Adiposity is linked to reproductive efficiency as adipose tissue (AT) is known to influence the hypothalamic-pituitary-gonadal axis, although the exact roles of AT remain poorly understood. Different cattle and pig breeds exhibit different adiposity and reproductive characteristics. The hypothesis is that differences in adiposity underpin the phenotypic differences in reproductive performance in these animals. To test this hypothesis, next generation RNA sequencing analysis was completed of AT from (i) cattle (Holstein, Hereford and Aberdeen Angus cross breeds enrolled in a dietary protein feed trial), (ii) European Large White and Asian Meishan pigs and (iii) African elephants. Differentially expressed genes (DEG) were determined in each species and validation completed utilising quantitative reverse transcriptase PCR (qRT-PCR). Results revealed significant DEG involved in adipogenesis and lipogenesis between (i) breeds, (ii) resulting from the dietary protein intervention and (iii) sexes and age groups. Fatty acid (FA) composition of AT from different cattle breeds and dietary protein levels was determined using gas chromatography and compared, although significant difference was not observed. Gene networks related to vitamins A and D suggest potential nutritional influences on adipogenesis and lipogenesis, consequently vitamin levels in feed may impact adiposity and thereby animal reproductive performance. Results from the elephants indicated disparity in energy storage and utilisation between sexes and age groups, thus this can provide baseline information to improve captive population management of these animals in different life stages. By exploiting comparative inferences from previous human and animal studies to the results obtained from this study, a positive influence of adiposity on fertility was identified. Future work should verify whether differential mRNA expression of genes of interest identified are reflected in protein expression. Also additional studies into the FA profiles of other biologically relevant tissues are warranted to enable comparison of FA uptake and usage between AT and other tissues.

In eukaryotic cells, the degradation of the mRNA poly(A) tail (deadenylation) is a crucial step in the regulation of gene expression. The Ccr4-Not complex is the major deadenylase enzyme involved in the mRNA deadenylation. The complex is composed by two subunits with ribonuclease activity (Caf1 and Ccr4) and at least six non-catalytic subunits. In vertebrate cells, the duplication of the catalytic subunits Caf1 (encoded by CNOT7 and CNOT8) and Ccr4 (encoded by CNOT6 and CNOT6L) lead to the heterogeneity of the complex. The non-catalytic subunits are organised in modules, each with a specific function, allowing the recruitment of the complex on specific mRNAs. Regulatory proteins interact with the deadenylase complex tethering it on specific mRNAs and activating mRNA degradation pathways and down regulating protein expression. This study discovered and explored the interactome of the catalytic subunits Caf1 and Ccr4 and detected new interacting partners that may recruit the deadenylase complex on specific mRNAs. Nowadays, genotyping patients using whole genome and next generation sequencing technologies, allows a wider but more accurate sight of the genomic contest of a specific disease. The alteration of the function or the structure of the Ccr4-Not complex in cancer was assessed investigating the structure and function of the variants of the components of the NOT-module (CNOT1, CNOT2 and CNOT3) and the nuclease sub complex (CNOT7, CNOT6L, CNOT1, and the regulatory protein BTG1) found in various types of cancers. Finally, phylogenetic analysis of 15 mammalian species identified different evolutionary rates acting on the paralogous deadenylase subunits.

‘Cutting out’ HIV-1 proviral DNA could potentially cure a person of the infection. Genome editing approaches have been proffered for eradicating the provirus in infected persons by activating all latent viral reservoirs for further antiretroviral therapy or for the excision of the proviral DNA from memory T- cells. Previous approaches to do this have used nuclease-based tools or reprogrammed tyrosine recombinases; the former presenting unpredictable therapeutic outcomes and the latter, lengthy design time for newer tool variants if viral mutability erodes their effectiveness. Unlike nuclease-based tools that only cut DNA and rely on host-mediated repair mechanisms, chimaeric recombinases (CRs) cut DNA and carry the inherent ability to re-ligate cut ends at the cleavage site. The modular domain architecture of small serine recombinases can be redesigned to mediate site-specific recombination on non-cognate sites, by replacing the C-terminal DNA binding domains (DBDs) of serine recombinases with programmable DBDs such as Zinc Finger (ZF) proteins, TAL effector proteins and CRISPR-dCas9. For HIV-1 proviral DNA excision, CR requirement for the interaction of two recombinase-bound sites, and the lack of necessity for host cell-encoded factors should maximize the fidelity and efficiency of provirus removal. In this work, the engineering and characterization of CRs with the specificity to recognize and promote site-specific recombination at highly conserved regions within the HIV-1 proviral DNA is explored. This research provides a solid proof-of-concept for the use of CRs to target divergent novel target sequences, expanding their applicability for applied genome editing and wider biotechnological applications.

The Gram-positive, anaerobic, acetogenic bacterium Clostridium autoethanogenum is regarded as an important biocatalyst in the current advancement of industrial gas fermentation. The ever-widening diversity of industrially-relevant acetogenic bacteria has inspired a rational approach into utilising industrial waste gases as a viable feedstock, with goals of mitigating greenhouse gas emissions, and supporting an alternative means of fuel and high-value chemical production. Combined with this effort, is the expanding repertoire of gene editing tools that have allowed for the improvement of gas fermentation processes and increased spectrum of fermentative products. Despite these advances, there remain many pertinent questions, which addressed, can further the understanding of metabolism and physiology in acetogenic bacteria. This includes cell-to-cell communication and signalling, Quorum Sensing. In this project, these questions are addressed through the study of the Agr QS system in C. autoethanogenum. Signalling peptide genes, agrD1 and agrD2 were disabled separately and more importantly, in tandem, which effectively abolished Agr signalling. Phenotypic characterisation of the double agrD mutants revealed a significant increase in ethanol at the expense of acetate output. Further observations exhibited a complete utilisation of the fructose carbon source, and the inability to fully re-assimilate CO2. These findings markedly contrasted with the wild type, and both single knock-out agrD mutants. Proteomics and enzyme activity analysis of the double AgrD mutant revealed a marked down-regulation of Wood-Ljungdahl pathway genes that included the CO2-assimilating, carbon monoxide dehydrogenase / acetyl-CoA synthase complex subunits and hydrogenases. An up-regulation of alcohol dehydrogenases was observed explaining ethanol increases, alongside an unexpected upregulation of bacterial micro-compartment clusters. These findings led to the hypothesis that the C. autoethanogenum Agr system influences the ancient Wood-Ljungdahl pathway, primarily as a means of survival by managing carbon-source utilisation and regulation.

Parasitic helminths are able to survive within their hosts due to their ability to dampen immune responses by secreting molecules with anti-inflammatory and tissue repair properties. Reflecting this, there is increasing evidence of an inverse correlation between parasitic worm infection and the incidence of allergic and autoimmune disorders on a global scale. Such epidemiological evidence has led to the “hygiene hypothesis” which postulates that the recent rapid eradication of parasitic worms in developed countries has resulted in unbalanced hyper-reactive immune systems and consequently, inflammatory disease. As “worm therapy” per se is not ideal, this in turn triggered the idea that exploiting the ability of helminth-derived “immunomodulators” to dampen pathological host inflammation would potentially allow identification of the key pathogenic events in models of human inflammatory disease and hence provide a starting point for development of new and safer therapeutics. Consistent with this, as a serendipitous side-effect of its anti-inflammatory actions, ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae exhibits therapeutic potential in mouse models of inflammatory disorders such as asthma, lupus and rheumatoid arthritis (RA). RA is a chronic autoimmune inflammatory disorder that affects 1 % of the population in industrialized countries, with no known cure. This disorder causes joint destruction and leads to reduced mobility and disability. Deregulation of T cell activation has long been considered to be a major force driving inflammation and thus to date, therapies have focused on systemic anti-inflammatory treatments, which generally leave individuals immunosuppressed and open to infection. Thus, interest has begun to focus on the role(s) that synovial fibroblasts (SF) in the joint play in the early onset of the disease, the maintenance of established inflammation and even in the spread of disease to unaffected joints. This reflects that despite not being part of the immune system, SF produce pro-inflammatory cytokines during the pathogenesis of RA and also directly mediate joint destruction by secreting matrix metalloproteinases (MMPs) that damage cartilage and bone. Indeed, there is increasing evidence that the local pro-inflammatory environment pertaining in the joints drives SF to become 3 imprinted pathogenic aggressors that initiate, drive and spread joint inflammation and bone resorption during development of collagen-induced arthritis (CIA), a mouse model of RA. Intriguingly, therefore, whilst it is established that protection afforded by ES-62 against joint inflammation and bone destruction in CIA is associated with reduced production of the pathogenic cytokine, IL-17 by  and CD4+ T cells, recent evidence suggested that ES-62 could also act directly to suppress the aggressive hyper-inflammatory phenotype of SF in the joint. The molecular mechanisms involved were not defined but interestingly, given that SF express the ES-62 target TLR4 and are the only cells in the joint to express the IL-22 receptor, the parasite product appeared to harness the inflammation-resolving and/or tissue repair actions of IL-22 to suppress SF responses during the established phase of disease. Thus the core goal of this thesis was to advance our fundamental understanding of how SF become imprinted pathogenic aggressors that initiate, drive and spread joint inflammation and bone resorption in the CIA mouse model, as a surrogate for the pathogenic events in the joints in RA. In particular, the primary major aim was to investigate the impact of the local pro-inflammatory environment pertaining during disease, specifically focusing on the signalling and epigenetic mechanisms by which IL-17 and IL-22 potentially (counter)regulate the pathogenic phenotype of SF. Complementing this, another major aim was to establish whether ES-62 acted directly to modulate the phenotype of SF and thus, to identify the key mechanisms by which ES-62 could prevent SF from promoting inflammation and bone destruction and in this way render them insensitive to pro-inflammatory signals. From a therapeutic point of view, being a large immunogenic molecule, ES-62 is not suitable for use in the clinic and thus candidate small molecule analogues (SMAs) of ES-62, based around its active PC moiety have been designed, some which mimic its therapeutic potential in a variety of inflammatory disorders. Thus, it was also important to address whether ES-62 and its SMAs were similarly able to affect SF and prevent their pathogenicity. These studies revealed that the microenvironment of the joint during induction and progression of CIA did indeed result in remodelling of the epigenetic landscape of SF and that such cell reprogramming was associated with the acquisition of a hyperinflammatory, tissue destructive phenotype. Such 4 reprogramming could be recapitulated in vitro, at least in part, by chronic exposure of normal SF to pro-inflammatory cytokines such as IL-17 and IL-1 pathogenic mediators that are found at high levels in the arthritic joint. Such reprogramming was dependent on ERK and STAT3 signalling converging on miR-155-mediated regulation of inflammatory networks via global DNA hypomethylation. ES-62 was able to counteract this by suppressing the levels of ERK, STAT3 and miR-155 signalling but rather surprisingly, this did not result in abrogation of this hypomethylated epigenetic landscape. Rather, whereas the SMA 12b appeared to act simply by preventing/reversing global DNA demethylation to suppress the induction of genes that drive pathogenesis in CIA, ES-62 induced further global DNA hypomethylation and modulation of the epigenetic landscape by inducing HDAC1: collectively these findings suggested that ES-62 might additionally induce (homeostatic) inflammation-resolving and tissue repair genes that would have translational impact in established disease. In any case, these studies suggest that the proposal to use the global DNA methylation status of RA patients as a biomarker of disease should be treated with caution.

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.

Yersinia pseudotuberculosis is a mammalian enteropathogen and is the direct ancestor of Y. pestis, the causative agent of the plague. For its pathogenicity, Y. pseudotuberculosis harbours a 70 kb virulence plasmid which encodes the components of the type three secretion system (T3SS) and effector proteins. These effectors serve to evade the host immune system and induce apoptosis of mammalian cells. Consistent with many Gram-negative bacteria, Yersinia facilitate cell: cell signalling through the production and sensing of N-acylhomoserine lactones (AHLs), which functions to mediate the expression of downstream target genes. This cell-cell communication is known as quorum sensing (QS) and is facilitated by two LuxI/R-type systems in Y. pseudotuberculosis: YtbI/R and YpsI/R, and several AHL molecules. Behaviours under QS control include motility, biofilm formation, clumping and the regulation of the T3SS. Recently, QS was reported to repress the T3SS whilst the T3SS attenuated biofilm formation on Caenorhabditis elegans. Colonising both the soil/water environment and the mammalian gut, Y. pseudotuberculosis exhibits a biphasic lifestyle whereby it exerts strict temperature-dependent control over the expression of pYV-encoded genes. The switch between these two lifestyles is govered by a pair of virulence regulators: LcrF is a transcriptional activator that targets pYV-encoded genes and is key for the assembly of the T3SS. Conversely, YmoA is a histone-like protein that represses transcription of lcrF through chromatin compaction. Considering the repression of the T3SS by QS, this study set out to investigate whether this regulation is mediated by a relationship between QS and LcrF/YmoA. By using chromosomal promoter:lux fusions, QS was identified to be an activator of YmoA at both 22oC and 37oC whilst a regulatory relationship between QS and LcrF was also identified. To investigate these links further, AHL profiling of the lcrF and ymoA mutants identified YmoA as a repressor of AHL biosynthesis whilst a very subtle repression was observed in ΔlcrF, suggesting that LcrF may influence AHL synthesis indirectly. Assessing the impact of LcrF and YmoA on the QS-mediated phenotypes of Yop secretion, biofilm formation and motility extended these observations. LcrF had no effect on any of the phenotypes examined supporting the hypothesis of either an indirect mode of regulation, or no regulation at all. In contrast, YmoA influenced both motility and biofilm formation. A decreased motility of ΔymoA was observed on both semi-solid agar and in liquid whereby both the speed and the percentage of motile cells was altered. This suggests an activating role of YmoA on motility. Interestingly, QS is known to repress motility therefore it is likely that YmoA-regulation of motility occurs irrespective of QS. Comparable to that of the QS synthase mutant (ΔypsI/ytbI), biofilm was attenuated in ΔymoA yet restored when cells were cured of the virulence plasmid supporting the hypothesis that the type three-secretion injectisome disrupts biofilm formation. This attenuation of biofilm formation in ΔymoA, in conjunction with the activation of ymoA by QS, led to the hypothesis that the repression of the T3SS by QS works through YmoA. Considering these results, evidence for an interaction between QS and virulence regulators LcrF and YmoA has been confirmed. We propose a model whereby YmoA is the missing link in the QS-mediated repression of the T3SS. Activation of YmoA by QS leads to increased repression of lcrF and subsequently, of the T3SS resulting in the de-repression of this system in the absence of QS.

Theileria annulata is an intracellular, tick-transmitted apicomplexan parasite, which causes tropical theileriosis in cattle. It undergoes a complex life cycle with several distinct stages occurring within the bovine host and tick vector. ApiAP2 proteins are key candidate transcription factors for regulation of stage specific gene expression across apicomplexans. They are differentially expressed in specific developmental stages and certain ApiAP2s bind specifically to unique DNA sequence motifs. Identification of stage-specific expression of putative transcriptional regulators, the motifs they bind to and potential target genes provided the rationale for this study to understand the molecular mechanisms that control stage differentiation to the merozoite in T. annulata. The results demonstrated that T. annulata ApiAP2s show marked differences in expression levels during the parasite life cycle. ApiAP2 target DNA motifs orthologous to those in Plasmodium and Cryptosporidium were also discovered in Theileria intergenic regions, indicating that the genes downstream are potential targets of Theileria ApiAP2s. These motifs were also found in upstream regions of up-regulated TaApiAP2 genes, suggesting possible auto-regulation and an interaction network of ApiAP2 transcription factors. Importantly ApiAP2 fusion proteins up-regulated during differentiation to the merozoite stage bound to their predicted specific DNA motifs validating that ApiAP2 DNA-binding domain structure is conserved across Apicomplexa genera. Evidence was also produced that AP2 proteins play important roles in steps that commit a cell to differentiate: TA13515D is the orthologue of the AP2G factor in Plasmodium that is a major regulator of gametocytogenesis: TA16485 may be involved in down-regulation of genes during merogony and expression of TA11145 at a higher level in a cell line competent for merogony relative to a line severelly attenuated indicated involvement in regulation of this differentiation step. Discovery of multiple nuclear factors binding to a 2x(A)CACAC(A) motif implicated in autoregulation of TA11145, together with phylogenetic evidence for a clade of related domains that bind this motif suggest that multiple competing ApiAP2s may operate to regulate stochastic commitment to merozoiteproduction. Based on this data an updated stage differentiation model has been generated, with up regulation of the TA11145 gene a key event. A C-box motif association with genes implicated in establishment of the transformed host cell (TashAT, SVSP) suggests it could be important for deregulation of this event as the parasite undergoes stage differentiation. In contrast the inverse G-box was found associated with genes up-regulated from merozoite to piroplasm. EMSA analysis of parasite nuclear extract with a G/Cbox motif probe showed that the motif is an active binding site for a stage regulated nuclear factor. Specific binding of candidate TA12015 protein to the G/C-box motif was unable to be confirmed. Taken together, these results provided evidence that ApiAP2 proteins are regulators of stage-specific gene expression in T.annulata. They also provide insight into probable ApiAP2 interaction networks and support the postulation of a differentiation mechanism conserved across the Apicomplexa. Finally, the data suggests that this mechanism is stochastic and is likely to occur via a positive feedback loop generating a threshold that commits the cell to differentiate to the next stage of the life cycle.

Mitophagy allows for the removal of damaged and dysfunctional mitochondria from the cell thereby attenuating any deleterious, potentially tumorigenic effects malfunctioning mitochondria may cause. Mitophagy is a specific from of macro-autophagy whereby mitochondria are selectively degraded. What controls this specificity is an area of active research. The translocation of various proteins such as PINK1 and PARKIN, to the mitochondria prior to mitophagy is thought to act as signals for recruitment of the autophagosome to the mitochondria. However what is the initiating signal for mitophagy that causes these proteins to act remains unclear. Damaged and dysfunctional mitochondria generate increased levels of reactive oxygen species and we hypothesized that these cause the oxidation of the mitochondrial membrane poly-unsaturated lipid, cardiolipin (CL), which acts as an indicator of mitochondrial health and as an initiating signal to the mitophagic machinery. Using human fibroblasts (derived from Barth’s syndrome patients) deficient in functional tafazzin (Taz), the enzyme responsible for CL maturation (poly-unsaturation), and control fibroblasts created by re-introducing a fully functional Taz gene into the parental Barth’s syndrome cells. The frequency at which mitophagy occurs in these deficient and revertant cell lines was analysed under different oxidative stress conditions, in conjunction with other factors known to affect the occurrence of mitophagy; such as mitochondrial morphology, dynamics, mass, membrane potential and function. We observed that not only were mitochondrial morphology, dynamics and function affected by the levels of polyunsaturated CL, but that indeed mitophagy is abrogated in cells lacking expression of functional TAZ and therefore lacking mature polyunsaturated CL. Further to this initial experiments have confirmed reduced levels of oxidized CL in the Barth’s syndrome cells, which combined with the evidence of reduced mitophagy suggests this could indeed be the initiating signal for mitophagy. Thus the data presented within this thesis provides evidence of the role of polyunsaturated CL, in mitophagy and suggests that through its oxidation it provides the initiating signal for mitophagy.