis has now demonstrated that these oral progenitors expressed numerous pluripotency markers and for the first time, that they could be reprogrammed to iPSCs utilising safer, non-integrating plasmids, thus increasing their potential for use in clinical applications. OMLP-iPSCs were positive for a number of pluripotent stem cell markers including SSEA-4, SSEA-5, TRA-1-60, TRA-1-81, Oct-4 and Sox-2. Moreover, their expression of early stage germ layer markers indicated their potential to differentiate into cell types of the mesoderm, endoderm and ectoderm. OMLP-PCs were also demonstrated within this Thesis to differentiate down an early neural lineage as evidenced by the presence of typical neural markers (Nestin, βIII tubulin, MAP-2 and NF-M). The presence of both ligand-gated and voltage-sensitive calcium channels indicated some limited potential functional phenotype. Unfortunately, utilising the same neural differentiation methodology, OMLP-iPSCs were not able to be similarly driven down a neural pathway. None-the-less this data suggests that OMLP-PCs and OMLP-iPSCs may hold great promise for a wide range of regenerative medicine applications

Replication of the genome is a central process in cellular life, which must be tightly regulated at the risk of genomic instability. DNA replication has been extensively studied in bacteria and eukaryotes, and recently, in archaea. Universally, DNA replication is started at specific genomic sites termed origins of replication, which are recognised by an initiator factor. While in both bacteria and archaea the initiator factor is a single protein, it is assumed that this role is performed in eukaryotes by a highly conserved six-subunit origin recognition complex (ORC). Recent phylogenetic studies, however, suggest that the presence of a six-subunit ORC might not be as conserved as initially believed. Trypanosoma brucei is a protozoan parasite in which little is known about nuclear DNA replication. To date, initiation of T. brucei DNA replication has been associated with a single factor, TbORC1/CDC6, though highly diverged interacting partners have been identified. To elucidate whether T. brucei possesses a diverged ORC-like complex, TbORC1/CDC6 and its known interacting partners, TbORC1B, TbORC4, Tb7980, Tb3120, and a novel factor, Tb1120, were analysed. First, the protein sequences of these factors were re-analysed, revealing varying degrees of conservation and divergence with other eukaryotes’ ORC proteins. Second, expression silencing by inducible-RNA interference (RNAi) of TbORC1/CDC6, TbORC1B, TbORC4, and Tb3120, in procyclic form (PCF) and bloodstream form (BSF) cells, confirmed their involvement in DNA replication. Third, subcellular localisation and dynamics of TbORC1/CDC6 and its interacting partners during the cell cycle of PCF and BSF cells was investigated by immunofluorescence, revealing TbORC1B to be the sole factor to display an apparent cell cycle-dependent localisation pattern, perhaps suggesting that it might be a DNA replication regulatory factor. Finally, immunoprecipitation and gel filtration assays support the existence of an ORC-like complex, apparently large enough to be composed of TbORC1/CDC6 and known interacting partners, and potentially, additional factors. TbORC1/CDC6-binding sites and origins of replication in T. brucei have been mapped in PCF cells. Like most eukaryotes, no specific sequence elements were found to define TbORC1/CDC6-binding sites or origins of replication, and the TbORC1/CDC6-binding sites outnumbered the mapped origins, which appeared to be activated at different times during S phase. It has been reported in other eukaryotes that different cell types activate different origins or the activation timing differs. Here, origins of replication were mapped in T. brucei PCF and BSF cells, revealing a pronounced inflexibility in origin usage in these two life cycle stages. Only one, notable genome-wide difference was found: in BSF cells, the single active variant surface glycoprotein (VSG) expression site was found to be early replicating, whereas all other silent VSG expression sites were late replicating; in PCF cells however, where all VSG expression sites are silenced, these were all late replicating. These data reveal a locus-specific link between DNA replication and transcription in T. brucei, which may relate to immune evasion. The genomes of T. brucei and related kinetoplastids are highly syntenic. Since most eukaryotic origins of replication are not defined by consensus DNA sequences, but appear instead to be defined by, among other features, chromatin context and status, origins were here mapped in both L. major and L. mexicana promastigotes in order to ask if common features could be found relative to T. brucei. Surprisingly, only a single origin could be found per Leishmania chromosome, in contrast with all eukaryotes examined to date, where each chromosome is replicated from multiple detectable origins. Origin-active loci in Leishmania were found to be distinguishable from related non-origin loci in terms of size, a characteristic not observed in T. brucei, although around 40% of the mapped origins are conserved in location relative to T. brucei. These data reveal pronounced differences in replication dynamics between the two genera, despite the considerable overlap in genome organisation.

The peptide hormone, ghrelin, exerts its physiological effects through a G-protein-coupled receptor called the ghrelin-R. The ghrelin-R displays a high degree of constitutive activity, signalling through the inositol phosphate pathway in the absence of bound agonist. TMs III and VI have been reported to be central to the activation of Family A GPCRs, with interactions between the two helices stabilising the ground state. During activation conformational rearrangements result in these interactions being broken, with new contacts forming and stabilising the active state. Investigation of the ghrelin-R constitutive activity gives an insight into the mechanisms involved in receptor activation. In this study the role of specific individual residues in the ghrelin-R has been investigated and the effect of disrupting or introducing intramolecular interactions was addressed. Site-directed mutagenesis and functional assays revealed that ghrelin-R constitutive activity can be increased and decreased with mutation of residues within the TM domains, specifically TMs III, VI and VII. The extracellular loops have been found to be involved in ligand binding and activation in a number of Family A GPCRs. The residues within ECL2 of the ghrelin-R were systematically mutated to alanine to determine their role. In particular, one residue, Asn196, was identified as being critical in ghrelin-R function and may be forming stabilising interactions which maintain ghrelin-R constitutive activity. The data presented in this thesis provide an insight into the structure and function of the ghrelin-R and the underlying molecular mechanisms of ghrelin-R constitutive activity.